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Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity
Bogerd, Hal P.; Kennedy, Edward M.; Whisnant, Adam W.
ABSTRACT Analysis of the incorporation of cellular microRNAs (miRNAs) into highly purified HIV-1 virions revealed that this largely, but not entirely, mirrored the level of miRNA expression in the producer CD4+ T cells. Specifically, of the 58 cellular miRNAs detected at significant levels in the producer cells, only 5 were found in virions at a level 2- to 4-fold higher than that predicted on the basis of random cytoplasmic sampling. Of note, these included two miRNAs, miR-155 and miR-92a, that were reported previously to at least weakly bind HIV-1 transcripts. To test whether miRNA binding to the HIV-1 genome can induce virion incorporation, artificial miRNA target sites were introduced into the viral genome and a 10- to 40-fold increase in the packaging of the cognate miRNAs into virions was then observed, leading to the recruitment of up to 1.6 miRNA copies per virion. Importantly, this high level of incorporation significantly inhibited HIV-1 virion infectivity. These results suggest that target sites for cellular miRNAs can inhibit RNA virus replication at two distinct steps, i.e., during infection and during viral gene expression, thus explaining why a range of different RNA viruses appear to have evolved to avoid cellular miRNA binding to their genome. PMID:28096489
Interactions of peptide triazole thiols with Env gp120 induce irreversible breakdown and inactivation of HIV-1 virions
Background We examined the underlying mechanism of action of the peptide triazole thiol, KR13 that has been shown previously to specifically bind gp120, block cell receptor site interactions and potently inhibit HIV-1 infectivity. Results KR13, the sulfhydryl blocked KR13b and its parent non-sulfhydryl peptide triazole, HNG156, induced gp120 shedding but only KR13 induced p24 capsid protein release. The resulting virion post virolysis had an altered morphology, contained no gp120, but retained gp41 that bound to neutralizing gp41 antibodies. Remarkably, HIV-1 p24 release by KR13 was inhibited by enfuvirtide, which blocks formation of the gp41 6-helix bundle during membrane fusion, while no inhibition of p24 release occurred for enfuvirtide-resistant virus. KR13 thus appears to induce structural changes in gp41 normally associated with membrane fusion and cell entry. The HIV-1 p24 release induced by KR13 was observed in several clades of HIV-1 as well as in fully infectious HIV-1 virions. Conclusions The antiviral activity of KR13 and its ability to inactivate virions prior to target cell engagement suggest that peptide triazole thiols could be highly effective in inhibiting HIV transmission across mucosal barriers and provide a novel probe to understand biochemical signals within envelope that are involved in membrane fusion. PMID:24330857
Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection
Höhne, Kristin; Businger, Ramona; van Nuffel, Anouk; Bolduan, Sebastian; Koppensteiner, Herwig; Baeyens, Ann; Vermeire, Jolien; Malatinkova, Eva; Verhasselt, Bruno; Schindler, Michael
The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca2+ influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4+ T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis. PMID:27383627
Antigenic Properties of the HIV Envelope on Virions in Solution
Mengistu, Meron; Lewis, George K.; Lakowicz, Joseph R.
The structural flexibility found in human immunodeficiency virus (HIV) envelope glycoproteins creates a complex relationship between antigenicity and sensitivity to antiviral antibodies. The study of this issue in the context of viral particles is particularly problematic as conventional virus capture approaches can perturb antigenicity profiles. Here, we employed a unique analytical system based on fluorescence correlation spectroscopy (FCS), which measures antibody-virion binding with all reactants continuously in solution. Panels of nine anti-envelope monoclonal antibodies (MAbs) and five virus types were used to connect antibody binding profiles with neutralizing activities. Anti-gp120 MAbs against the 2G12 or b12 epitope, which marks functional envelope structures, neutralized viruses expressing CCR5-tropic envelopes and exhibited efficient virion binding in solution. MAbs against CD4-induced (CD4i) epitopes considered hidden on functional envelope structures poorly bound these viruses and were not neutralizing. Anti-gp41 MAb 2F5 was neutralizing despite limited virion binding. Similar antigenicity patterns occurred on CXCR4-tropic viruses, except that anti-CD4i MAbs 17b and 19e were neutralizing despite little or no virion binding. Notably, anti-gp120 MAb PG9 and anti-gp41 MAb F240 bound to both CCR5-tropic and CXCR4-tropic viruses without exerting neutralizing activity. Differences in the virus production system altered the binding efficiencies of some antibodies but did not enhance antigenicity of aberrant gp120 structures. Of all viruses tested, only JRFL pseudoviruses showed a direct relationship between MAb binding efficiency and neutralizing potency. Collectively, these data indicate that the antigenic profiles of free HIV particles generally favor the exposure of functional over aberrant gp120 structures. However, the efficiency of virion-antibody interactions in solution inconsistently predicts neutralizing activity in vitro. PMID:24284318
Antibodies elicited by yeast glycoproteins recognize HIV-1 virions and potently neutralize virions with high mannose N-glycans
Zhang, Hong; Fu, Hu; Luallen, Robert J.; Liu, Bingfen; Lee, Fang-Hua; Doms, Robert W.; Geng, Yu
The glycan shield on the human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein has drawn attention as a target for HIV-1 vaccine design given that an increasing number of potent and broadly neutralizing antibodies (bNAbs) recognize epitopes entirely or partially comprised of high mannose type N-linked glycans. In an attempt to generate immunogens that target the glycan shield of HIV-1, we previously engineered a triple mutant (TM) strain of Saccharomyces cerevisiae that results in exclusive presentation of high mannose type N-glycans, and identified five TM yeast glycoproteins that support strong binding of 2G12, a bNAb that targets a cluster of high mannose glycans on the gp120 subunit of Env. Here, we further analyzed the antigenicity and immunogenicity of these proteins in inducing anti-HIV responses. Our study demonstrated that the 2G12-reactive TM yeast glycoproteins efficiently bound to recently identified bNAbs including PGT125–130 and PGT135 that recognize high mannose glycan-dependent epitopes. Immunization of rabbits with a single TM yeast glycoprotein (Gp38 or Pst1), when conjugated to a promiscuous T-cell epitope peptide and coadministered with a Toll-like receptor 2 agonist, induced glycan-specific HIV-1 Env cross-reactive antibodies. The immune sera bound to both synthetic mannose oligosaccharides and gp120 proteins from a broad range of HIV-1 strains. The purified antibodies recognized and captured virions that contain both complex- and high mannose-type of N-glycans, and potently neutralized virions from different HIV-1 clades but only when the virions were enforced to retain high mannose N-glycans. This study provides insights into the elicitation of anti-carbohydrate, HIV-1 Env-cross reactive antibodies with a heterologous glycoprotein and may have applications in the design and administration of immunogens that target the viral glycan shield for development of an effective HIV-1 vaccine. PMID:26277072
Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses
McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T.; Dennison, S. Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S. Munir; Haynes, Barton F.; Tomaras, Georgia D.
Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine-induced
Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.
Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D
Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine-induced
An oligosaccharide-based HIV-1 2G12 mimotope vaccine induces carbohydrate-specific antibodies that fail to neutralize HIV-1 virions.
Joyce, Joseph G; Krauss, Isaac J; Song, Hong C; Opalka, David W; Grimm, Karen M; Nahas, Deborah D; Esser, Mark T; Hrin, Renee; Feng, Meizhen; Dudkin, Vadim Y; Chastain, Michael; Shiver, John W; Danishefsky, Samuel J
The conserved oligomannose epitope, Man(9)GlcNAc(2), recognized by the broadly neutralizing human mAb 2G12 is an attractive prophylactic vaccine candidate for the prevention of HIV-1 infection. We recently reported total chemical synthesis of a series of glycopeptides incorporating one to three copies of Man(9)GlcNAc(2) coupled to a cyclic peptide scaffold. Surface plasmon resonance studies showed that divalent and trivalent, but not monovalent, compounds were capable of binding 2G12. To test the efficacy of the divalent glycopeptide as an immunogen capable of inducing a 2G12-like neutralizing antibody response, we covalently coupled the molecule to a powerful immune-stimulating protein carrier and evaluated immunogenicity of the conjugate in two animal species. We used a differential immunoassay to demonstrate induction of high levels of carbohydrate-specific antibodies; however, these antibodies showed poor recognition of recombinant gp160 and failed to neutralize a panel of viral isolates in entry-based neutralization assays. To ascertain whether antibodies produced during natural infection could recognize the mimetics, we screened a panel of HIV-1-positive and -negative sera for binding to gp120 and the synthetic antigens. We present evidence from both direct and competitive binding assays that no significant recognition of the glycopeptides was observed, although certain sera did contain antibodies that could compete with 2G12 for binding to recombinant gp120.
The choreography of HIV-1 proteolytic processing and virion assembly.
Lee, Sook-Kyung; Potempa, Marc; Swanstrom, Ronald
HIV-1 has been the target of intensive research at the molecular and biochemical levels for >25 years. Collectively, this work has led to a detailed understanding of viral replication and the development of 24 approved drugs that have five different targets on various viral proteins and one cellular target (CCR5). Although most drugs target viral enzymatic activities, our detailed knowledge of so much of the viral life cycle is leading us into other types of inhibitors that can block or disrupt protein-protein interactions. Viruses have compact genomes and employ a strategy of using a small number of proteins that can form repeating structures to enclose space (i.e. condensing the viral genome inside of a protein shell), thus minimizing the need for a large protein coding capacity. This creates a relatively small number of critical protein-protein interactions that are essential for viral replication. For HIV-1, the Gag protein has the role of a polyprotein precursor that contains all of the structural proteins of the virion: matrix, capsid, spacer peptide 1, nucleocapsid, spacer peptide 2, and p6 (which contains protein-binding domains that interact with host proteins during budding). Similarly, the Gag-Pro-Pol precursor encodes most of the Gag protein but now includes the viral enzymes: protease, reverse transcriptase (with its associated RNase H activity), and integrase. Gag and Gag-Pro-Pol are the substrates of the viral protease, which is responsible for cleaving these precursors into their mature and fully active forms (see Fig. 1A).
Purification of HIV-1 virions by subtilisin digestion or CD45 immunoaffinity depletion for biochemical studies.
Ott, David E
The presence of cellular proteins outside and inside retroviruses can indicate the roles they play in viral biology. However, experiments examining retroviruses can be complicated by the contamination of even highly purified virion preparations with nonviral particles (either microvesicles or exosomes). Two useful methods have been developed that can remove contaminating particles from virus stocks to produce highly pure virus preparations. One approach, the subtilisin digestion procedure, enzymatically removes the proteins outside the virions. While this method is well suited for the analysis of the interior proteins in the virions, it removes the extracellular domains of the integral membrane proteins on the virion. To preserve the proteins on the exterior of the virion for biochemical studies, a CD45 immunoaffinity depletion procedure that removes vesicles by capture with antibody-linked microbeads is employed. These methods allow for the isolation of highly purified virion preparations that are suitable for a wide variety of experiments, including the biochemical characterization of cellular proteins both on and in HIV virions, examination of virion/cell interactions, and imaging of virions.
High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis.
Chen, Jianbo; Nikolaitchik, Olga; Singh, Jatinder; Wright, Andrew; Bencsics, Craig E; Coffin, John M; Ni, Na; Lockett, Stephen; Pathak, Vinay K; Hu, Wei-Shau
A long-standing question in retrovirus biology is how RNA genomes are distributed among virions. In the studies presented in this report, we addressed this issue by directly examining HIV-1 RNAs in virions using a modified HIV-1 genome that contained recognition sites for BglG, an antitermination protein in the Escherichia coli bgl operon, which was coexpressed with a fragment of BglG RNA binding protein fused to a fluorescent protein. Our results demonstrate that the majority of virions (>90%) contain viral RNAs. We also coexpressed HIV-1 genomes containing binding sites for BglG or the bacteriophage MS2 coat protein along with 2 fluorescent protein-tagged RNA binding proteins. This method allows simultaneously labeling and discrimination of 2 different RNAs at single-RNA-detection sensitivity. Using this strategy, we obtained physical evidence that virions contain RNAs derived from different parental viruses (heterozygous virion) at ratios expected from a random distribution, and we found that this ratio can be altered by changing the dimerization sequences. Our studies of heterozygous virions also support a generally accepted but unproven assumption that most particles contain 1 dimer. This study provides answers to long-standing questions in HIV-1 biology and illustrates the power and sensitivity of the 2-RNA labeling method, which can also be adapted to analyze various issues of RNA biogenesis including the detection of different RNAs in live cell imaging.
HIV-1 Integrase Binds the Viral RNA Genome and Is Essential during Virion Morphogenesis.
Kessl, Jacques J; Kutluay, Sebla B; Townsend, Dana; Rebensburg, Stephanie; Slaughter, Alison; Larue, Ross C; Shkriabai, Nikoloz; Bakouche, Nordine; Fuchs, James R; Bieniasz, Paul D; Kvaratskhelia, Mamuka
While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here, we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity, as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild-type, but not escape mutant, virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs.
M2BP inhibits HIV-1 virion production in a vimentin filaments-dependent manner
Wang, Qin; Zhang, Xiaolin; Han, Yuling; Wang, Xinlu; Gao, Guangxia
M2BP (also called 90K) is an interferon-stimulated gene product that is upregulated in HIV-1 infection. A recent study revealed that M2BP reduces the infectivity of HIV-1 by inhibiting the processing of the viral envelope protein. Here we report that in addition to reducing viral infectivity, M2BP inhibits HIV-1 virion production. We provide evidence showing that M2BP inhibits HIV-1 Gag trafficking to the plasma membrane in a vimentin-dependent manner. When vimentin filaments were collapsed by treating cells with acrylamide or by overexpression of a dominant-negative mutant of vimentin, M2BP inhibition of HIV-1 virion production was significantly relieved. We further show that M2BP interacts with both HIV-1 Gag and vimentin and thereby mediates their interactions. We propose that M2BP traps HIV-1 Gag to vimentin filaments to inhibit the transportation of HIV-1 Gag to the plasma membrane. These findings uncover a novel mechanism by which a host antiviral factor inhibits HIV-1 virion production. PMID:27604950
Evidence for direct association of Vpr and matrix protein p17 within the HIV-1 virion.
Sato, A; Yoshimoto, J; Isaka, Y; Miki, S; Suyama, A; Adachi, A; Hayami, M; Fujiwara, T; Yoshie, O
Vpr is one of the auxiliary proteins of HIV-1 and is selectively incorporated into the virion by a process involving the C-terminal p6 portion of the Gag precursor Pr55. Vpr and the matrix protein p17 are the components of the viral preintegration complex and appear to play important roles in the nuclear transport of proviral DNA in nondividing cells. In the present study, we have demonstrated by coimmunoprecipitation experiments that Vpr associates with matrix protein p17 but not with capsid protein p24 within the HIV-1 virion. Experiments employing the yeast two-hybrid GAL4 assay for protein-protein interactions also demonstrated a direct association between Vpr and the C-terminal region of matrix protein p17. Association of Vpr and the matrix protein p17 within the mature virion is consistent with their collaborative role in the nuclear transportation of the viral preintegration complex in nondividing cells such as macrophages.
Modulation of HIV-1 infectivity by MAPK, a virion-associated kinase.
Jacqué, J M; Mann, A; Enslen, H; Sharova, N; Brichacek, B; Davis, R J; Stevenson, M
Infection of a cell by human immunodeficiency virus type 1 (HIV-1) results in the formation of a reverse transcription complex in which viral nucleic acids are synthesized. Efficient disengagement of the reverse transcription complex from the cell membrane and subsequent nuclear translocation require phosphorylation of reverse transcription complex components by a virion-associated kinase. In this study, we identify the virion-associated kinase as mitogen-activated protein kinase (ERK/MAPK). Upon density gradient fractionation, MAPK, but not its activating kinase MEK, co-sedimented with viral particles. Expression of a constitutively active, but not kinase-inactive, MEK1 in virus producer cells was able to activate virion-associated MAPK in trans. Stimulation of virion-associated MAPK activity in trans by the mitogen phorbol myristate acetate (PMA) increased viral infectivity. Conversely, suppression of virion-associated MAPK by specific inhibitors of the MAPK cascade markedly impaired viral infectivity. These studies demonstrate regulation of an early step in HIV-1 infection by the host cell MAPK signal transduction pathway. PMID:9564043
Virion-incorporated alpha-enolase suppresses the early stage of HIV-1 reverse transcription.
Kishimoto, Naoki; Iga, Nozomi; Yamamoto, Kengo; Takamune, Nobutoki; Misumi, Shogo
Human immunodeficiency virus type-1 (HIV-1) particles contain not only viral-encoded but also host-encoded proteins. Interestingly, several studies showed that host proteins play a critical role in viral infectivity, replication and/or immunoreactivity in the next target cells. Here, we show that alpha-enolase (ENO1) is incorporated into HIV-1 virions and the virion-incorporated ENO1 prevents the early stage of HIV-1 reverse transcription. We found that viral particles contain two isoforms of ENO1 with different isoelectric points by two-dimensional electrophoresis. Suppression of ENO1 expression by RNA interference in the HIV-1 producer cells decreased ENO1 incorporation into virions without altering the packaging of viral structural proteins and viral production but increased viral infectivity. Although the low-level-ENO1-packaging virus maintained comparable levels of reverse transcriptase activity, viral genomic RNA and tRNA(Lys3) packaging to the control virus, its levels of early cDNA products of reverse transcription were higher than those of the control virus. In contrast, the high-level-ENO1-packaging virus, which was produced from ENO1-overexpressing cells, showed decreased infectivity and the levels of early cDNA products. Taken together, these findings reveal a novel function of ENO1 as a negative regulation factor targeting HIV-1 reverse transcription.
Estimating the fraction of progeny virions that must incorporate APOBEC3G for suppression of productive HIV-1 infection
Thangavelu, Pulari U.; Gupta, Vipul; Dixit, Narendra M.
The contest between the host factor APOBEC3G (A3G) and the HIV-1 protein Vif presents an attractive target of intervention. The extent to which the A3G–Vif interaction must be suppressed to tilt the balance in favor of A3G remains unknown. We employed stochastic simulations and mathematical modeling of the within-host dynamics and evolution of HIV-1 to estimate the fraction of progeny virions that must incorporate A3G to render productive infection unsustainable. Using three different approaches, we found consistently that a transition from sustained infection to suppression of productive infection occurred when the latter fraction exceeded ∼0.8. The transition was triggered by A3G-induced hypermutations that led to premature stop codons compromising viral production and was consistent with driving the basic reproductive number, R{sub 0}, below unity. The fraction identified may serve as a quantitative guideline for strategies targeting the A3G–Vif axis. - Highlights: • We perform simulations and mathematical modeling of the role of APOBEC3G in suppressing HIV-1 infection. • In three distinct ways, we estimate that when over 80% of progeny virions carry APOBEC3G, productive HIV-1 infection would be suppressed. • Our estimate of this critical fraction presents quantitative guidelines for strategies targeting the APOBEC3G–Vif axis.
Gp120 on HIV-1 Virions Lacks O-Linked Carbohydrate.
Stansell, Elizabeth; Panico, Maria; Canis, Kevin; Pang, Poh-Choo; Bouché, Laura; Binet, Daniel; O'Connor, Michael-John; Chertova, Elena; Bess, Julian; Lifson, Jeffrey D; Haslam, Stuart M; Morris, Howard R; Desrosiers, Ronald C; Dell, Anne
As HIV-1-encoded envelope protein traverses the secretory pathway, it may be modified with N- and O-linked carbohydrate. When the gp120s of HIV-1 NL4-3, HIV-1 YU2, HIV-1 Bal, HIV-1 JRFL, and HIV-1 JRCSF were expressed as secreted proteins, the threonine at consensus position 499 was found to be O-glycosylated. For SIVmac239, the corresponding threonine was also glycosylated when gp120 was recombinantly expressed. Similarly-positioned, highly-conserved threonines in the influenza A virus H1N1 HA1 and H5N1 HA1 envelope proteins were also found to carry O-glycans when expressed as secreted proteins. In all cases, the threonines were modified predominantly with disialylated core 1 glycans, together with related core 1 and core 2 structures. Secreted HIV-1 gp140 was modified to a lesser extent with mainly monosialylated core 1 O-glycans, suggesting that the ectodomain of the gp41 transmembrane component may limit the accessibility of Thr499 to glycosyltransferases. In striking contrast to these findings, gp120 on purified virions of HIV-1 Bal and SIV CP-MAC lacked any detectable O-glycosylation of the C-terminal threonine. Our results indicate the absence of O-linked carbohydrates on Thr499 as it exists on the surface of virions and suggest caution in the interpretation of analyses of post-translational modifications that utilize recombinant forms of envelope protein.
Quantitative Correlation between Infectivity and Gp120 Density on HIV-1 Virions Revealed by Optical Trapping Virometry.
DeSantis, Michael C; Kim, Jin H; Song, Hanna; Klasse, Per Johan; Cheng, Wei
The envelope glycoprotein (Env) gp120/gp41 is required for HIV-1 infection of host cells. Although in general it has been perceived that more Env gives rise to higher infectivity, the precise quantitative dependence of HIV-1 virion infectivity on Env density has remained unknown. Here we have developed a method to examine this dependence. This method involves 1) production of a set of single-cycle HIV-1 virions with varied density of Env on their surface, 2) site-specific labeling of Env-specific antibody Fab with a fluorophore at high efficiency, and 3) optical trapping virometry to measure the number of gp120 molecules on individual HIV-1 virions. The resulting gp120 density per virion is then correlated with the infectivity of the virions measured in cell culture. In the presence of DEAE-dextran, the polycation known to enhance HIV-1 infectivity in cell culture, virion infectivity follows gp120 density as a sigmoidal dependence and reaches an apparent plateau. This quantitative dependence can be described by a Hill equation, with a Hill coefficient of 2.4 ± 0.6. In contrast, in the absence of DEAE-dextran, virion infectivity increases monotonically with gp120 density and no saturation is observed under the experimental conditions. These results provide the first quantitative evidence that Env trimers cooperate on the virion surface to mediate productive infection by HIV-1. Moreover, as a result of the low number of Env trimers on individual virions, the number of additional Env trimers per virion that is required for the optimal infectivity will depend on the inclusion of facilitating agents during infection.
Capillarity-induced disassembly of virions in carbon nanotubes
NASA Astrophysics Data System (ADS)
Fan, Xiaobin; Barclay, J. Elaine; Peng, Wenchao; Li, Yang; Li, Xianyu; Zhang, Guoliang; Evans, David J.; Zhang, Fengbao
Studying the transport and fate of viruses through nanochannels is of great importance. By using the nanochannel of a carbon nanotube (CNT) as an ideal model, we evaluated the possibility of capillarity-induced viral transport through a closely fitting nanochannel and explored the mechanisms involved. It is shown both experimentally and theoretically that Cowpea mosaic virus can enter CNTs by capillarity. However, when introduced into a nanotube the protein capsid may disassemble. During the initial capillary filling stage, anomalous needle-shaped high pressure exists in the centre of the nanotube's entrance. This high pressure, combining with the significant negative pressure within the nanotube, may account for the disassembly of the virions.
Unusual features of protein interaction in human immunodeficiency virus (HIV) virions.
Bukrinskaya, A G; Sharova, N K
The treatment of HIV-1 virions with ionic and nonionic detergents (NP 40, octylglucoside, Na deoxycholate) resulted in an effect unusual for enveloped viruses: instead of solubilization of glycoproteins, the core protein p24 was solubilized while envelope glycoproteins with other structural proteins were found in subviral particles. These data are consistent with a model of HIV structural organization in which glycoproteins are included in the matrix formed by the protein p 17 and suggest that p24 is neither involved in the matrix nor closely bound to any viral proteins.
TRIM5α associates with proteasomal subunits in cells while in complex with HIV-1 virions
Background The TRIM5 proteins are cellular restriction factors that prevent retroviral infection in a species-specific manner. Multiple experiments indicate that restriction activity requires accessory host factors, including E2-enzymes. To better understand the mechanism of restriction, we conducted yeast-two hybrid screens to identify proteins that bind to two TRIM5 orthologues. Results The only cDNAs that scored on repeat testing with both TRIM5 orthologues were the proteasome subunit PSMC2 and ubiquitin. Using co-immunoprecipitation assays, we demonstrated an interaction between TRIM5α and PSMC2, as well as numerous other proteasome subunits. Fluorescence microscopy revealed co-localization of proteasomes and TRIM5α cytoplasmic bodies. Forster resonance energy transfer (FRET) analysis indicated that the interaction between TRIM5 and PSMC2 was direct. Previous imaging experiments demonstrated that, when cells are challenged with fluorescently-labeled HIV-1 virions, restrictive TRIM5α orthologues assemble cytoplasmic bodies around incoming virion particles. Following virus challenge, we observed localization of proteasome subunits to rhTRIM5α cytoplasmic bodies that contained fluorescently labeled HIV-1 virions. Conclusions Taken together, the results presented here suggest that localization of the proteasome to TRIM5α cytoplasmic bodies makes an important contribution to TRIM5α-mediated restriction. PMID:22078707
Immunomagnetic quantitative immuno-PCR for detection of less than one HIV-1 virion.
Barletta, Janet; Bartolome, Amelita; Constantine, Niel T
Methods that allow the accurate and reliable detection of ultra-low molecular levels of proteins using techniques such as quantitative immuno-PCR (qIPCR) have demonstrated numerous technical difficulties. Protein detection methods lose specificity when the protein target is immersed within a matrix of thousands of molecules having wide ranges of concentrations. In addition, sensitivities are limited because of high background signals. To validate the performance of an immunomagnetic bead qIPCR method designed to remove the 'matrix' effect for HIV-1 p24 antigen detection, regression analyses were performed using samples from patients infected with HIV-1 diluted to approximately 100-1000, 10-100, 1-10, and 0.1-1.0 HIV-1 p24 Ag molecules/reaction. The number of HIV-1 p24 Ag molecules was derived from quantified HIV-1 RNA determinations. The modified immunomagnetic qIPCR bead assay demonstrated a limit of quantification of 10-100 HIV-1 p24 molecules per reaction, with an average correlation coefficient of 0.948+/-0.028 over a 4-log dynamic range. This method detects less than one HIV-1 virion (a limit of detection unreported previously for HIV-1), and thus, has the potential to identify HIV-1 infection and monitor the dynamics of the disease course earlier than nucleic acid methods. The immunomagnetic qIPCR bead assay is a simple and inexpensive method for ultra-low protein detection of infectious agents, toxins, and cancer markers at a level unrecognized previously using any enzymatic or molecular method.
Prostaglandin E2 Reduces the Release and Infectivity of New Cell-Free Virions and Cell-To-Cell HIV-1 Transfer
Serramía, María Jesús; Martínez-Bonet, Marta; Muñoz-Fernández, María Ángeles
Background The course of human immunodeficiency virus type-1 (HIV-1) infection is influenced by a complex interplay between viral and host factors. HIV infection stimulates several proinflammatory genes, such as cyclooxigense-2 (COX-2), which leads to an increase in prostaglandin (PG) levels in the plasma of HIV-1-infected patients. These genes play an indeterminate role in HIV replication and pathogenesis. The effect of prostaglandin E2 (PGE2) on HIV infection is quite controversial and even contradictory, so we sought to determine the role of PGE2 and the signal transduction pathways involved in HIV infection to elucidate possible new targets for antiretrovirals. Results Our results suggest that PGE2 post-infection treatment acts in the late stages of the viral cycle to reduce HIV replication. Interestingly, viral protein synthesis was not affected, but a loss of progeny virus production was observed. No modulation of CD4 CXCR4 and CCR5 receptor expression, cell proliferation, or activation after PGE2 treatment was detected. Moreover, PGE2 induced an increase in intracellular cAMP (cyclic AMP) levels through the EP2/EP4 receptors. PGE2 effects were mimicked by dbcAMP and by a specific Epac (exchange protein directly activated by cyclic AMP) agonist, 8-Cpt-cAMP. Treatment with PGE2 increased Rap1 activity, decreased RhoA activity and subsequently reduced the polymerization of actin by approximately 30% compared with untreated cells. In connection with this finding, polarized viral assembly platforms enriched in Gag were disrupted, altering HIV cell-to-cell transfer and the infectivity of new virions. Conclusions Our results demonstrate that PGE2, through Epac and Rap activation, alters the transport of newly synthesized HIV-1 components to the assembly site, reducing the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer. PMID:24586238
The phosphorylation of HIV-1 Gag by atypical protein kinase C facilitates viral infectivity by promoting Vpr incorporation into virions
Background Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. The Gag C-terminal p6 domain contains short sequence motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. Gag p6 has also been found to be phosphorylated during HIV-1 infection and this event may affect virus replication. However, the kinase that directs the phosphorylation of Gag p6 toward virus replication remains to be identified. In our present study, we identified this kinase using a proteomic approach and further delineate its role in HIV-1 replication. Results A proteomic approach was designed to systematically identify human protein kinases that potently interact with HIV-1 Gag and successfully identified 22 candidates. Among this panel, atypical protein kinase C (aPKC) was found to phosphorylate HIV-1 Gag p6. Subsequent LC-MS/MS and immunoblotting analysis with a phospho-specific antibody confirmed both in vitro and in vivo that aPKC phosphorylates HIV-1 Gag at Ser487. Computer-assisted structural modeling and a subsequent cell-based assay revealed that this phosphorylation event is necessary for the interaction between Gag and Vpr and results in the incorporation of Vpr into virions. Moreover, the inhibition of aPKC activity reduced the Vpr levels in virions and impaired HIV-1 infectivity of human primary macrophages. Conclusion Our current results indicate for the first time that HIV-1 Gag phosphorylation on Ser487 is mediated by aPKC and that this kinase may regulate the incorporation of Vpr into HIV-1 virions and thereby supports virus infectivity. Furthermore, aPKC inhibition efficiently suppresses HIV-1 infectivity in macrophages. aPKC may therefore be an intriguing therapeutic target for HIV-1 infection. PMID:24447338
Human Papillomavirus (HPV) virion induced cancer and subfertility, two sides of the same coin.
Depuydt, T; Beert, J; Bosmans, E; Salembier, G
In the natural history of HPV infections, the HPV virions can induce two different pathways, namely the infec- tious virion producing pathway and the clonal transforming pathway. An overview is given of the burden that is associated with HPV infections that can both lead to cervical cancer and/or temporal subfertility. That HPV infections cause serious global health burden due to HPV-associated cancers is common knowledge, but that it is also responsible for a substantial part of idiopathic subfertility is greatly underestimated. The bulk of the detected HPV DNA whether in men or women is however infectious from origin. Because the dissociation between HPV viruses and HPV virions or infection and disease remains difficult for clinicians as well as for HPV detection, we propose a review of the different effects caused by the two different HPV virion induced pathways, and highlight the mechanisms that are responsible for causing transient subfertility and cancer.
Human Papillomavirus (HPV) virion induced cancer and subfertility, two sides of the same coin
Depuydt, T; Beert, J; Bosmans, E; Salembier, G
Abstract In the natural history of HPV infections, the HPV virions can induce two different pathways, namely the infec- tious virion producing pathway and the clonal transforming pathway. An overview is given of the burden that is associated with HPV infections that can both lead to cervical cancer and/or temporal subfertility. That HPV infections cause serious global health burden due to HPV-associated cancers is common knowledge, but that it is also responsible for a substantial part of idiopathic subfertility is greatly underestimated. The bulk of the detected HPV DNA whether in men or women is however infectious from origin. Because the dissociation between HPV viruses and HPV virions or infection and disease remains difficult for clinicians as well as for HPV detection, we propose a review of the different effects caused by the two different HPV virion induced pathways, and highlight the mechanisms that are responsible for causing transient subfertility and cancer. PMID:28210481
How do cell-free HIV virions avoid infecting dead-end host cells and cell fragments?
Lyengar, Sujatha; Schwartz, David H
HIV faces the challenge of identifying and entering suitable host cells (i.e. activated and viable) among a wide array of receptor-positive but unsuitable targets. Lymph nodes contain resting cells, activated cells destined for apoptosis within 24 h, and cell fragments, all of which represent replicative dead ends. We postulate that 1) HIV virions have evolved the ability to probe the internal status of potential host cells from the external cell membrane by assessing the ability of cells to co-cap CD4 and chemokine receptors, and 2) the requirement for dual receptor binding in a concerted manner by three gp120 molecules is the molecular mechanism by which virions stochastically ensure high density co-capping of receptors. Cell-associated HIV accomplishes the same selective process by targeting cells capable of participating in immunological synapse formation.
Concentration and purification of HIV-1 virions by microfluidic separation of superparamagnetic nanoparticles
Chen, Grace Dongqing; Alberts, Catharina Johanna
The low concentration and complex sample matrix of many clinical and environmental viral samples presents a significant challenge in the development of low cost, point-of-care viral assays. To address this problem, we investigated the use of a microfluidic passive magnetic separator combined with on-chip mixer to both purify and concentrate whole particle HIV-1 virions. Virus-containing plasma samples are first mixed to allow specific binding of the viral particles with antibody-conjugated superparamagnetic nanoparticles, and several passive mixer geometries were assessed for their mixing efficiencies. The virus-nanoparticle complexes are then separated from the plasma in a novel magnetic separation chamber, where packed micron-sized ferromagnetic particles serve as high magnetic gradient concentrators for an externally applied magnetic field. Thereafter, a viral lysis buffer was flowed through the chip and the released HIV proteins were assayed off-chip. Viral protein extraction efficiencies of 62% and 45% were achieved at 10uL/min and 30uL/min throughputs respectively. More importantly, an 80-fold concentration was observed for an initial sample volume of 1mL, and a 44-fold concentration for an initial sample volume of 0.5mL. The system is broadly applicable to microscale sample preparation of any viral sample and can be used for nucleic acid extraction as well as 40–80 fold enrichment of target viruses. PMID:19954210
Mechanism of Enhanced HIV Restriction by Virion Coencapsidated Cytidine Deaminases APOBEC3F and APOBEC3G.
Ara, Anjuman; Love, Robin P; Follack, Tyson B; Ahmed, Khawaja A; Adolph, Madison B; Chelico, Linda
The APOBEC3 (A3) enzymes, A3G and A3F, are coordinately expressed in CD4(+) T cells and can become coencapsidated into HIV-1 virions, primarily in the absence of the viral infectivity factor (Vif). A3F and A3G are deoxycytidine deaminases that inhibit HIV-1 replication by inducing guanine-to-adenine hypermutation through deamination of cytosine to form uracil in minus-strand DNA. The effect of the simultaneous presence of both A3G and A3F on HIV-1 restriction ability is not clear. Here, we used a single-cycle infectivity assay and biochemical analyses to determine if coencapsidated A3G and A3F differ in their restriction capacity from A3G or A3F alone. Proviral DNA sequencing demonstrated that compared to each A3 enzyme alone, A3G and A3F, when combined, had a coordinate effect on hypermutation. Using size exclusion chromatography, rotational anisotropy, and in vitro deamination assays, we demonstrate that A3F promotes A3G deamination activity by forming an A3F/G hetero-oligomer in the absence of RNA which is more efficient at deaminating cytosines. Further, A3F caused the accumulation of shorter reverse transcripts due to decreasing reverse transcriptase efficiency, which would leave single-stranded minus-strand DNA exposed for longer periods of time, enabling more deamination events to occur. Although A3G and A3F are known to function alongside each other, these data provide evidence for an A3F/G hetero-oligomeric A3 with unique properties compared to each individual counterpart.
Enhancing Virion Tethering by BST2 Sensitizes Productively and Latently HIV-infected T cells to ADCC Mediated by Broadly Neutralizing Antibodies
Pham, Tram N. Q.; Lukhele, Sabelo; Dallaire, Frédéric; Perron, Gabrielle; Cohen, Éric A.
Binding of anti-HIV antibodies (Abs) to envelope (Env) glycoproteins on infected cells can mark them for elimination via antibody-dependent cell-mediated cytotoxicity (ADCC). BST2, a type I interferon (IFN)-stimulated restriction factor that anchors nascent Env-containing virions at the surface of infected cells has been shown to enhance ADCC functions. In a comprehensive analysis of ADCC potency by neutralizing anti-HIV Abs (NAbs), we show in this study that NAbs are capable of mediating ADCC against HIV-infected T cells with 3BNC117, PGT126 and PG9 being most efficient. We demonstrate that HIV-induced BST2 antagonism effectively attenuates Ab binding and ADCC responses mediated by all classes of NAbs that were tested. Interestingly, IFNα treatment can reverse this effect in a BST2-dependent manner. Importantly, while reactivated latent T cell lines display some susceptibility to ADCC mediated by broadly NAbs, inactivating BST2 viral countermeasures and/or exogenous IFNα augment their elimination. Overall, our findings support the notion that NAbs can induce ADCC. They highlight that while BST2 antagonism by HIV promotes ADCC evasion, strategies aimed at restoring BST2 restriction could improve anti-HIV responses and potentially provide a means to eliminate reactivated cells in latent reservoirs. PMID:27853288
HIV-1 gag proteins in virions and in infected cell fractions.
Sharova, N K; Grigor'ev, V B; Bukrinskaya, A G
The relation of the initial products of the HIV-1 gag gene to the final products was determined in virus samples and cell fractions of infected H9 and Jurkat-tat cell cultures. The proteins were identified by immunoblotting with pooled sera from AIDS patients or monoclonal antibodies. The proportion in the virions of gag precursor proteins and the products of their proteolytic cleavage varied according to the maturity of the virus particles as determined by electron microscopy. The distribution of viral gag proteins in the cell fractions was determined 2, 4, and 24 h after infection. Treatment of cells with cycloheximide to block de novo protein synthesis did not significantly affect the results. Gag proteins containing the N terminus of the precursor p55 (including p55, the intermediate precursors p41(45) and p39, and mature protein p17) were found in the cell nuclei up to 24 h after infection. The major core protein p24 was located in the cytoplasmic fraction. These data strongly suggest that gag precursors from the p55 N terminus and the matrix protein p17 enter the infected cell separately from the major core protein p24, or become separated from it in the cytoplasm.
Probing the HIV-1 genomic RNA trafficking pathway and dimerization by genetic recombination and single virion analyses.
Moore, Michael D; Nikolaitchik, Olga A; Chen, Jianbo; Hammarskjöld, Marie-Louise; Rekosh, David; Hu, Wei-Shau
Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking.
Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding
Panico, Maria; Bouché, Laura; Binet, Daniel; O'Connor, Michael-John; Rahman, Dinah; Pang, Poh-Choo; Canis, Kevin; North, Simon J.; Desrosiers, Ronald C.; Chertova, Elena; Keele, Brandon F.; Bess, Julian W.; Lifson, Jeffrey D.; Haslam, Stuart M.; Dell, Anne; Morris, Howard R.
The surface envelope glycoprotein (SU) of Human immunodeficiency virus type 1 (HIV-1), gp120SU plays an essential role in virus binding to target CD4+ T-cells and is a major vaccine target. Gp120 has remarkably high levels of N-linked glycosylation and there is considerable evidence that this "glycan shield" can help protect the virus from antibody-mediated neutralization. In recent years, however, it has become clear that gp120 glycosylation can also be included in the targets of recognition by some of the most potent broadly neutralizing antibodies. Knowing the site-specific glycosylation of gp120 can facilitate the rational design of glycopeptide antigens for HIV vaccine development. While most prior studies have focused on glycan analysis of recombinant forms of gp120, here we report the first systematic glycosylation site analysis of gp120 derived from virions produced by infected T lymphoid cells and show that a single site is exclusively substituted with complex glycans. These results should help guide the design of vaccine immunogens. PMID:27604319
Cocaine Enhances HIV-1–Induced CD4+ T-Cell Apoptosis
Pandhare, Jui; Addai, Amma B.; Mantri, Chinmay K.; Hager, Cynthia; Smith, Rita M.; Barnett, Louis; Villalta, Fernando; Kalams, Spyros A.; Dash, Chandravanu
Substance abuse is a major barrier in eradication of the HIV epidemic because it serves as a powerful cofactor for viral transmission, disease progression, and AIDS-related mortality. Cocaine, one of the commonly abused drugs among HIV-1 patients, has been suggested to accelerate HIV disease progression. However, the underlying mechanism remains largely unknown. Therefore, we tested whether cocaine augments HIV-1–associated CD4+ T-cell decline, a predictor of HIV disease progression. We examined apoptosis of resting CD4+ T cells from HIV-1–negative and HIV-1–positive donors in our study, because decline of uninfected cells plays a major role in HIV-1 disease progression. Treatment of resting CD4+ T cells with cocaine (up to 100 μmol/L concentrations) did not induce apoptosis, but 200 to 1000 μmol/L cocaine induced apoptosis in a dose-dependent manner. Notably, treatment of CD4+ T cells isolated from healthy donors with both HIV-1 virions and cocaine significantly increased apoptosis compared with the apoptosis induced by cocaine or virions alone. Most important, our biochemical data suggest that cocaine induces CD4+ T-cell apoptosis by increasing intracellular reactive oxygen species levels and inducing mitochondrial depolarization. Collectively, our results provide evidence of a synergy between cocaine and HIV-1 on CD4+ T-cell apoptosis that may, in part, explain the accelerated disease observed in HIV-1–infected drug abusers. PMID:24486327
The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.
van Bel, Nikki; van der Velden, Yme; Bonnard, Damien; Le Rouzic, Erwann; Das, Atze T; Benarous, Richard; Berkhout, Ben
The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.
Cocaine enhances HIV-1-induced CD4(+) T-cell apoptosis: implications in disease progression in cocaine-abusing HIV-1 patients.
Pandhare, Jui; Addai, Amma B; Mantri, Chinmay K; Hager, Cynthia; Smith, Rita M; Barnett, Louis; Villalta, Fernando; Kalams, Spyros A; Dash, Chandravanu
Substance abuse is a major barrier in eradication of the HIV epidemic because it serves as a powerful cofactor for viral transmission, disease progression, and AIDS-related mortality. Cocaine, one of the commonly abused drugs among HIV-1 patients, has been suggested to accelerate HIV disease progression. However, the underlying mechanism remains largely unknown. Therefore, we tested whether cocaine augments HIV-1-associated CD4(+) T-cell decline, a predictor of HIV disease progression. We examined apoptosis of resting CD4(+) T cells from HIV-1-negative and HIV-1-positive donors in our study, because decline of uninfected cells plays a major role in HIV-1 disease progression. Treatment of resting CD4(+) T cells with cocaine (up to 100 μmol/L concentrations) did not induce apoptosis, but 200 to 1000 μmol/L cocaine induced apoptosis in a dose-dependent manner. Notably, treatment of CD4(+) T cells isolated from healthy donors with both HIV-1 virions and cocaine significantly increased apoptosis compared with the apoptosis induced by cocaine or virions alone. Most important, our biochemical data suggest that cocaine induces CD4(+) T-cell apoptosis by increasing intracellular reactive oxygen species levels and inducing mitochondrial depolarization. Collectively, our results provide evidence of a synergy between cocaine and HIV-1 on CD4(+) T-cell apoptosis that may, in part, explain the accelerated disease observed in HIV-1-infected drug abusers.
Effects of HCV on Basal and Tat-Induced HIV LTR Activation
Sengupta, Satarupa; Powell, Eleanor; Kong, Ling; Blackard, Jason T.
Hepatitis C virus (HCV) co-infection occurs in ∼30–40% of the HIV-infected population in the US. While a significant body of research suggests an adverse effect of HIV on HCV replication and disease progression, the impact of HCV on HIV infection has not been well studied. Increasing data suggest that hepatocytes and other liver cell populations can serve as reservoirs for HIV replication. Therefore, to gain insight into the impact of HCV on HIV, the effects of the HCV Core protein and infectious hepatitis C virions were evaluated on basal and Tat-induced activation of the HIV long terminal repeat (LTR) in hepatocytes. The HIV LTR was highly induced by the HIV transactivator protein Tat in hepatocytes. Activation varied according to the number of NF-kB binding sites present in the LTRs from different HIV subtypes. Involvement of the NF-kB binding pathway in LTR activation was demonstrated using an NF-kB inhibitor and deletion of the NF-kB binding sites. TNFα, a pro-inflammatory cytokine that plays an important role in HIV pathogenesis, also induced LTR activity in hepatocytes. However, HIV LTR activity was suppressed in hepatocytes in the presence of HCV Core protein, and the suppressive effect persisted in the presence of TNFα. In contrast, infectious hepatitis C virions upregulated HIV LTR activation and gene transcription. Core-mediated suppression remained unaltered in the presence of HCV NS3/4A protein, suggesting the involvement of other viral/cellular factors. These findings have significant clinical implications as they imply that HCV could accelerate HIV disease progression in HIV/HCV co-infected patients. Such analyses are important to elucidate the mechanisms by which these viruses interact and could facilitate the development of more effective therapies to treat HIV/HCV co-infection. PMID:23762271
Enhanced expression of HIV and SIV vaccine antigens in the structural gene region of live attenuated rubella viral vectors and their incorporation into virions.
Virnik, Konstantin; Ni, Yisheng; Berkower, Ira
Despite the urgent need for an HIV vaccine, its development has been hindered by virus variability, weak immunogenicity of conserved epitopes, and limited durability of the immune response. For other viruses, difficulties with immunogenicity were overcome by developing live attenuated vaccine strains. However, there is no reliable method of attenuation for HIV, and an attenuated strain would risk reversion to wild type. We have developed rubella viral vectors, based on the live attenuated vaccine strain RA27/3, which are capable of expressing important HIV and SIV vaccine antigens. The rubella vaccine strain has demonstrated safety, immunogenicity, and long lasting protection in millions of children. Rubella vectors combine the growth and immunogenicity of live rubella vaccine with the antigenicity of HIV or SIV inserts. This is the first report showing that live attenuated rubella vectors can stably express HIV and SIV vaccine antigens at an insertion site located within the structural gene region. Unlike the Not I site described previously, the new site accommodates a broader range of vaccine antigens without interfering with essential viral functions. In addition, antigens expressed at the structural site were controlled by the strong subgenomic promoter, resulting in higher levels and longer duration of antigen expression. The inserts were expressed as part of the structural polyprotein, processed to free antigen, and incorporated into rubella virions. The rubella vaccine strain readily infects rhesus macaques, and these animals will be the model of choice for testing vector growth in vivo and immunogenicity.
HIV-1 Vpr reactivates latent HIV-1 provirus by inducing depletion of class I HDACs on chromatin
Romani, Bizhan; Kamali Jamil, Razieh; Hamidi-Fard, Mojtaba; Rahimi, Pooneh; Momen, Seyed Bahman; Aghasadeghi, Mohammad Reza; Allahbakhshi, Elham
HIV-1 Vpr is an accessory protein that induces proteasomal degradation of multiple proteins. We recently showed that Vpr targets class I HDACs on chromatin for proteasomal degradation. Here we show that Vpr induces degradation of HDAC1 and HDAC3 in HIV-1 latently infected J-Lat cells. Degradation of HDAC1 and HDAC3 was also observed on the HIV-1 LTR and as a result, markers of active transcription were recruited to the viral promoter and induced viral activation. Knockdown of HDAC1 and HDAC3 activated the latent HIV-1 provirus and complementation with HDAC3 inhibited Vpr-induced HIV-1 reactivation. Viral reactivation and degradation of HDAC1 and HDAC3 was conserved among Vpr proteins of HV-1 group M. Serum Vpr isolated from patients or the release of virion-incorporated Vpr from viral lysates also activated HIV-1 in latently infected cell lines and PBMCs from HIV-1 infected patients. Our results indicate that Vpr counteracts HIV-1 latency by inducing proteasomal degradation of HDAC1 and 3 leading to reactivation of the viral promoter. PMID:27550312
RhoA Signaling Is Required for Respiratory Syncytial Virus-Induced Syncytium Formation and Filamentous Virion Morphology
Gower, Tara L.; Pastey, Manoj K.; Peeples, Mark E.; Collins, Peter L.; McCurdy, Lewis H.; Hart, Timothy K.; Guth, Alex; Johnson, Teresa R.; Graham, Barney S.
Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants, the elderly, and immunocompromised adults. RSV infection of HEp-2 cells induces the activation of RhoA, a small GTPase. We therefore asked whether RhoA signaling is important for RSV replication or syncytium formation. The treatment of HEp-2 cells with Clostridium botulinum C3, an enzyme that ADP-ribosylates and specifically inactivates RhoA, inhibited RSV-induced syncytium formation and cell-to-cell fusion, although similar levels of PFU were released into the medium and viral protein expression levels were equivalent. Treatment with another inhibitor of RhoA signaling, the Rho kinase inhibitor Y-27632, yielded similar results. Scanning electron microscopy of C3-treated infected cells showed reduced numbers of single blunted filaments, in contrast to the large clumps of long filaments in untreated infected cells. These data suggest that RhoA signaling is associated with filamentous virus morphology, cell-to-cell fusion, and syncytium formation but is dispensable for the efficient infection and production of infectious virus in vitro. Next, we developed a semiquantitative method to measure spherical and filamentous virus particles by using sucrose gradient velocity sedimentation. Fluorescence and transmission electron microscopy confirmed the separation of spherical and filamentous forms of infectious virus into two identifiable peaks. The C3 treatment of RSV-infected cells resulted in a shift to relatively more spherical virions than those from untreated cells. These data suggest that viral filamentous protuberances characteristic of RSV infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play a role in initiating cell-to-cell fusion. PMID:15827147
Rapid dissociation of HIV-1 from cultured cells severely limits infectivity assays, causes the inactivation ascribed to entry inhibitors, and masks the inherently high level of infectivity of virions.
Platt, Emily J; Kozak, Susan L; Durnin, James P; Hope, Thomas J; Kabat, David
By using immunofluorescence microscopy to observe and analyze freshly made HIV-1 virions adsorbed onto cells, we found that they are inherently highly infectious, rather than predominantly defective as previously suggested. Surprisingly, polycations enhance titers 20- to 30-fold by stabilizing adsorption and preventing a previously undescribed process of rapid dissociation, strongly implying that infectivity assays for many viruses are limited not only by inefficient virus diffusion onto cells but also by a postattachment race between entry and dissociation. This kinetic competition underlies inhibitory effects of CCR5 antagonists and explains why adaptive HIV-1 mutations overcome many cell entry limitations by accelerating entry.
Virus-Induced Tubules: A Vehicle for Spread of Virions into Ovary Oocyte Cells of an Insect Vector
Liao, Zhenfeng; Mao, Qianzhuo; Li, Jiajia; Lu, Chengcong; Wu, Wei; Chen, Hongyan; Chen, Qian; Jia, Dongsheng; Wei, Taiyun
Many arthropod-borne viruses are persistently propagated and transovarially transmitted by female insect vectors through eggs, but the mechanism remains poorly understood. Insect oocytes are surrounded by a layer of follicular cells, which are connected to the oocyte through actin-based microvilli. Here, we demonstrate that a plant reovirus, rice gall dwarf virus (RGDV), exploits virus-containing tubules composed of viral non-structural protein Pns11 to pass through actin-based junctions between follicular cells or through actin-based microvilli from follicular cells into oocyte of its leafhopper vector Recilia dorsalis, thus overcoming transovarial transmission barriers. We further determine that the association of Pns11 tubules with actin-based cellular junctions or microvilli of the ovary is mediated by a specific interaction between Pns11 and actin. Interestingly, RGDV can replicate and assemble progeny virions in the oocyte cytoplasm. The destruction of the tubule assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene strongly inhibits transovarial transmission of RGDV by its vectors. For the first time, we show that a virus can exploit virus-induced tubule as a vehicle to overcome the transovarial transmission barrier by insect vectors. PMID:28382031
Virus-Induced Tubules: A Vehicle for Spread of Virions into Ovary Oocyte Cells of an Insect Vector.
Liao, Zhenfeng; Mao, Qianzhuo; Li, Jiajia; Lu, Chengcong; Wu, Wei; Chen, Hongyan; Chen, Qian; Jia, Dongsheng; Wei, Taiyun
Many arthropod-borne viruses are persistently propagated and transovarially transmitted by female insect vectors through eggs, but the mechanism remains poorly understood. Insect oocytes are surrounded by a layer of follicular cells, which are connected to the oocyte through actin-based microvilli. Here, we demonstrate that a plant reovirus, rice gall dwarf virus (RGDV), exploits virus-containing tubules composed of viral non-structural protein Pns11 to pass through actin-based junctions between follicular cells or through actin-based microvilli from follicular cells into oocyte of its leafhopper vector Recilia dorsalis, thus overcoming transovarial transmission barriers. We further determine that the association of Pns11 tubules with actin-based cellular junctions or microvilli of the ovary is mediated by a specific interaction between Pns11 and actin. Interestingly, RGDV can replicate and assemble progeny virions in the oocyte cytoplasm. The destruction of the tubule assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene strongly inhibits transovarial transmission of RGDV by its vectors. For the first time, we show that a virus can exploit virus-induced tubule as a vehicle to overcome the transovarial transmission barrier by insect vectors.
Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce β-chemokines
Liao, Hua-Xin; Alam, S. Munir; Scearce, Richard M.; Plonk, M. Kelly; Kozink, Daniel M.; Drinker, Mark S.; Zhang, Ruijun; Xia, Shi-Mao; Sutherland, Laura L.; Tomaras, Georgia D.; Giles, Ian P.; Kappes, John C.; Ochsenbauer-Jambor, Christina; Edmonds, Tara G.; Soares, Melina; Barbero, Gustavo; Forthal, Donald N.; Landucci, Gary; Chang, Connie; King, Steven W.; Kavlie, Anita; Denny, Thomas N.; Hwang, Kwan-Ki; Chen, Pojen P.; Thorpe, Philip E.; Montefiori, David C.
Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to ∼10 µg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1α and MIP-1β. The release of these β-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes. PMID:20368576
Lentiviral Delivery of HIV-1 Vpr Protein Induces Apoptosis in Transformed Cells
NASA Astrophysics Data System (ADS)
Stewart, Sheila A.; Poon, Betty; Jowett, Jeremy B. M.; Xie, Yiming; Chen, Irvin S. Y.
Most current anticancer therapies act by inducing tumor cell stasis followed by apoptosis. HIV-1 Vpr effectively induces apoptosis of T cells after arrest of cells at a G2/M checkpoint. Here, we investigated whether this property of Vpr could be exploited for use as a potential anticancer agent. As a potentially safer alternative to transfer of genes encoding Vpr, we developed a method to efficiently introduce Vpr protein directly into cells. Vpr packaged into HIV-1 virions lacking a genome induced efficient cell cycle arrest and apoptosis. Introduction of Vpr into tumor cell lines of various tissue origin, including those bearing predisposing mutations in p53, XPA, and hMLH1, induced cell cycle arrest and apoptosis with high efficiency. Significantly, apoptosis mediated by virion-associated Vpr was more effective on rapidly dividing cells compared with slow-growing cells, thus, in concept, providing a potential differential effect between some types of tumor cells and surrounding normal cells. This model system provides a rationale and proof of concept for the development of potential cancer therapeutic agents based on the growth-arresting and apoptotic properties of Vpr.
HIV transcription is induced in dying cells
Woloschak, G.E.; Chang-Liu, Chin-Mei; Schreck, S. |; Panozzo, J.; Libertin, C.R.
Using HeLa cells stably transfected with an HIV-LTR-CAT construct, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. {gamma} rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that {gamma}-ray-induced apoptotic death requires functional p53, which is not present in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture. Doses which caused over 99% cell killing induced HIV-LTR transcription maximally, demonstrating that cells that will go on to die by 14 days are the cells expressing HIV-LTR-CAT.
Swine adipose stromal cells loaded with recombinant bovine herpesvirus 4 virions expressing a foreign antigen induce potent humoral immune responses in pigs.
Donofrio, Gaetano; Taddei, Simone; Franceschi, Valentina; Capocefalo, Antonio; Cavirani, Sandro; Martinelli, Nicola; Ottonello, Simone; Ferrari, Maura
Increasingly effective vaccination strategies are needed to counteract the high incidence of contagious diseases associated with intensive swine breeding. Recombinant viral vaccines are a promising new avenue in this direction. Key features of viral vectors suitable for immunoprophylaxis are safety, ease of manipulation and the ability to replicate in a variety of hosts. Most of the above requirements are met by bovine herpesvirus 4 (BoHV-4), a non-pathogenic dsDNA virus capable of infecting a broad range of cell types in vitro. Here we report the results of an exploratory study using an engineered BoHV-4 virus (eBoHV-4) expressing two unrelated glycoprotein antigens from bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1), to assess the potential of recombinant BoHV-4 as a self-adjuvanted immunogen in pigs. Free eBoHV-4 virions and virions preloaded into homologous swine adipose-derived stromal cells (SADSC) were tested. Neither virus formulation elicited neutralizing anti-BoHV-4 antibodies, nor any disease symptom, yet both induced specific immune responses against the heterologous antigens. However, a much earlier (18 vs 28 days post-infection) and more robust neutralizing response against BVDV and BoHV-1 viruses was elicited by eBoHV-4-preinfected SADSCs compared to free virions. The data validate BoHV-4 as a safe and effective heterologous antigen carrier/producer and identify SADSCs as helpful tools for the formulation of increasingly efficacious recombinant immunogens for pig vaccination.
Guanylate Binding Protein (GBP) 5 Is an Interferon-Inducible Inhibitor of HIV-1 Infectivity.
Krapp, Christian; Hotter, Dominik; Gawanbacht, Ali; McLaren, Paul J; Kluge, Silvia F; Stürzel, Christina M; Mack, Katharina; Reith, Elisabeth; Engelhart, Susanne; Ciuffi, Angela; Hornung, Veit; Sauter, Daniel; Telenti, Amalio; Kirchhoff, Frank
Guanylate binding proteins (GBPs) are an interferon (IFN)-inducible subfamily of guanosine triphosphatases (GTPases) with well-established activity against intracellular bacteria and parasites. Here we show that GBP5 potently restricts HIV-1 and other retroviruses. GBP5 is expressed in the primary target cells of HIV-1, where it impairs viral infectivity by interfering with the processing and virion incorporation of the viral envelope glycoprotein (Env). GBP5 levels in macrophages determine and inversely correlate with infectious HIV-1 yield over several orders of magnitude, which may explain the high donor variability in macrophage susceptibility to HIV. Antiviral activity requires Golgi localization of GBP5, but not its GTPase activity. Start codon mutations in the accessory vpu gene from macrophage-tropic HIV-1 strains conferred partial resistance to GBP5 inhibition by increasing Env expression. Our results identify GBP5 as an antiviral effector of the IFN response and may explain the increased frequency of defective vpu genes in primary HIV-1 strains.
HIV-1 protease-induced apoptosis
Background Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. Results Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. Conclusion In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3. PMID:24886575
Lymphotropic Virions Affect Chemokine Receptor-Mediated Neural Signaling and Apoptosis: Implications for Human Immunodeficiency Virus Type 1-Associated Dementia
Zheng, Jialin; Ghorpade, Anuja; Niemann, Douglas; Cotter, Robin L.; Thylin, Michael R.; Epstein, Leon; Swartz, Jennifer M.; Shepard, Robin B.; Liu, Xiaojuan; Nukuna, Adeline; Gendelman, Howard E.
Chemokine receptors pivotal for human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes and macrophages (CCR3, CCR5, and CXCR4) are expressed on neural cells (microglia, astrocytes, and/or neurons). It is these cells which are damaged during progressive HIV-1 infection of the central nervous system. We theorize that viral coreceptors could effect neural cell damage during HIV-1-associated dementia (HAD) without simultaneously affecting viral replication. To these ends, we studied the ability of diverse viral strains to affect intracellular signaling and apoptosis of neurons, astrocytes, and monocyte-derived macrophages. Inhibition of cyclic AMP, activation of inositol 1,4,5-trisphosphate, and apoptosis were induced by diverse HIV-1 strains, principally in neurons. Virions from T-cell-tropic (T-tropic) strains (MN, IIIB, and Lai) produced the most significant alterations in signaling of neurons and astrocytes. The HIV-1 envelope glycoprotein, gp120, induced markedly less neural damage than purified virions. Macrophage-tropic (M-tropic) strains (ADA, JR-FL, Bal, MS-CSF, and DJV) produced the least neural damage, while 89.6, a dual-tropic HIV-1 strain, elicited intermediate neural cell damage. All T-tropic strain-mediated neuronal impairments were blocked by the CXCR4 antibody, 12G5. In contrast, the M-tropic strains were only partially blocked by 12G5. CXCR4-mediated neuronal apoptosis was confirmed in pure populations of rat cerebellar granule neurons and was blocked by HA1004, an inhibitor of calcium/calmodulin-dependent protein kinase II, protein kinase A, and protein kinase C. Taken together, these results suggest that progeny HIV-1 virions can influence neuronal signal transduction and apoptosis. This process occurs, in part, through CXCR4 and is independent of CD4 binding. T-tropic viruses that traffic in and out of the brain during progressive HIV-1 disease may play an important role in HAD neuropathogenesis. PMID:10482576
RNA-Dependent RNA Polymerase Activity in Influenza Virions
Penhoet, Edward; Miller, Henry; Doyle, Michael; Blatti, Stanley
An RNA-dependent RNA polymerase activity has been detected in purified preparations of influenza virus. In contrast to the replicase activity induced in influenza-infected cells, the virion-associated enzyme has an absolute requirement for Mn++. Most of the RNA synthesized in vitro is complementary to virion RNA. PMID:5288388
Cyclin F/FBXO1 Interacts with HIV-1 Viral Infectivity Factor (Vif) and Restricts Progeny Virion Infectivity by Ubiquitination and Proteasomal Degradation of Vif Protein through SCF(cyclin F) E3 Ligase Machinery.
Augustine, Tracy; Chaudhary, Priyanka; Gupta, Kailash; Islam, Sehbanul; Ghosh, Payel; Santra, Manas Kumar; Mitra, Debashis
Cyclin F protein, also known as FBXO1, is the largest among all cyclins and oscillates in the cell cycle like other cyclins. Apart from being a G2/M cyclin, cyclin F functions as the substrate-binding subunit of SCF(cyclin F) E3 ubiquitin ligase. In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4(+) T cells, we observed down-regulation of the cyclin F gene (CCNF). Later, using gene overexpression and knockdown studies, we identified cyclin F as negatively influencing HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that cyclin F negatively regulates the expression of viral protein Vif (viral infectivity factor) at the protein level. We also identified a novel host-pathogen interaction between cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a cyclin F-specific amino acid motif in the C-terminal region of Vif indicated rescue of the protein from cyclin F-mediated down-regulation. Subsequently, we showed that Vif is a novel substrate of the SCF(cyclin F) E3 ligase, where cyclin F mediates the ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we showed that cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate that cyclin F is a novel F-box protein that functions as an intrinsic cellular regulator of HIV-1 Vif and has a negative regulatory effect on the maintenance of viral infectivity by restoring APOBEC3G expression.
HIV transcription is induced with cell killing
Woloschak, G.E.; Schreck, S.; Chang-Liu, Chin Mei; Panozzo, J.; Libertin, C.R.
Previous work has shown that HeLa cells stably transfected with an HIV-LTR-CAT construct are induced to express chloramphenicol acetyl transferase (CAT) following exposure to DNA-damaging agents such as ultraviolet radiation, {gamma} rays, neutrons, and others. In this report, the authors demonstrate that this induction of HIV-LTR transcription occurs when stably transfected HeLa cells are exposed to agents which mediate cell killing, such as UV radiation, electroporation of sucrose buffer, prolonged heating, and low and high pH. Cells cultured following UV exposure demonstrated a peak in CAT expression that is evidence in viable (but not necessarily cell division-competent) cells 24 h after exposure; this inductive response continued until at least 72 h after exposure. HIV-LTR induction was dose-dependent, and the amount of CAT transcription induced was correlated with the amount of cell killing that occurred in the culture. Other agents which caused no cell killing (such as heat-shock for up to 2 h, treatment with metronidazole, exposure to sunlight, vitamin C treatment, and others) had no effect on HIV-LTR induction. These results suggest that HIV transcription is induced as a consequence of the turn on of a cellular death or apoptotic pathway.
A rev1-vpu polymorphism unique to HIV-1 subtype A and C strains impairs envelope glycoprotein expression from rev-vpu-env cassettes and reduces virion infectivity in pseudotyping assays
Kraus, Matthias H.; Parrish, Nicholas F.; Shaw, Katharina S.; Decker, Julie M.; Keele, Brandon F.; Salazar-Gonzalez, Jesus F.; Grayson, Truman; McPherson, David T.; Ping, Li-Hua; Anderson, Jeffrey A.; Swanstrom, Ronald; Williamson, Carolyn; Shaw, George M.; Hahn, Beatrice H.
Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01{sub A}/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism.
HIV transcription is induced in dying cells
Woloschak, G.E.; Chang-Liu, Chin-Mei; Schreck, S. |
Using HeLa cells stably transfected with an HIV-LTR-CAT construct, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. {gamma} rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that {gamma}-ray-induced apoptotic death requires functional p53, which is not present in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture. 14 refs., 4 figs., 1 tab.
Nanoparticle-based flow virometry for the analysis of individual virions
Arakelyan, Anush; Fitzgerald, Wendy; Margolis, Leonid; Grivel, Jean-Charles
While flow cytometry has been used to analyze the antigenic composition of individual cells, the antigenic makeup of viral particles is still characterized predominantly in bulk. Here, we describe a technology, "flow virometry," that can be used for antigen detection on individual virions. The technology is based on binding magnetic nanoparticles to virions, staining the virions with monoclonal antibodies, separating the formed complexes with magnetic columns, and characterizing them with flow cytometers. We used this technology to study the distribution of two antigens (HLA-DR and LFA-1) that HIV-1 acquires from infected cells among individual HIV-1 virions. Flow virometry revealed that the antigenic makeup of virions from a single preparation is heterogeneous. This heterogeneity could not be detected with bulk analysis of viruses. Moreover, in two preparations of the same HIV-1 produced by different cells, the distribution of antigens among virions was different. In contrast, HIV-1 of two different HIV-1 genotypes replicating in the same cells became somewhat antigenically similar. This nanotechnology allows the study of virions in bodily fluids without virus propagation and in principle is not restricted to the analysis of HIV, but can be applied to the analysis of the individual surface antigenic makeup of any virus. PMID:23925291
HIV transcription is induced with cell killing
Woloschak, G.E.; Schreck, S.; Chang-Liu, Chin-Mei; Panozzo, J.; Libertin, C.R.
In this report, we demonstrate that this induction of HIV-LTR transcription occurs when stably transfected HeLa cells are exposed to agents which mediate cell killing, such as UV radiation, electroporation of sucrose buffer, prolonged heating, and low and high pH. Cells cultured following UV exposure demonstrated a peak in CAT expression that is evident in viable (but not necessarily cell division-competent) cells 24 h after exposure; this inductive response continued until at least 72 h after exposure. HIV-LTR induction was dose-dependent, and the amount of CAT transcription induced was correlated with the amount of cell killing that occurred in the culture.
Neutralizing antibodies to HIV-1 induced by immunization
McCoy, Laura E.
Most neutralizing antibodies act at the earliest steps of viral infection and block interaction of the virus with cellular receptors to prevent entry into host cells. The inability to induce neutralizing antibodies to HIV has been a major obstacle to HIV vaccine research since the early days of the epidemic. However, in the past three years, the definition of a neutralizing antibody against HIV has been revolutionized by the isolation of extremely broad and potent neutralizing antibodies from HIV-infected individuals. Considerable hurdles remain for inducing neutralizing antibodies to a protective level after immunization. Meanwhile, novel technologies to bypass the induction of antibodies are being explored to provide prophylactic antibody-based interventions. This review addresses the challenge of inducing HIV neutralizing antibodies upon immunization and considers notable recent advances in the field. A greater understanding of the successes and failures for inducing a neutralizing response upon immunization is required to accelerate the development of an effective HIV vaccine. PMID:23401570
Herpesvirus gB-induced fusion between the virion envelope and outer nuclear membrane during virus egress is regulated by the viral US3 kinase.
Wisner, Todd W; Wright, Catherine C; Kato, Akihisa; Kawaguchi, Yasushi; Mou, Fan; Baines, Joel D; Roller, Richard J; Johnson, David C
Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.
Recent progress in HIV vaccines inducing mucosal immune responses.
Pavot, Vincent; Rochereau, Nicolas; Lawrence, Philip; Girard, Marc P; Genin, Christian; Verrier, Bernard; Paul, Stéphane
In spite of several attempts over many years at developing a HIV vaccine based on classical strategies, none has convincingly succeeded to date. As HIV is transmitted primarily by the mucosal route, particularly through sexual intercourse, understanding antiviral immunity at mucosal sites is of major importance. An ideal vaccine should elicit HIV-specific antibodies and mucosal CD8⁺ cytotoxic T-lymphocyte (CTL) as a first line of defense at a very early stage of HIV infection, before the virus can disseminate into the secondary lymphoid organs in mucosal and systemic tissues. A primary focus of HIV preventive vaccine research is therefore the induction of protective immune responses in these crucial early stages of HIV infection. Numerous approaches are being studied in the field, including building upon the recent RV144 clinical trial. In this article, we will review current strategies and briefly discuss the use of adjuvants in designing HIV vaccines that induce mucosal immune responses.
Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation.
Delviks-Frankenberry, Krista A; Nikolaitchik, Olga A; Burdick, Ryan C; Gorelick, Robert J; Keele, Brandon F; Hu, Wei-Shau; Pathak, Vinay K
Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10-5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10-21 and1 × 10-11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic
Amino acid substitutions in the human immunodeficiency virus type 1 gp120 V3 loop that change viral tropism also alter physical and functional properties of the virion envelope.
Willey, R L; Theodore, T S; Martin, M A
The third variable (V3) region within the gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) has been reported to be an important determinant of viral tropism. In this study a series of isogenic recombinant HIV-1 viruses, containing V3 regions from fresh isolates, were examined to ascertain if a relationship exists between viral tropism and specific properties of the virion-associated envelope. All of the viruses were able to infect CD4+ primary lymphocytes, although with different infection kinetics. Several recombinants, however, were unable to infect a continuous CD4+ T-cell line permissive for the parental virus and exhibited a marked decrease in the kinetics of virion-associated gp120 binding to a soluble form of CD4. A known macrophage-tropic HIV-1 isolate, also unable to infect the T-cell line, bound CD4 with similarly slow reaction kinetics. Although the inability to infect T-cell lines is a commonly observed property of macrophage-tropic isolates of HIV-1, the loss of T-cell line tropism by the V3 recombinants was not accompanied by a substantial infectivity for monocyte-derived macrophages, as monitored by reverse transcriptase production. Additional analyses of the recombinant virion gp120s indicated that most of the V3 substitutions increased the inherent stability of the virion gp120-gp41 envelope complex. These results indicate that V3-induced alterations in viral tropism are associated with changes in physical and functional properties of the virion envelope. Images PMID:7515973
The localization of a heterologous displayed antigen in the baculovirus-budded virion determines the type and strength of induced adaptive immune response.
Tavarone, Eugenia; Molina, Guido Nicolás; Amalfi, Sabrina; Peralta, Andrea; Molinari, Paula; Taboga, Oscar
In the search of strategies of presentation of heterologous antigens to elicit humoral or cellular immune responses that modulate and properly potentiate each type of response, researchers have been studying baculovirus (BV) as vaccine vectors with promising results. For some years, several research groups explored different antigen presentation approaches using the BV AcNPV by expressing polypeptides on the surface of budded virions or by de novo synthesis of heterologous antigens by transduction of mammalian cells. In the case of expression on the surface of budded virions, for example, researchers have expressed polypeptides in peplomers as GP64 glycoprotein fusions or distributed throughout the entire surface by fusions to portions of the G protein of vesicular stomatitis virus, VSV. Recently, our group developed the strategy of cross-presentation of antigens by fusions of GP64 to the capsid protein VP39 (capsid display) for the generation of cytotoxic responses. While the different strategies showed to be effective in raising immune responses, the individuality of each analysis makes difficult the comparison of the results. Here, by comparing the different strategies, we show that localization of the model antigen ovalbumin (OVA) strongly determined the quality and intensity of the adaptive response to the heterologous antigen. Furthermore, surface display favored humoral responses, whereas capsid display favored cytotoxic responses. Finally, capsid display showed a much more efficient strategy to activate CD8-mediated responses than transduction. The incorporation of adjuvants in baculovirus formulations dramatically diminished the immunostimulatory properties of baculovirus.
Guanylate binding protein 5: Impairing virion infectivity by targeting retroviral envelope glycoproteins.
Hotter, Dominik; Sauter, Daniel; Kirchhoff, Frank
Guanylate binding proteins (GBPs) are interferon-inducible cellular factors that belong to the superfamily of guanosine triphosphatases (GTPases) and play important roles in the cell-intrinsic defense against bacteria, protozoa and viruses. In a recent report in Cell Host & Microbe, we identify GBP5 as novel restriction factor of HIV-1 that reduces the infectivity of progeny virions by interfering with processing and incorporation of the viral envelope (Env) glycoprotein. The inhibitory activity of GBP5 requires C-terminal isoprenylation, mediating Golgi-association, but not its GTPase function. Notably, GBP5 expression levels vary considerably in human macrophages and inversely correlate with infectious virus yield. We demonstrate that GBP5 can be evaded by an unusual tradeoff mechanism: Naturally occurring mutations in the start codon of the viral accessory gene vpu attenuate GBP5 inhibition by increasing Env expression at the cost of Vpu function. Whether direct counteraction mechanisms or more subtle changes balancing Vpu and Env expression also affect HIV-1 inhibition by GBP5 remains to be clarified. Other open questions are whether GBP5 restricts HIV-1 in CD4(+) T cells and if other GBP family members also decrease infectivity of HIV and/or additional enveloped viruses.
Guanylate binding protein 5: Impairing virion infectivity by targeting retroviral envelope glycoproteins
Hotter, Dominik; Sauter, Daniel; Kirchhoff, Frank
ABSTRACT Guanylate binding proteins (GBPs) are interferon-inducible cellular factors that belong to the superfamily of guanosine triphosphatases (GTPases) and play important roles in the cell-intrinsic defense against bacteria, protozoa and viruses. In a recent report in Cell Host & Microbe, we identify GBP5 as novel restriction factor of HIV-1 that reduces the infectivity of progeny virions by interfering with processing and incorporation of the viral envelope (Env) glycoprotein. The inhibitory activity of GBP5 requires C-terminal isoprenylation, mediating Golgi-association, but not its GTPase function. Notably, GBP5 expression levels vary considerably in human macrophages and inversely correlate with infectious virus yield. We demonstrate that GBP5 can be evaded by an unusual tradeoff mechanism: Naturally occurring mutations in the start codon of the viral accessory gene vpu attenuate GBP5 inhibition by increasing Env expression at the cost of Vpu function. Whether direct counteraction mechanisms or more subtle changes balancing Vpu and Env expression also affect HIV-1 inhibition by GBP5 remains to be clarified. Other open questions are whether GBP5 restricts HIV-1 in CD4+ T cells and if other GBP family members also decrease infectivity of HIV and/or additional enveloped viruses. PMID:27275775
HIV transcription is induced with some forms of cell killing
Woloschak, G.E.; Schreck, S.; Panozzo, J.; Chang-Liu, C.-M.; Libertin, C.R.
Using HeLa cells stably transfected with an HIV-LTR-CAT construct`, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. {Gamma} rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that {gamma}-ray-induced apoptotic death requires function p53, which is missing in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture.
BST2/CD317 counteracts human coronavirus 229E productive infection by tethering virions at the cell surface
Wang, Shiu-Mei; Huang, Kuo-Jung; Wang, Chin-Tien
Bone marrow stromal antigen 2 (BST2), an interferon-inducible antiviral factor, has been shown to block the release of various enveloped viruses from cells. It has also been identified as an innate immune system component. Most enveloped viruses subject to BST2 restriction bud at the plasma membrane. Here we report our findings that (a) the production of human coronavirus 229E (HCoV-229E) progeny viruses, whose budding occurs at the ER-Golgi intermediate compartment (ERGIC), markedly decreases in the presence of BST2; and (b) BST2 knockdown expression results in enhanced HCoV-229E virion production. Electron microscopy analyses indicate that HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. Our results suggest that BST2 exerts a broad blocking effect against enveloped virus release, regardless of whether budding occurs at the plasma membrane or intracellular compartments. - Highlights: • BST2 knockdown expression results in enhanced HCoV-229E egress. • HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. • HCoV-229E infection at high MOI can significantly downregulate HeLa BST2 and rescue HIV-1 egress.
HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers.
Sanders, Rogier W; van Gils, Marit J; Derking, Ronald; Sok, Devin; Ketas, Thomas J; Burger, Judith A; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne C; Julien, Jean-Philippe; Rakasz, Eva G; Seaman, Michael S; Guttman, Miklos; Lee, Kelly K; Klasse, Per Johan; LaBranche, Celia; Schief, William R; Wilson, Ian A; Overbaugh, Julie; Burton, Dennis R; Ward, Andrew B; Montefiori, David C; Dean, Hansi; Moore, John P
A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505 SOSIP.664, induced NAbs potently against the sequence-matched tier 2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (tier 1) viruses. Tier 2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas tier 1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous tier 2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for the development of HIV-1 vaccines aimed at inducing bNAbs.
HIV-1-induced AIDS in monkeys.
Hatziioannou, Theodora; Del Prete, Gregory Q; Keele, Brandon F; Estes, Jacob D; McNatt, Matthew W; Bitzegeio, Julia; Raymond, Alice; Rodriguez, Anthony; Schmidt, Fabian; Mac Trubey, C; Smedley, Jeremy; Piatak, Michael; KewalRamani, Vineet N; Lifson, Jeffrey D; Bieniasz, Paul D
Primate lentiviruses exhibit narrow host tropism, reducing the occurrence of zoonoses but also impairing the development of optimal animal models of AIDS. To delineate the factors limiting cross-species HIV-1 transmission, we passaged a modified HIV-1 in pigtailed macaques that were transiently depleted of CD8(+) cells during acute infection. During adaptation over four passages in macaques, HIV-1 acquired the ability to antagonize the macaque restriction factor tetherin, replicated at progressively higher levels, and ultimately caused marked CD4(+) T cell depletion and AIDS-defining conditions. Transient treatment with an antibody to CD8 during acute HIV-1 infection caused rapid progression to AIDS, whereas untreated animals exhibited an elite controller phenotype. Thus, an adapted HIV-1 can cause AIDS in macaques, and stark differences in outcome can be determined by immunological perturbations during early infection.
HIV-1 envelope glycoprotein immunogens to induce broadly neutralizing antibodies.
Sliepen, Kwinten; Sanders, Rogier W
The long pursuit for a vaccine against human immunodeficiency virus 1 (HIV-1) has recently been boosted by a number of exciting developments. An HIV-1 subunit vaccine ideally should elicit potent broadly neutralizing antibodies (bNAbs), but raising bNAbs by vaccination has proved extremely difficult because of the characteristics of the HIV-1 envelope glycoprotein complex (Env). However, the isolation of bNAbs from HIV-1-infected patients demonstrates that the human humoral immune system is capable of making such antibodies. Therefore, a focus of HIV-1 vaccinology is the elicitation of bNAbs by engineered immunogens and by using vaccination strategies aimed at mimicking the bNAb maturation pathways in HIV-infected patients. Important clues can also be taken from the successful subunit vaccines against hepatitis B virus and human papillomavirus. Here, we review the different types of HIV-1 immunogens and vaccination strategies that are being explored in the search for an HIV-1 vaccine that induces bNAbs.
Noninfectious X4 but not R5 human immunodeficiency virus type 1 virions inhibit humoral immune responses in human lymphoid tissue ex vivo
NASA Technical Reports Server (NTRS)
Fitzgerald, Wendy; Sylwester, Andrew W.; Grivel, Jean-Charles; Lifson, Jeffrey D.; Margolis, Leonid B.
Ex vivo human immunodeficiency virus type 1 (HIV-1) infection of human lymphoid tissue recapitulates some aspects of in vivo HIV-1 infection, including a severe depletion of CD4(+) T cells and suppression of humoral immune responses to recall antigens or to polyclonal stimuli. These effects are induced by infection with X4 HIV-1 variants, whereas infection with R5 variants results in only mild depletion of CD4(+) T cells and no suppression of immune responses. To study the mechanisms of suppression of immune responses in this ex vivo system, we used aldrithiol-2 (AT-2)-inactivated virions that have functional envelope glycoproteins but are not infectious and do not deplete CD4(+) T cells in human lymphoid tissues ex vivo. Nevertheless, AT-2-inactivated X4 (but not R5) HIV-1 virions, even with only a brief exposure, inhibit antibody responses in human lymphoid tissue ex vivo, similarly to infectious virus. This phenomenon is mediated by soluble immunosuppressive factor(s) secreted by tissue exposed to virus.
The HIV-1 Vpu protein induces apoptosis in Drosophila via activation of JNK signaling.
Marchal, Christelle; Vinatier, Gérald; Sanial, Matthieu; Plessis, Anne; Pret, Anne-Marie; Limbourg-Bouchon, Bernadette; Théodore, Laurent; Netter, Sophie
The genome of the human immunodeficiency virus type 1 (HIV-1) encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu), in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin-proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated.We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs) which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1). Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/β-TrCP protein, as in mammals, both SLIMB/βTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK) pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.
The HIV-1 Vpu Protein Induces Apoptosis in Drosophila via Activation of JNK Signaling
Marchal, Christelle; Vinatier, Gérald; Sanial, Matthieu; Plessis, Anne; Pret, Anne-Marie; Limbourg-Bouchon, Bernadette; Théodore, Laurent; Netter, Sophie
The genome of the human immunodeficiency virus type 1 (HIV-1) encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu), in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin–proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated. We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs) which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1). Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/β-TrCP protein, as in mammals, both SLIMB/βTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK) pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway. PMID:22479597
HIV-infected microglia mediate cathepsin B induced neurotoxicity
Zenón, Frances; Cantres-Rosario, Yisel; Adiga, Radhika; Gonzalez, Mariangeline; Rodriguez-Franco, Eillen; Langford, Dianne; Melendez, Loyda M.
BACKGROUND HIV-1-infected mononuclear phagocytes release soluble factors that affect the homeostasis in tissue. HIV-1 can prompt metabolic encephalopathy with the addition of neuronal dysfunction and apoptosis. Recently, we reported that HIV-1 enhances the expression and secretion of bioactive cathepsin B in monocyte-derived macrophages, ultimately contributing to neuronal apoptosis. In this research, we request if microglia respond to HIV infection similarly by modifying the expression, secretion, neurotoxic potential of cathepsin B and the in vivo relevance of these findings. METHODS HIV-ADA infected human primary microglia and CHME-5 were assessed for expression and activity of cathepsin B, its inhibitors, cystatins B and C, and neurotoxicity associated with these changes. Human primary neurons were exposed to supernatants from HIV-infected and uninfected microglia in the presence of cathepsin B inhibitors and apoptosis was assessed by TUNEL. Microglial expression of cathepsin B was validated in brain tissue from HIVE patients. RESULTS HIV-infected microglia secreted significantly greater levels of cathepsin B, cystatin B, and cystatin C compared to uninfected cells. Increased apoptosis was observed in neurons exposed to supernatants from HIV-1 infected microglia at days 12 post-infection. The cathepsin B inhibitor CA-074 and cathepsin B antibody prevented neuronal apoptosis. Increased microglia-derived cathepsin B, cystatin B, and cystatin C and caspase-3+ neurons were detected in HIVE brains compared to controls. CONCLUSIONS Our results suggest that HIV-1-induced cathepsin B production in microglia contributes to neuronal apoptosis and may be an important factor in neuronal death associated with HIVE. PMID:26092112
Rigid proteins and softening of biological membranes-with application to HIV-induced cell membrane softening.
Agrawal, Himani; Zelisko, Matthew; Liu, Liping; Sharma, Pradeep
A key step in the HIV-infection process is the fusion of the virion membrane with the target cell membrane and the concomitant transfer of the viral RNA. Experimental evidence suggests that the fusion is preceded by considerable elastic softening of the cell membranes due to the insertion of fusion peptide in the membrane. What are the mechanisms underpinning the elastic softening of the membrane upon peptide insertion? A broader question may be posed: insertion of rigid proteins in soft membranes ought to stiffen the membranes not soften them. However, experimental observations perplexingly appear to show that rigid proteins may either soften or harden membranes even though conventional wisdom only suggests stiffening. In this work, we argue that regarding proteins as merely non-specific rigid inclusions is flawed, and each protein has a unique mechanical signature dictated by its specific interfacial coupling to the surrounding membrane. Predicated on this hypothesis, we have carried out atomistic simulations to investigate peptide-membrane interactions. Together with a continuum model, we reconcile contrasting experimental data in the literature including the case of HIV-fusion peptide induced softening. We conclude that the structural rearrangements of the lipids around the inclusions cause the softening or stiffening of the biological membranes.
Rigid proteins and softening of biological membranes—with application to HIV-induced cell membrane softening
NASA Astrophysics Data System (ADS)
Agrawal, Himani; Zelisko, Matthew; Liu, Liping; Sharma, Pradeep
A key step in the HIV-infection process is the fusion of the virion membrane with the target cell membrane and the concomitant transfer of the viral RNA. Experimental evidence suggests that the fusion is preceded by considerable elastic softening of the cell membranes due to the insertion of fusion peptide in the membrane. What are the mechanisms underpinning the elastic softening of the membrane upon peptide insertion? A broader question may be posed: insertion of rigid proteins in soft membranes ought to stiffen the membranes not soften them. However, experimental observations perplexingly appear to show that rigid proteins may either soften or harden membranes even though conventional wisdom only suggests stiffening. In this work, we argue that regarding proteins as merely non-specific rigid inclusions is flawed, and each protein has a unique mechanical signature dictated by its specific interfacial coupling to the surrounding membrane. Predicated on this hypothesis, we have carried out atomistic simulations to investigate peptide-membrane interactions. Together with a continuum model, we reconcile contrasting experimental data in the literature including the case of HIV-fusion peptide induced softening. We conclude that the structural rearrangements of the lipids around the inclusions cause the softening or stiffening of the biological membranes.
BCG vaccination induces HIV target cell activation in HIV-exposed infants in a randomized trial
Gasper, Melanie A.; Hesseling, Anneke C.; Mohar, Isaac; Myer, Landon; Azenkot, Tali; Passmore, Jo-Ann S.; Hanekom, Willem; Cotton, Mark F.; Crispe, I. Nicholas; Sodora, Donald L.; Jaspan, Heather B.
BACKGROUND. Bacillus Calmette-Guérin (BCG) vaccine is administered at birth to protect infants against tuberculosis throughout Africa, where most perinatal HIV-1 transmission occurs. We examined whether BCG vaccination alters the levels of activated HIV target T cells in HIV-exposed South African infants. METHODS. HIV-exposed infants were randomized to receive routine (at birth) or delayed (at 8 weeks) BCG vaccination. Activated and CCR5-expressing peripheral blood CD4+ T cell, monocyte, and NK cell frequencies were evaluated by flow cytometry and immune gene expression via PCR using Biomark (Fluidigm). RESULTS. Of 149 infants randomized, 92% (n = 137) were retained at 6 weeks: 71 in the routine BCG arm and 66 in the delayed arm. Routine BCG vaccination led to a 3-fold increase in systemic activation of HIV target CD4+CCR5+ T cells (HLA-DR+CD38+) at 6 weeks (0.25% at birth versus 0.08% in delayed vaccination groups; P = 0.029), which persisted until 8 weeks of age when the delayed arm was vaccinated. Vaccination of the infants in the delayed arm at 8 weeks resulted in a similar increase in activated CD4+CCR5+ T cells. The increase in activated T cells was associated with increased levels of MHC class II transactivator (CIITA), IL12RB1, and IFN-α1 transcripts within peripheral blood mononuclear cells but minimal changes in innate cells. CONCLUSION. BCG vaccination induces immune changes in HIV-exposed infants, including an increase in the proportion of activated CCR5+CD4+ HIV target cells. These findings provide insight into optimal BCG vaccine timing to minimize the risks of HIV transmissions to exposed infants while preserving potential benefits conferred by BCG vaccination. TRIAL REGISTRATION. ClinicalTrials.gov NCT02062580. FUNDING. This trial was sponsored by the Elizabeth Glaser Pediatric AIDS Foundation (MV-00-9-900-01871-0-00) and the Thrasher Foundation (NR-0095); for details, see Acknowledgments.
Toll-Like Receptor 7 Agonist GS-9620 Induces HIV Expression and HIV-Specific Immunity in Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy.
Tsai, Angela; Irrinki, Alivelu; Kaur, Jasmine; Cihlar, Tomas; Kukolj, George; Sloan, Derek D; Murry, Jeffrey P
Antiretroviral therapy can suppress HIV replication to undetectable levels but does not eliminate latent HIV, thus necessitating lifelong therapy. Recent efforts to target this persistent reservoir have focused on inducing the expression of latent HIV so that infected cells may be recognized and eliminated by the immune system. Toll like receptor (TLR) activation stimulates antiviral immunity and has been shown to induce HIV from latently infected cells. Activation of TLR7 leads to the production of several stimulatory cytokines, including type I interferons (IFNs). In this study, we show that the selective TLR7 agonist, GS-9620, induced HIV in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals on suppressive antiretroviral therapy. GS-9620 increased extracellular HIV RNA 1.5-2-fold through a mechanism that required type I IFN signaling. GS-9620 also activated HIV-specific T cells and enhanced antibody-mediated clearance of HIV-infected cells. Activation by GS-9620 in combination with HIV peptide stimulation increased CD8 T cell degranulation, production of intracellular cytokines, and cytolytic activity. T cell activation was again dependent on type I IFNs produced by plasmacytoid dendritic cells. GS-9620 induced phagocytic cell maturation and improved effector-mediated killing of HIV-infected CD4 T cells by the HIV envelope-specific broadly neutralizing antibody PGT121. Collectively, these data show that GS-9620 can activate HIV production and improve the effector functions that target latently infected cells. GS-9620 may effectively complement orthogonal therapies designed to stimulate antiviral immunity, such as therapeutic vaccines or broadly neutralizing antibodies. Clinical studies are underway to determine if GS-9620 can target HIV reservoirs.IMPORTANCE Though antiretroviral therapies effectively suppress viral replication, they do not eliminate integrated proviral DNA. This stable intermediate of viral infection is persistently
Toll-Like Receptor 7 Agonist GS-9620 Induces HIV Expression and HIV-Specific Immunity in Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy
Tsai, Angela; Irrinki, Alivelu; Kaur, Jasmine; Cihlar, Tomas; Kukolj, George
ABSTRACT Antiretroviral therapy can suppress HIV replication to undetectable levels but does not eliminate latent HIV, thus necessitating lifelong therapy. Recent efforts to target this persistent reservoir have focused on inducing the expression of latent HIV so that infected cells may be recognized and eliminated by the immune system. Toll-like receptor (TLR) activation stimulates antiviral immunity and has been shown to induce HIV from latently infected cells. Activation of TLR7 leads to the production of several stimulatory cytokines, including type I interferons (IFNs). In this study, we show that the selective TLR7 agonist GS-9620 induced HIV in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals on suppressive antiretroviral therapy. GS-9620 increased extracellular HIV RNA 1.5- to 2-fold through a mechanism that required type I IFN signaling. GS-9620 also activated HIV-specific T cells and enhanced antibody-mediated clearance of HIV-infected cells. Activation by GS-9620 in combination with HIV peptide stimulation increased CD8 T cell degranulation, production of intracellular cytokines, and cytolytic activity. T cell activation was again dependent on type I IFNs produced by plasmacytoid dendritic cells. GS-9620 induced phagocytic cell maturation and improved effector-mediated killing of HIV-infected CD4 T cells by the HIV envelope-specific broadly neutralizing antibody PGT121. Collectively, these data show that GS-9620 can activate HIV production and improve the effector functions that target latently infected cells. GS-9620 may effectively complement orthogonal therapies designed to stimulate antiviral immunity, such as therapeutic vaccines or broadly neutralizing antibodies. Clinical studies are under way to determine if GS-9620 can target HIV reservoirs. IMPORTANCE Though antiretroviral therapies effectively suppress viral replication, they do not eliminate integrated proviral DNA. This stable intermediate of viral infection is
Feline Immunodeficiency Virus Envelope Glycoproteins Antagonize Tetherin through a Distinctive Mechanism That Requires Virion Incorporation
Guevara, Rebekah B.; Marcano, Adriana C.; Saenz, Dyana T.; Fadel, Hind J.; Rogstad, Daniel K.
ABSTRACT BST2/tetherin inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved specific antagonists (Vpu, Nef, and Env). Here we characterized tetherin proteins of species representing both branches of the order Carnivora. Comparison of tiger and cat (Feliformia) to dog and ferret (Caniformia) genes demonstrated that the tiger and cat share a start codon mutation that truncated most of the tetherin cytoplasmic tail early in the Feliformia lineage (19 of 27 amino acids, including the dual tyrosine motif). Alpha interferon (IFN-α) induced tetherin and blocked feline immunodeficiency virus (FIV) replication in lymphoid and nonlymphoid feline cells. Budding of bald FIV and HIV particles was blocked by carnivore tetherins. However, infectious FIV particles were resistant, and spreading FIV replication was uninhibited. Antagonism mapped to the envelope glycoprotein (Env), which rescued FIV from carnivore tetherin restriction when expressed in trans but, in contrast to known antagonists, did not rescue noncognate particles. Also unlike the primate lentiviral antagonists, but similar to the Ebola virus glycoprotein, FIV Env did not reduce intracellular or cell surface tetherin levels. Furthermore, FIV-enveloped FIV particles actually required tetherin for optimal release from cells. The results show that FIV Envs mediate a distinctive tetherin evasion. Well adapted to a phylogenetically ancient tetherin tail truncation in the Felidae, it requires functional virion incorporation of Env, and it shields the budding particle without downregulating plasma membrane tetherin. Moreover, FIV has evolved dependence on this protein: particles containing FIV Env need tetherin for optimal release from the cell, while Env− particles do not. IMPORTANCE HIV-1 antagonizes the restriction factor tetherin with the accessory protein Vpu, while HIV-2 and the filovirus Ebola use their envelope (Env) glycoproteins for this purpose. It turns out that the FIV
Immunotherapy of HIV-infected patients with Gc protein-derived macrophage activating factor (GcMAF).
Yamamoto, Nobuto; Ushijima, Naofumi; Koga, Yoshihiko
Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by alpha-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV-infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years.
Drug induced superinfection in HIV and the evolution of drug resistance.
Leontiev, Vladimir V; Maury, Wendy J; Hadany, Lilach
The rapid evolution of HIV drug resistance is a major cause of AIDS treatment failure. Superinfection, the infection of an already infected cell by additional virions, can be a major factor contributing to the evolution of drug resistance. However, the pattern and consequences of superinfection in HIV populations are far from fully understood. In this paper we study the implications of the fact that superinfection is regulated by HIV. We propose that superinfection is negatively associated with the success of the virus, so that more successful viruses are less likely to allow superinfection. We use computational models to investigate the effect that regulated superinfection would have on the evolution of drug resistance in HIV population. We find that regulated, fitness-associated superinfection can provide a distinct advantage to the virus in adapting to anti-HIV drugs in comparison with unregulated superinfection. Based on the results of the computational models and on current biological evidence, we suggest that the mechanism of fitness-associated regulation of coinfection in HIV is plausible, and that its investigation can lead to new ways to fight viral drug resistance.
Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates.
Moore, J P; McKeating, J A; Huang, Y X; Ashkenazi, A; Ho, D D
Primary isolates of human immunodeficiency virus type 1 (HIV-1) are much less sensitive to neutralization by soluble CD4 (sCD4) and sCD4-immunoglobulin (Ig) chimeras (CD4-IgG) than are HIV-1 strains adapted to growth in cell culture. We demonstrated that there are significant reductions (10- to 30-fold) in the binding of sCD4 and CD4-IgG to intact virions of five primary isolates compared with sCD4-sensitive, cell culture-adapted isolates RF and IIIB. However, soluble envelope glycoproteins (gp120) derived from the primary isolate virions, directly by detergent solubilization or indirectly by recombinant DNA technology, differed in affinity from RF and IIIB gp120 by only one- to threefold. The reduced binding of sCD4 to these primary isolate virions must therefore be a consequence of the tertiary or quaternary structure of the envelope glycoproteins in their native, oligomeric form on the viral surface. In addition, the rate and extent of sCD4-induced gp120 shedding from these primary isolates was lower than that from RF. We suggest that reduced sCD4 binding and increased gp120 retention together account for the relative resistance of these primary isolates to neutralization by sCD4 and CD4-IgG and that virions of different HIV-1 isolates vary both in the mechanism of sCD4 binding and in subsequent conformational changes in their envelope glycoproteins. PMID:1727487
Suppression of APOBEC3-mediated restriction of HIV-1 by Vif
Feng, Yuqing; Baig, Tayyba T.; Love, Robin P.; Chelico, Linda
The APOBEC3 restriction factors are a family of deoxycytidine deaminases that are able to suppress replication of viruses with a single-stranded DNA intermediate by inducing mutagenesis and functional inactivation of the virus. Of the seven human APOBEC3 enzymes, only APOBEC3-D, -F, -G, and -H appear relevant to restriction of HIV-1 in CD4+ T cells and will be the focus of this review. The restriction of HIV-1 occurs most potently in the absence of HIV-1 Vif that induces polyubiquitination and degradation of APOBEC3 enzymes through the proteasome pathway. To restrict HIV-1, APOBEC3 enzymes must be encapsidated into budding virions. Upon infection of the target cell during reverse transcription of the HIV-1 RNA into (-)DNA, APOBEC3 enzymes deaminate cytosines to form uracils in single-stranded (-)DNA regions. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite to the uracils thereby inducing C/G to T/A mutations that can functionally inactivate HIV-1. APOBEC3G is the most studied APOBEC3 enzyme and it is known that Vif attempts to thwart APOBEC3 function not only by inducing its proteasomal degradation but also by several degradation-independent mechanisms, such as inhibiting APOBEC3G virion encapsidation, mRNA translation, and for those APOBEC3G molecules that still become virion encapsidated, Vif can inhibit APOBEC3G mutagenic activity. Although most Vif variants can induce efficient degradation of APOBEC3-D, -F, and -G, there appears to be differential sensitivity to Vif-mediated degradation for APOBEC3H. This review examines APOBEC3-mediated HIV restriction mechanisms, how Vif acts as a substrate receptor for a Cullin5 ubiquitin ligase complex to induce degradation of APOBEC3s, and the determinants and functional consequences of the APOBEC3 and Vif interaction from a biological and biochemical perspective. PMID:25206352
Creatine protects against mitochondrial dysfunction associated with HIV-1 Tat-induced neuronal injury
Stevens, Patrick R.; Gawryluk, Jeremy W.; Hui, Liang; Chen, Xuesong; Geiger, Jonathan D.
HIV-1 infected individuals are living longer but experiencing a prevalence rate of over 50% for HIV-1 associated neurocognitive disorders (HAND) for which no effective treatment is available. Viral and cellular factors secreted by HIV-1 infected cells leads to neuronal injury and HIV-1 Tat continues to be implicated in the pathogenesis of HAND. Here we tested the hypothesis that creatine protected against HIV-1 Tat-induced neuronal injury by preventing mitochondrial bioenergetic crisis and/or redox catastrophe. Creatine blocked HIV-1 Tat1-72-induced increases in neuron cell death and synaptic area loss. Creatine protected against HIV-1 Tat-induced decreases in ATP. Creatine and creatine plus HIV-1 Tat increased cellular levels of creatine, and creatine plus HIV-1 Tat further decreased ratios of phosphocreatine to creatine observed with creatine or HIV-1 Tat treatments alone. Additionally, creatine protected against HIV-1 Tat-induced mitochondrial hypopolarization and HIV-1 Tat-induced mitochondrial permeability transition pore opening. Thus, creatine may be a useful adjunctive therapy against HAND. PMID:25613139
Naturally Occurring Fc-Dependent Antibody From HIV-Seronegative Individuals Promotes HIV-Induced IFN-α Production
Lum, Thomas; Green, Jon A.
A majority of adults without HIV infection and with a low risk of HIV-exposure have plasma IgG antibodies that enhance the rate and magnitude of HIV-induced interferon alpha (IFN-α) production. Fc-dependent IgG-HIV complexes induce IFN-α rapidly and in high titers in response to HIV concentrations that are too low to otherwise stimulate an effective IFN-α response. IFN-α promoting antibody (IPA) counters HIV-specific inhibition of IFN-α production, and compensates for the inherent delay in IFN-α production common to HIV infection and other viruses. Naturally occurring IPA has the potential to initiate a potent IFN-α response early in the course of HIV mucosal invasion in time to terminate infection prior to the creation of a pool of persistently infected cells. The current study adds IPA as a mediator of an Fc-dependent antiviral state capable of preventing HIV infection. PMID:27881846
High Dose Atorvastatin Decreases Cellular Markers of Immune Activation Without Affecting HIV-1 RNA Levels: Results of a Double-Blind Randomized Placebo Controlled Clinical Trial
cholesterol -depleting agents, such as 3-hydroxy- 3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors ( statins ), reduces HIV-1 particle production [5...Furthermore, virions derived from cholesterol -depleted cells demonstrate reduced infectivity in vitro [5]. In addition, statins have dem- onstrated...studies revealed that substantial statin - induced decreases in cholesterol resulted in declines in HIV-1 production [5]. We therefore chose the highest
HIV Tat protein inhibits hERG K+ channels: a potential mechanism of HIV infection induced LQTs.
Bai, Yun-Long; Liu, Hui-Bin; Sun, Bo; Zhang, Ying; Li, Qi; Hu, Chao-Wei; Zhu, Jiu-Xin; Gong, Dong-Mei; Teng, Xue; Zhang, Qin; Yang, Bao-Feng; Dong, De-Li
HIV-infected patients have a high prevalence of long QT syndrome (LQTs). hERG K(+) channel encoded by human ether-a-go-go related gene contributes to IKr K(+) currents responsible for the repolarization of cardiomyocytes. Inhibition of hERG K(+) channels leads to LQTs. HIV Tat protein, the virus transactivator protein, plays a pivotal role in AIDS. The aim of the present study is to examine the effects of HIV Tat protein on hERG K(+) channels stably expressed in HEK293 cells. The hERG K(+) currents were recorded by whole-cell patch-clamp technique and the hERG channel expression was measured by real-time PCR and Western blot techniques. HIV Tat protein at 200 ng/ml concentration showed no acute effect on hERG currents, but HIV Tat protein (200 ng/ml) incubation for 24 h significantly inhibited hERG currents. In HIV Tat incubated cells, the inactivation and the recovery time from inactivation of hERG channels were significantly changed. HIV Tat protein incubation (200 ng/ml) for 24h had no effect on the hERG mRNA expression, but dose-dependently inhibited hERG protein expression. The MTT assay showed that HIV Tat protein at 50 ng/ml and 200 ng/ml had no effect on the cell viability. HIV Tat protein increased reactive oxygen species (ROS) generation and the inhibition of hERG channel protein expression by HIV Tat protein was prevented by antioxidant tempol. HIV Tat protein in vivo treatment reduced IKr currents and prolonged action potential duration of guinea pig cardiomyocytes. We conclude that HIV Tat protein inhibits hERG K(+) currents through the inhibition of hERG protein expression, which might be the potential mechanism of HIV infection induced LQTs.
Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase
Sherrill-Mix, Scott; Hwang, Young; Eilers, Grant; McDanal, Charlene; Wang, Ping; Temelkoff, David
The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization. PMID:27935939
Virion basic phosphoprotein from human cytomegalovirus contains O-linked N-acetylglucosamine.
Benko, D M; Haltiwanger, R S; Hart, G W; Gibson, W
A 149-kDa virion protein of human strains of cytomegalovirus is the principal acceptor for galactose added in vitro by bovine milk galactosyltransferase. Peptide comparisons with other biochemical characteristics of the galactosylated protein identified it as the virus-encoded basic phosphoprotein. This protein is an abundant constituent of the virion and is located in the tegument region, between the capsid and the envelope, rather than in the envelope layer with the recognized virion glycoproteins. The galactosylated carbohydrate was resistant to a commercial preparation of endoglycosidase F but was sensitive to removal by alkali-induced beta-elimination, indicating an O-linkage to the protein. Chromatographic and electrophoretic determinations identified the beta-eliminated material as the alditol of Gal beta 1-4GlcNAc, establishing that the human cytomegalovirus virion basic phosphoprotein contains single O-linked residues of N-acetylglucosamine. Images PMID:2833746
The Interferon-Inducible Host Factor Bone Marrow Stromal Antigen 2/Tetherin Restricts Virion Release, but Is It Actually a Viral Restriction Factor?
Andrew, Amy
Viruses face a variety of obstacles when infecting a new host. The past few years have brought exciting new insights into the function of restriction factors, which form part of the host's innate immune system. One of the most recently identified restriction factors is bone marrow stromal antigen 2 (BST-2)/tetherin. BST-2 is an interferon-inducible gene whose expression dramatically reduces the release of viruses from infected cells. This effect of BST-2 is not specific to human immunodeficiency virus but affects a broad range of enveloped viruses. Since the identification of BST-2 as a restriction factor in 2008, much progress has been made in understanding the molecular properties and functional characteristics of this host factor. The goal of this review was to provide an update on our current understanding of the role of BST-2 in regulating virus release and to discuss its role in controlling virus spread during productive infection with special emphasis on human immunodeficiency virus-1. PMID:21166593
Glutathione is required for efficient production of infectious picornavirus virions
Smith, Allen D. . E-mail: email@example.com; Dawson, Harry . E-mail: firstname.lastname@example.com
Glutathione is an intracellular reducing agent that helps maintain the redox potential of the cell and is important for immune function. The drug L-buthionine sulfoximine (BSO) selectively inhibits glutathione synthesis. Glutathione has been reported to block replication of HIV, HSV-1, and influenza virus, whereas cells treated with BSO exhibit increased replication of Sendai virus. Pre-treatment of HeLa cell monolayers with BSO inhibited replication of CVB3, CVB4, and HRV14 with viral titers reduced by approximately 6, 5, and 3 log{sub 1}, respectively. The addition of glutathione ethyl ester, but not dithiothreitol or 2-mercaptoethanol, to the culture medium reversed the inhibitory effect of BSO. Viral RNA and protein synthesis were not inhibited by BSO treatment. Fractionation of lysates from CVB3-infected BSO-treated cells on cesium chloride and sucrose gradients revealed that empty capsids but not mature virions were being produced. The levels of the 5S and 14S assembly intermediates, however, were not affected by BSO treatment. These results demonstrate that glutathione is important for production of mature infectious picornavirus virions.
Conserved and host-specific features of influenza virion architecture.
Hutchinson, Edward C; Charles, Philip D; Hester, Svenja S; Thomas, Benjamin; Trudgian, David; Martínez-Alonso, Mónica; Fodor, Ervin
Viruses use virions to spread between hosts, and virion composition is therefore the primary determinant of viral transmissibility and immunogenicity. However, the virions of many viruses are complex and pleomorphic, making them difficult to analyse in detail. Here we address this by identifying and quantifying virion proteins with mass spectrometry, producing a complete and quantified model of the hundreds of host-encoded and viral proteins that make up the pleomorphic virions of influenza viruses. We show that a conserved influenza virion architecture is maintained across diverse combinations of virus and host. This 'core' architecture, which includes substantial quantities of host proteins as well as the viral protein NS1, is elaborated with abundant host-dependent features. As a result, influenza virions produced by mammalian and avian hosts have distinct protein compositions. Finally, we note that influenza virions share an underlying protein composition with exosomes, suggesting that influenza virions form by subverting microvesicle production.
HIV Vaccine-Induced Sero-Reactivity: A Challenge for Trial Participants, Researchers, and Physicians
Voronin, Yegor; Zinszner, Helene; Karg, Carissa; Brooks, Katie; Coombs, Robert; Hural, John; Holt, Renee; Fast, Pat; Allen, Mary; Allen, Mary; Busch, Michael; Fast, Pat; Fruth, Ulrich; Golding, Hana; Khurana, Surender; Mulenga, Joseph; Peel, Sheila; Schito, Marco; Voronin, Yegor; Barnabas, Nomampondo; Bentsen, Christopher; Graham, Barney; Gray, Glenda; Levin, Andrew; McCluskey, Margaret; O'Connell, Robert; Snow, Bill; Ware, Mark
Antibody-inducing vaccines are a major focus in the preventive HIV vaccine field. Because the most common tests for HIV infection rely on detecting antibodies to HIV, they may also detect antibodies induced by a candidate HIV vaccine. The detection of vaccine-induced antibodies to HIV by serological tests is most commonly referred to as vaccine-induced sero-reactivity (VISR). VISR can be misinterpreted as a sign of HIV infection in a healthy study participant. In a participant who has developed vaccine-induced antibodies, accurate diagnosis of HIV infection (or lack thereof) may require specialized tests and algorithms (differential testing) that are usually not available in community settings. Organizations sponsoring clinical testing of preventive HIV vaccine candidates have an ethical obligation not only to inform healthy volunteers about the potential problems associated with participating in a clinical trial but also to help manage any resulting issues. This article explores the scope of VISR-related issues that become increasingly prevalent as the search for an effective HIV vaccine continues and will be paramount once a preventive vaccine is deployed. We also describe ways in which organizations conducting HIV vaccine trials have addressed these issues and outline areas where more work is needed. PMID:25649349
A new approach to an AIDS vaccine: creating antibodies to HIV vif will enable apobec3G to turn HIV-infection into a benign problem.
Jeffrey Fessel, W
For a decade, attempts to produce a vaccine that prevents HIV infection have been fruitless, and fresh approaches are required. Apobec3G is a natural defensin and a cytidine deaminase. Apobec3G induces a high rate of dC to dU mutation in the first minus strand of cDNA, causing degradation throughout the HIV genome that renders the virus effete. The viral infectivity factor (vif) of HIV is essential for efficient replication of that virus. Vif binds to apobec3G and induces its polyubiquitination, which enables HIV to evade apobec3G. This suggests that a vif-based vaccine which induced anti-vif antibodies, would prevent the neutralizing action of vif upon apobec3G. Then, with HIV-vif ineffective, apobec3G could act without hindrance to create a less aggressive, non-lethal HIV infection. Mutated vif impedes HIV infection. Slow progressors with vif 132S had 4-fold lower viral loads than those with vif 132R; and introducing vif 132S into HIV-1 caused a 5-fold decrease in viral replication. And in the absence of vif, HIV virions accumulate multiple defects in structural, enzymatic, and regulatory viral proteins. The success of a vif-based vaccine depends upon (1) a vif-antibody response, and (2) vif antibodies entering the cells that harbor HIV. First, antibodies to vif have been seen in frequencies ranging between 25% and 100% in patients infected with HIV-1. Second, transport of anti-vif antibodies into cells might occur via several mechanisms. Likeliest is that in viremic persons, antibodies would attach to cell-free virions which would piggyback the antibodies into CD4+ cells. Alternatively, a fusion protein between vif and a cell-surface receptor, e.g., CD4 or CCR5, might be used as vaccine antigen. Also, anti-vif antibodies might internalize after ligation of HIV virions budding on the cell surface, in the same way as monoclonal antibodies against porcine pseudorabies virus induced viral glycoproteins on the cell surface to internalize. Finally, monoclonal
HIV-1 VACCINES. Diversion of HIV-1 vaccine-induced immunity by gp41-microbiota cross-reactive antibodies.
Williams, Wilton B; Liao, Hua-Xin; Moody, M Anthony; Kepler, Thomas B; Alam, S Munir; Gao, Feng; Wiehe, Kevin; Trama, Ashley M; Jones, Kathryn; Zhang, Ruijun; Song, Hongshuo; Marshall, Dawn J; Whitesides, John F; Sawatzki, Kaitlin; Hua, Axin; Liu, Pinghuang; Tay, Matthew Z; Seaton, Kelly E; Shen, Xiaoying; Foulger, Andrew; Lloyd, Krissey E; Parks, Robert; Pollara, Justin; Ferrari, Guido; Yu, Jae-Sung; Vandergrift, Nathan; Montefiori, David C; Sobieszczyk, Magdalena E; Hammer, Scott; Karuna, Shelly; Gilbert, Peter; Grove, Doug; Grunenberg, Nicole; McElrath, M Juliana; Mascola, John R; Koup, Richard A; Corey, Lawrence; Nabel, Gary J; Morgan, Cecilia; Churchyard, Gavin; Maenza, Janine; Keefer, Michael; Graham, Barney S; Baden, Lindsey R; Tomaras, Georgia D; Haynes, Barton F
An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy.
Topical oestrogen keratinises the human foreskin and may help prevent HIV infection.
Pask, Andrew J; McInnes, Kerry J; Webb, David R; Short, Roger V
With the growing incidence of HIV, there is a desperate need to develop simple, cheap and effective new ways of preventing HIV infection. Male circumcision reduces the risk of infection by about 60%, probably because of the removal of the Langerhans cells which are abundant in the inner foreskin and are the primary route by which HIV enters the penis. Langerhans cells form a vital part of the body's natural defence against HIV and only cause infection when they are exposed to high levels of HIV virions. Rather than removing this natural defence mechanism by circumcision, it may be better to enhance it by thickening the layer of keratin overlying the Langerhans cells, thereby reducing the viral load to which they are exposed. We have investigated the ability of topically administered oestrogen to induce keratinization of the epithelium of the inner foreskin. Histochemically, the whole of the foreskin is richly supplied with oestrogen receptors. The epithelium of the inner foreskin, like the vagina, responds within 24 hours to the topical administration of oestriol by keratinization, and the response persists for at least 5 days after the cessation of the treatment. Oestriol, a cheap, readily available natural oestrogen metabolite, rapidly keratinizes the inner foreskin, the site of HIV entry into the penis. This thickening of the overlying protective layer of keratin should reduce the exposure of the underlying Langerhans cells to HIV virions. This simple treatment could become an adjunct or alternative to surgical circumcision for reducing the incidence of HIV infection in men.
Nonneutralizing Antibodies Induced by the HIV-1 gp41 NHR Domain Gain Neutralizing Activity in the Presence of the HIV Fusion Inhibitor Enfuvirtide: a Potential Therapeutic Vaccine Strategy.
Wang, Qian; Bi, Wenwen; Zhu, Xiaojie; Li, Haoyang; Qi, Qianqian; Yu, Fei; Lu, Lu; Jiang, Shibo
A key barrier against developing preventive and therapeutic human immunodeficiency virus (HIV) vaccines is the inability of viral envelope glycoproteins to elicit broad and potent neutralizing antibodies. However, in the presence of fusion inhibitor enfuvirtide, we show that the nonneutralizing antibodies induced by the HIV type 1 (HIV-1) gp41 N-terminal heptad repeat (NHR) domain (N63) exhibit potent and broad neutralizing activity against laboratory-adapted HIV-1 strains, including the drug-resistant variants, and primary HIV-1 isolates with different subtypes, suggesting the potential of developing gp41-targeted HIV therapeutic vaccines.
Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells
Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah
(IR) increases HIV-1 transcription in latently-infected cells. • IR enhances activating effect of bryostatin 1 on HIV-1 transcription in monocytes. • IR induces apoptosis in HIV-1 infected cells via phosphorylation of p53 Ser46. • IR of HIV-1 infected humanized mice increases HIV-1 RNA in plasma, lung and brain.
Cocaine enhances HIV-1 gp120-induced lymphatic endothelial dysfunction in the lung
Zhang, Xuefeng; Jiang, Susan; Yu, Jinlong; Kuzontkoski, Paula M; Groopman, Jerome E
Pulmonary complications are common in both AIDS patients and cocaine users. We addressed the cellular and molecular mechanisms by which HIV and cocaine may partner to induce their deleterious effects. Using primary lung lymphatic endothelial cells (L-LECs), we examined how cocaine and HIV-1 gp120, alone and together, modulate signaling and functional properties of L-LECs. We found that brief cocaine exposure activated paxillin and induced cytoskeletal rearrangement, while sustained exposure increased fibronectin (FN) expression, decreased Robo4 expression, and enhanced the permeability of L-LEC monolayers. Moreover, incubating L-LECs with both cocaine and HIV-1 gp120 exacerbated hyperpermeability, significantly enhanced apoptosis, and further impaired in vitro wound healing as compared with cocaine alone. Our studies also suggested that the sigma-1 receptor (Sigma-1R) and the dopamine-4 receptor (D4R) are involved in cocaine-induced pathology in L-LECs. Seeking clinical correlation, we found that FN levels in sera and lung tissue of HIV+ donors were significantly elevated as compared to HIV− donors. Our in vitro data demonstrate that cocaine and HIV-1 gp120 induce dysfunction and damage of lung lymphatics, and suggest that cocaine use may exacerbate pulmonary edema and fibrosis associated with HIV infection. Continued exploration of the interplay between cocaine and HIV should assist the design of therapeutics to ameliorate HIV-induced pulmonary disorders within the drug using population. PMID:26311830
Development of benzimidazole derivatives to inhibit HIV-1 replication through protecting APOBEC3G protein.
Pan, Ting; He, Xin; Chen, Bing; Chen, Hui; Geng, Guannan; Luo, Haihua; Zhang, Hui; Bai, Chuan
Human APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, A3G) is a potent restriction factor against human immunodeficiency virus type 1 (HIV-1) by inducing hypermutation of G to A in viral genome after its incorporation into virions. HIV-1 Vif (Virion Infectivity Factor) counteracts A3G by inducing ubiquitination and proteasomal degradation of A3G protein. Vif-A3G axis therefore is a promising therapeutic target of HIV-1. Here we report the screening, synthesis and SAR studies of benzimidazole derivatives as potent inhibitors against HIV-1 replication via protecting A3G protein. Based on the steep SAR of the benzimidazole scaffold, we identified compound 14 and 26 which provided the best potency, with IC50 values of 3.45 nM and 58.03 nM respectively in the anti-HIV-1 replication assay in H9 cells. Compound 14 and 26 also afforded protective effects on A3G protein level. Both compounds have been proved to be safe in acute toxicological studies. Taken together, we suggest that these two benzimidazole derivatives can be further developed as a new category of anti-HIV-1 leads.
Increased iron export by ferroportin induces restriction of HIV-1 infection in sickle cell disease
Kumari, Namita; Ammosova, Tatiana; Diaz, Sharmin; Lin, Xionghao; Niu, Xiaomei; Ivanov, Andrey; Jerebtsova, Marina; Dhawan, Subhash; Oneal, Patricia; Nekhai, Sergei
The low incidence of HIV-1 infection in patients with sickle cell disease (SCD) and inhibition of HIV-1 replication in vitro under the conditions of low intracellular iron or heme treatment suggests a potential restriction of HIV-1 infection in SCD. We investigated HIV-1 ex vivo infection of SCD peripheral blood mononuclear cells (PBMCs) and found that HIV-1 replication was inhibited at the level of reverse transcription (RT) and transcription. We observed increased expression of heme and iron-regulated genes, previously shown to inhibit HIV-1, including ferroportin, IKBα, HO-1, p21, and SAM domain and HD domain-containing protein 1 (SAMHD1). HIV-1 inhibition was less pronounced in hepcidin-treated SCD PBMCs and more pronounced in the iron or iron chelators treated, suggesting a key role of iron metabolism. In SCD PBMCs, labile iron levels were reduced and protein levels of ferroportin, HIF-1α, IKBα, and HO-1 were increased. Hemin treatment induced ferroportin expression and inhibited HIV-1 in THP-1 cells, mimicking the HIV-1 inhibition in SCD PBMCs, especially as hepcidin similarly prevented HIV-1 inhibition. In THP-1 cells with knocked down ferroportin, IKBα, or HO-1 genes but not HIF-1α or p21, HIV-1 was not inhibited by hemin. Activity of SAMHD1-regulatory CDK2 was decreased, and SAMHD1 phosphorylation was reduced in SCD PBMCs and hemin-treated THP-1 cells, suggesting SAMHD1-mediated HIV-1 restriction in SCD. Our findings point to ferroportin as a trigger of HIV-1 restriction in SCD settings, linking reduced intracellular iron levels to the inhibition of CDK2 activity, reduction of SAMHD1 phosphorylation, increased IKBα expression, and inhibition of HIV-1 RT and transcription. PMID:28203649
Early events in herpes simplex virus type 1 infection: photosensitivity of fluorescein isothiocyanate-treated virions
DeLuca, N.; Bzik, D.; Person, S.; Snipes, W.
Herpes simplex virus type 1 is photosensitized by treatment with fluorescein isothiocyanate (FITC). The inactivation of FITC-treated virions upon subsequent exposure to light is inhibited by the presence of sodium azide, suggesting the involvement of singlet oxygen in the process. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that treatment with FITC plus light induces crosslinks in viral envelope glycoproteins. Treatment of virions with high concentrations of FITC (50 ..mu..g/ml) plus light causes a reduction in the adsorption of the virus to monolayers of human embryonic lung cells. For lower concentrations of FITC (10 ..mu..g/ml) plus light, treated virions adsorb to the host cells, but remain sensitive to light until entry occurs. The loss of light sensitivity coincides with the development of resistance to antibodies. These results are most consistent with a mechanism of entry for herpes simplex virus involving fusion of the viral membrane with the plasma membrane of the host cell.
Virion Background and Efficiency of Virion Incorporation Determine Susceptibility of Simian Immunodeficiency Virus Env-Driven Viral Entry to Inhibition by IFITM Proteins
Wrensch, Florian; Hoffmann, Markus; Gärtner, Sabine; Nehlmeier, Inga; Winkler, Michael
ABSTRACT Interferon-induced transmembrane proteins (IFITMs) can inhibit the cellular entry of several enveloped viruses, including simian immunodeficiency virus (SIV). The blockade of SIV by IFITMs is isolate specific, raising the question of which parameters impact sensitivity to IFITM. We show that the virion context in which SIV-Env is presented and the efficiency of virion incorporation determine Env susceptibility to inhibition by IFITMs. Thus, determinants other than the nature of the envelope protein can impact the IFITM sensitivity of viral entry. IMPORTANCE The host cell-encoded IFITM proteins can block viral entry and are an important component of the innate defenses against viral infection. However, the determinants controlling whether a virus is susceptible to blockade by IFITM proteins are incompletely understood. Our study shows that the amount of envelope proteins incorporated into virions as well as the nature of the virion particle itself can impact the sensitivity of viral entry to IFITMs. These results show for the first time that determinants other than the viral envelope protein can impact sensitivity to IFITM and have implications for the interpretation of previously published data on inhibition of viruses by IFITM proteins. Moreover, our findings might help to define the mechanism underlying the antiviral activity of IFITM proteins. PMID:27807233
Impaired NK Cell Activation and Chemotaxis toward Dendritic Cells Exposed to Complement-Opsonized HIV-1.
Ellegård, Rada; Crisci, Elisa; Andersson, Jonas; Shankar, Esaki M; Nyström, Sofia; Hinkula, Jorma; Larsson, Marie
Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection.
Structural Lability of Barley Stripe Mosaic Virus Virions
Semenyuk, Pavel I.; Abashkin, Dmitry A.; Kalinina, Natalya O.; Arutyunyan, Alexsandr M.; Solovyev, Andrey G.; Dobrov, Eugeny N.
Virions of Barley stripe mosaic virus (BSMV) were neglected for more than thirty years after their basic properties were determined. In this paper, the physicochemical characteristics of BSMV virions and virion-derived viral capsid protein (CP) were analyzed, namely, the absorption and intrinsic fluorescence spectra, circular dichroism spectra, differential scanning calorimetry curves, and size distributions by dynamic laser light scattering. The structural properties of BSMV virions proved to be intermediate between those of Tobacco mosaic virus (TMV), a well-characterized virus with rigid rod-shaped virions, and flexuous filamentous plant viruses. The BSMV virions were found to be considerably more labile than expected from their rod-like morphology and a distant sequence relation of the BSMV and TMV CPs. The circular dichroism spectra of BSMV CP subunits incorporated into the virions, but not subunits of free CP, demonstrated a significant proportion of beta-structure elements, which were proposed to be localized mostly in the protein regions exposed on the virion outer surface. These beta-structure elements likely formed during virion assembly can comprise the N- and C-terminal protein regions unstructured in the non-virion CP and can mediate inter-subunit interactions. Based on computer-assisted structure modeling, a model for BSMV CP subunit structural fold compliant with the available experimental data was proposed. PMID:23613760
CCTTT-repeat polymorphism of the inducible nitric oxide synthase is not associated with HIV pathogenesis
HERSBERGER, M; BONHOEFFER, S; RAMPINI, S K; OPRAVIL, M; MARTI-JAUN, J; TELENTI, A; HÄNSELER, E; LEDERGERBER, B; Speck, R F
Nitric oxide (NO) produced by the inducible form of nitric oxide synthase (iNOS) has bactericidal and virocidal effects. Although NO synthesis and iNOS expression in macrophages affect several aspects of human immunodeficiency virus (HIV) type-1 pathogenesis, their role in HIV disease remains largely unknown. In humans, the expression of iNOS is influenced by a functional CCTTT-repeat polymorphism in the promoter region of the gene. We investigated the association of this polymorphism with HIV pathogenesis in naive HIV-infected patients before the initiation of antiretroviral therapy. The allele frequencies of the iNOS CCTTT-repeat polymorphism were assessed by PCR in 857 patients from the Swiss HIV Cohort Study, including rapid progressors and long-term nonprogressors, and in 240 healthy volunteers. In HIV-infected patients, the initial viral load and the decline in total CD4 cells was calculated to estimate disease progression. Allele frequencies of the iNOS CCTTT-repeat polymorphism were similar between the HIV-infected and noninfected blood donors. In treatment-naive HIV-positive patients, there was no association of the iNOS polymorphism with viral load or with the course of CD4 cells. Regulation of iNOS expression by the functional CCTTT-polymorphism does not modify HIV pathogenesis. PMID:15320907
Plasmacytoid dendritic cells promote HIV-1–induced group 3 innate lymphoid cell depletion
Zhang, Zheng; Cheng, Liang; Zhao, Juanjuan; Li, Guangming; Zhang, Liguo; Chen, Weiwei; Nie, Weiming; Reszka-Blanco, Natalia J.; Wang, Fu-Sheng; Su, Lishan
Group 3 innate lymphoid cells (ILC3s) have demonstrated roles in promoting antibacterial immunity, maintaining epithelial barrier function, and supporting tissue repair. ILC3 alterations are associated with chronic inflammation and inflammatory disease; however, the characteristics and relevant regulatory mechanisms of this cell population in HIV-1 infection are poorly understood due in part to a lack of a robust model. Here, we determined that functional human ILC3s develop in lymphoid organs of humanized mice and that persistent HIV-1 infection in this model depletes ILC3s, as observed in chronic HIV-1–infected patients. In HIV-1–infected mice, effective antiretroviral therapy reversed the loss of ILC3s. HIV-1–dependent reduction of ILC3s required plasmacytoid dendritic cells (pDCs), IFN-I, and the CD95/FasL pathway, as targeted depletion or blockade of these prevented HIV-1–induced ILC3 depletion in vivo and in vitro, respectively. Finally, we determined that HIV-1 infection induces CD95 expression on ILC3s via a pDC- and IFN-I–dependent mechanism that sensitizes ILC3s to undergo CD95/FasL-mediated apoptosis. We conclude that chronic HIV-1 infection depletes ILC3s through pDC activation, induction of IFN-I, and CD95-mediated apoptosis. PMID:26301812
HIV infection and HERV expression: a review
The human genome contains multiple copies of retrovirus genomes known as endogenous retroviruses (ERVs) that have entered the germ-line at some point in evolution. Several of these proviruses have retained (partial) coding capacity, so that a number of viral proteins or even virus particles are expressed under various conditions. Human ERVs (HERVs) belong to the beta-, gamma-, or spuma- retrovirus groups. Endogenous delta- and lenti- viruses are notably absent in humans, although endogenous lentivirus genomes have been found in lower primates. Exogenous retroviruses that currently form a health threat to humans intriguingly belong to those absent groups. The best studied of the two infectious human retroviruses is the lentivirus human immunodeficiency virus (HIV) which has an overwhelming influence on its host by infecting cells of the immune system. One HIV-induced change is the induction of HERV transcription, often leading to induced HERV protein expression. This review will discuss the potential HIV-HERV interactions. Several studies have suggested that HERV proteins are unlikely to complement defective HIV virions, nor is HIV able to package HERV transcripts, probably due to low levels of sequence similarity. It is unclear whether the expression of HERVs has a negative, neutral, or positive influence on HIV-AIDS disease progression. A positive effect was recently reported by the specific expression of HERVs in chronically HIV-infected patients, which results in the presentation of HERV-derived peptides to CD8+ T-cells. These cytotoxic T-cells were not tolerant to HERV peptides, as would be expected for self-antigens, and consequently lysed the HIV-infected, HERV-presenting cells. This novel mechanism could control HIV replication and result in a low plasma viral load. The possibility of developing a vaccination strategy based on these HERV peptides will be discussed. PMID:22248111
Antigenic Properties of the Human Immunodeficiency Virus Envelope Glycoprotein Gp120 on Virions Bound to Target Cells
Mengistu, Meron; Ray, Krishanu; Lewis, George K.; DeVico, Anthony L.
The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of
Stimulation of Liver X Receptor Has Potent Anti-HIV Effects in a Humanized Mouse Model of HIV Infection
Ramezani, Ali; Dubrovsky, Larisa; Pushkarsky, Tatiana; Sviridov, Dmitri; Karandish, Sara; Raj, Dominic S.; Fitzgerald, Michael L.
Previous studies demonstrated that liver X receptor (LXR) agonists inhibit human immunodeficiency virus (HIV) replication by upregulating cholesterol transporter ATP-binding cassette A1 (ABCA1), suppressing HIV production, and reducing infectivity of produced virions. In this study, we extended these observations by analyzing the effect of the LXR agonist T0901317 [N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide] on the ongoing HIV infection and investigating the possibility of using LXR agonist for pre-exposure prophylaxis of HIV infection in a humanized mouse model. Pre-exposure of monocyte-derived macrophages to T0901317 reduced susceptibility of these cells to HIV infection in vitro. This protective effect lasted for up to 4 days after treatment termination and correlated with upregulated expression of ABCA1, reduced abundance of lipid rafts, and reduced fusion of the cells with HIV. Pre-exposure of peripheral blood leukocytes to T0901317 provided only a short-term protection against HIV infection. Treatment of HIV-exposed humanized mice with LXR agonist starting 2 weeks postinfection substantially reduced viral load. When eight humanized mice were pretreated with LXR agonist prior to HIV infection, five animals were protected from infection, two had viral load at the limit of detection, and one had viral load significantly reduced relative to mock-treated controls. T0901317 pretreatment also reduced HIV-induced dyslipidemia in infected mice. In conclusion, these results reveal a novel link between LXR stimulation and cell resistance to HIV infection and suggest that LXR agonists may be good candidates for development as anti-HIV agents, in particular for pre-exposure prophylaxis of HIV infection. PMID:26126533
Stimulation of Liver X Receptor Has Potent Anti-HIV Effects in a Humanized Mouse Model of HIV Infection.
Ramezani, Ali; Dubrovsky, Larisa; Pushkarsky, Tatiana; Sviridov, Dmitri; Karandish, Sara; Raj, Dominic S; Fitzgerald, Michael L; Bukrinsky, Michael
Previous studies demonstrated that liver X receptor (LXR) agonists inhibit human immunodeficiency virus (HIV) replication by upregulating cholesterol transporter ATP-binding cassette A1 (ABCA1), suppressing HIV production, and reducing infectivity of produced virions. In this study, we extended these observations by analyzing the effect of the LXR agonist T0901317 [N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide] on the ongoing HIV infection and investigating the possibility of using LXR agonist for pre-exposure prophylaxis of HIV infection in a humanized mouse model. Pre-exposure of monocyte-derived macrophages to T0901317 reduced susceptibility of these cells to HIV infection in vitro. This protective effect lasted for up to 4 days after treatment termination and correlated with upregulated expression of ABCA1, reduced abundance of lipid rafts, and reduced fusion of the cells with HIV. Pre-exposure of peripheral blood leukocytes to T0901317 provided only a short-term protection against HIV infection. Treatment of HIV-exposed humanized mice with LXR agonist starting 2 weeks postinfection substantially reduced viral load. When eight humanized mice were pretreated with LXR agonist prior to HIV infection, five animals were protected from infection, two had viral load at the limit of detection, and one had viral load significantly reduced relative to mock-treated controls. T0901317 pretreatment also reduced HIV-induced dyslipidemia in infected mice. In conclusion, these results reveal a novel link between LXR stimulation and cell resistance to HIV infection and suggest that LXR agonists may be good candidates for development as anti-HIV agents, in particular for pre-exposure prophylaxis of HIV infection.
Interaction with the p6 Domain of the Gag Precursor Mediates Incorporation into Virions of Vpr and Vpx Proteins from Primate Lentiviruses
Selig, L.; Pages, J.-C.; Tanchou, V.; Prévéral, S.; Berlioz-Torrent, C.; Liu, L. X.; Erdtmann, L.; Darlix, J.-L.; Benarous, R.; Benichou, S.
Vpr and Vpx proteins from human and simian immunodeficiency viruses (HIV and SIV) are incorporated into virions in quantities equivalent to those of the viral Gag proteins. We demonstrate here that Vpr and Vpx proteins from distinct lineages of primate lentiviruses were able to bind to their respective Gag precursors. The capacity of HIV type 1 (HIV-1) Vpr mutants to bind to Pr55Gag was correlated with their incorporation into virions. Molecular analysis of these interactions revealed that they required the C-terminal p6 domain of the Gag precursors. While the signal for HIV-1 Vpr binding lies in the leucine triplet repeat region of the p6 domain reported to be essential for incorporation, SIVsm Gag lacking the equivalent region still bound to SIVsm Vpr and Vpx, indicating that the determinants for Gag binding are located upstream of this region of the p6 domain. Binding to Gag cleavage products showed that HIV-1 Vpr interacted directly with the nucleocapsid protein (NC), whereas SIVsm Vpr and Vpx did not interact with NC but with the p6 protein. These results (i) reveal differences between HIV-1 and SIVsm for the p6 determinants required for Vpr and Vpx binding to Gag and (ii) suggest that HIV-1 Vpr and SIVsm Vpr and Vpx interact with distinct cleavage products of the precursor following proteolytic processing in the virions. PMID:9847364
Scaling, crumpled wires, and genome packing in virions
NASA Astrophysics Data System (ADS)
de Holanda, V. H.; Gomes, M. A. F.
The packing of a genome in virions is a topic of intense current interest in biology and biological physics. The area is dominated by allometric scaling relations that connect, e.g., the length of the encapsulated genome and the size of the corresponding virion capsid. Here we report scaling laws obtained from extensive experiments of packing of a macroscopic wire within rigid three-dimensional spherical and nonspherical cavities that can shed light on the details of the genome packing in virions. We show that these results obtained with crumpled wires are comparable to those from a large compilation of biological data from several classes of virions.
Pharmacological, behavioural and mechanistic analysis of HIV-1 gp120 induced painful neuropathy.
Wallace, Victoria C J; Blackbeard, Julie; Pheby, Timothy; Segerdahl, Andrew R; Davies, Meirion; Hasnie, Fauzia; Hall, Susan; McMahon, Stephen B; Rice, Andrew S C
A painful neuropathy is frequently observed in people living with human immunodeficiency virus type 1 (HIV-1). The HIV coat protein, glycoprotein 120 (gp120), implicated in the pathogenesis of neurological disorders associated with HIV, is capable of initiating neurotoxic cascades via an interaction with the CXCR4 and/or CCR5 chemokine receptors, which may underlie the pathogenesis of HIV-associated peripheral neuropathic pain. In order to elucidate the mechanisms underlying HIV-induced painful peripheral neuropathy, we have characterised pathological events in the peripheral and central nervous system following application of HIV-1 gp120 to the rat sciatic nerve. Perineural HIV-1 gp120 treatment induced a persistent mechanical hypersensitivity (44% decrease from baseline), but no alterations in sensitivity to thermal or cold stimuli, and thigmotactic (anxiety-like) behaviour in the open field. The mechanical hypersensitivity was sensitive to systemic treatment with gabapentin, morphine and the cannabinoid WIN 55,212-2, but not with amitriptyline. Immunohistochemical studies reveal: decreased intraepidermal nerve fibre density, macrophage infiltration into the peripheral nerve at the site of perineural HIV-1 gp120; changes in sensory neuron phenotype including expression of activating transcription factor 3 (ATF3) in 27% of cells, caspase-3 in 25% of cells, neuropeptide Y (NPY) in 12% of cells and galanin in 13% of cells and a spinal gliosis. These novel findings suggest that this model is not only useful for the elucidation of mechanisms underlying HIV-1-related peripheral neuropathy but may prove useful for preclinical assessment of drugs for the treatment of HIV-1 related peripheral neuropathic pain.
Pharmacological, behavioural and mechanistic analysis of HIV-1 gp120 induced painful neuropathy
Wallace, Victoria C.J.; Blackbeard, Julie; Pheby, Timothy; Segerdahl, Andrew R.; Davies, Meirion; Hasnie, Fauzia; Hall, Susan; McMahon, Stephen B.; Rice, Andrew S.C.
A painful neuropathy is frequently observed in people living with human immunodeficiency virus type 1 (HIV-1). The HIV coat protein, glycoprotein 120 (gp120), implicated in the pathogenesis of neurological disorders associated with HIV, is capable of initiating neurotoxic cascades via an interaction with the CXCR4 and/or CCR5 chemokine receptors, which may underlie the pathogenesis of HIV-associated peripheral neuropathic pain. In order to elucidate the mechanisms underlying HIV-induced painful peripheral neuropathy, we have characterised pathological events in the peripheral and central nervous system following application of HIV-1 gp120 to the rat sciatic nerve. Perineural HIV-1 gp120 treatment induced a persistent mechanical hypersensitivity (44% decrease from baseline), but no alterations in sensitivity to thermal or cold stimuli, and thigmotactic (anxiety-like) behaviour in the open field. The mechanical hypersensitivity was sensitive to systemic treatment with gabapentin, morphine and the cannabinoid WIN 55,212-2, but not with amitriptyline. Immunohistochemical studies reveal: decreased intraepidermal nerve fibre density, macrophage infiltration into the peripheral nerve at the site of perineural HIV-1 gp120; changes in sensory neuron phenotype including expression of activating transcription factor 3 (ATF3) in 27% of cells, caspase-3 in 25% of cells, neuropeptide Y (NPY) in 12% of cells and galanin in 13% of cells and a spinal gliosis. These novel findings suggest that this model is not only useful for the elucidation of mechanisms underlying HIV-1-related peripheral neuropathy but may prove useful for preclinical assessment of drugs for the treatment of HIV-1 related peripheral neuropathic pain. PMID:17433546
HIV Protease Inhibitor-Induced Cathepsin Modulation Alters Antigen Processing and Cross-Presentation.
Kourjian, Georgio; Rucevic, Marijana; Berberich, Matthew J; Dinter, Jens; Wambua, Daniel; Boucau, Julie; Le Gall, Sylvie
Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells, but the persistence of chronic infections calls for new approaches to modulate immune recognition. Ag cross-presentation, the process by which pathogen Ags are internalized, degraded, and presented by MHC class I, is crucial to prime CD8 T cell responses. The original degradation of Ags is performed by pH-dependent endolysosomal cathepsins. In this article, we show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin activities in human APCs, dendritic cells and macrophages, and CD4 T cells, three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin hydrolytic activities: directly on cathepsins and indirectly on their regulators by inhibiting Akt kinase activities, reducing NADPH oxidase 2 activation, and lowering phagolysosomal reactive oxygen species production and pH, which led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence-dependent manner. They altered cross-presentation of Ags by dendritic cells to epitope-specific T cells and T cell-mediated killing. HIV PI-induced modulation of Ag processing partly changed the MHC self-peptidome displayed by primary human cells. This first identification, to our knowledge, of prescription drugs modifying the regulation of cathepsin activities and the MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond HIV.
Highly potent, synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo.
Beans, Elizabeth J; Fournogerakis, Dennis; Gauntlett, Carolyn; Heumann, Lars V; Kramer, Rainer; Marsden, Matthew D; Murray, Danielle; Chun, Tae-Wook; Zack, Jerome A; Wender, Paul A
Highly active antiretroviral therapy (HAART) decreases plasma viremia below the limits of detection in the majority of HIV-infected individuals, thus serving to slow disease progression. However, HAART targets only actively replicating virus and is unable to eliminate latently infected, resting CD4(+) T cells. Such infected cells are potentially capable of reinitiating virus replication upon cessation of HAART, thus leading to viral rebound. Agents that would eliminate these reservoirs, when used in combination with HAART, could thus provide a strategy for the eradication of HIV. Prostratin is a preclinical candidate that induces HIV expression from latently infected CD4(+) T cells, potentially leading to their elimination through a virus-induced cytopathic effect or host anti-HIV immunity. Here, we report the synthesis of a series of designed prostratin analogs and report in vitro and ex vivo studies of their activity relevant to induction of HIV expression. Members of this series are up to 100-fold more potent than the preclinical lead (prostratin) in binding to cell-free PKC, and in inducing HIV expression in a latently infected cell line and prostratin-like modulation of cell surface receptor expression in primary cells from HIV-negative donors. Significantly, selected members were also tested for HIV induction in resting CD4(+) T cells isolated from infected individuals receiving HAART and were found to exhibit potent induction activity. These more potent agents and by extension related tunable analogs now accessible through the studies described herein should facilitate research and preclinical advancement of this strategy for HIV/AIDS eradication.
Ethambutol induced toxic optic neuropathy in HIV positive patients
Mustak, Hamzah; Rogers, Graeme; Cook, Colin
AIM To determine whether HIV and the use of antiretroviral therapy is a risk factor for the development of ethambutol toxic optic neuropathy. To describe the clinical course of ethambutol toxic optic neuropathy in patients with HIV and to identify prognostic factors. METHODS The case notes of 14 consecutive patients referred to the neuro-ophthalmology clinic were reviewed. Data regarding HIV status, antiretroviral therapy, visual function, ethambutol therapy dosage, and ethambutol therapy duration were collected and analysed. RESULTS Eleven of the 14 patients were HIV positive. Ten of the HIV positive patients were receiving antiretroviral therapy. The mean dose of ethambutol was 17.25mg/kg/day. No statistically significant difference in mean dose, duration of therapy, age or CD4 count was found between those who showed visual improvement and those who did not. Delay in presentation of more than one month post symptom onset was correlated with poor visual outcome (P=0.001). CONCLUSION HIV and, perhaps more importantly, the potential mitochondrial toxic effects of Nucleoside analogue reverse transcriptase inhibitors (NRTIs) may be a risk factor for the development of toxic optic neuropathy from ethambutol therapy via a multiple hit effect. Delay in presentation results in poor visual outcome. Regular monitoring is recommended for HIV positive patients receiving antiretrovirals and requiring ethambutol therapy in order to avoid permanent visual loss. PMID:23991394
Designed, synthetically accessible bryostatin analogues potently induce activation of latent HIV reservoirs in vitro
NASA Astrophysics Data System (ADS)
Dechristopher, Brian A.; Loy, Brian A.; Marsden, Matthew D.; Schrier, Adam J.; Zack, Jerome A.; Wender, Paul A.
Bryostatin is a unique lead in the development of potentially transformative therapies for cancer, Alzheimer's disease and the eradication of HIV/AIDS. However, the clinical use of bryostatin has been hampered by its limited supply, difficulties in accessing clinically relevant derivatives, and side effects. Here, we address these problems through the step-economical syntheses of seven members of a new family of designed bryostatin analogues using a highly convergent Prins-macrocyclization strategy. We also demonstrate for the first time that such analogues effectively induce latent HIV activation in vitro with potencies similar to or better than bryostatin. Significantly, these analogues are up to 1,000-fold more potent in inducing latent HIV expression than prostratin, the current clinical candidate for latent virus induction. This study provides the first demonstration that designed, synthetically accessible bryostatin analogues could serve as superior candidates for the eradication of HIV/AIDS through induction of latent viral reservoirs in conjunction with current antiretroviral therapy.
Drug-Induced Reactivation of Apoptosis Abrogates HIV-1 Infection
Hanauske-Abel, Hartmut M.; Saxena, Deepti; Palumbo, Paul E.; Hanauske, Axel-Rainer; Luchessi, Augusto D.; Cambiaghi, Tavane D.; Hoque, Mainul; Spino, Michael; Gandolfi, Darlene D'Alliessi; Heller, Debra S.; Singh, Sukhwinder; Park, Myung Hee; Cracchiolo, Bernadette M.; Tricta, Fernando; Connelly, John; Popowicz, Anthony M.; Cone, Richard A.; Holland, Bart; Pe'ery, Tsafi; Mathews, Michael B.
HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal
Lifetime Induced Abortion: A Comparison between Women Living and Not Living with HIV
Pilecco, Flávia Bulegon; Teixeira, Luciana Barcellos; Vigo, Álvaro; Dewey, Michael E.; Knauth, Daniela Riva
Background Studies aimed at understanding the association between induced abortion and HIV are scarce and differ on the direction of the association. This paper aims to show the prevalence of induced abortion in a sample of pregnancies of women living and not living with HIV/Aids, determining variables associated with pregnancy termination and linked to the life course of women and to the specific context of the pregnancy. Methods Data came from a cross-sectional study, using interviewer-administered questionnaire, developed with women that attended public health services in Porto Alegre, Brazil. A generalized estimating equation model with logit link measured the association between determinants and abortion. Findings The final sample was composed of 684 women living with HIV/Aids (2,039 pregnancies) and 639 women not living with HIV/Aids (1,539 pregnancies). The prevalence of induced abortion among pregnancies in women living with HIV/Aids was 6.5%, while in women not living with HIV/Aids was 2.9%. Among women living with HIV/Aids, the following were associated with induced abortion in the multivariable analysis: being older, having a higher education level, having had more sexual partners (i.e., variables linked to the life course of women), having had children prior to the index pregnancy and living with a sexual partner during pregnancy (i.e., variables linked to the context of each pregnancy). On the other hand, among women not living with HIV/Aids, only having a higher education level and having had more sexual partners (i.e., determinants linked to the life course of women) were associated with voluntary pregnancy termination in multivariable analysis. Conclusion Although determinants are similar between women living and not living with HIV/Aids, prevalence of induced abortion is higher among pregnancies in women living with HIV/Aids, pointing to their greater social vulnerability and to the need for public policy to address prevention and treatment of HIV
Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells
NASA Technical Reports Server (NTRS)
Plymale, D. R.; Tang, D. S.; Comardelle, A. M.; Fermin, C. D.; Lewis, D. E.; Garry, R. F.
BACKGROUND: Data currently available on HIV-1-induced cytopathology is unclear regarding the mechanism of cell killing. OBJECTIVE: To clarify the extent to which apoptosis or necrosis is involved in HIV-1-induced cell death in view of conflicting existing data. METHODS: T lymphoblastoid cells or peripheral blood mononuclear cells were infected by various strains of HIV-1 and the numbers of apoptotic or necrotic cells were quantified at various times after infection using video-image analysis techniques; the results were compared with the amount of fragmented DNA using a quantitative method. Measurement of mitochondrial transmembrane potential (deltapsi(m)) and intracellular calcium concentrations [Ca2+]i was performed with fluorescent probes and fluorescence concentration analysis (FCA). RESULTS: Although lymphoblastoid and monocytoid cells acutely infected by HIV-1 had increased levels of fragmented DNA, a marker of apoptotic cell death, few (<12%) had condensed chromatin and fragmented nuclei, the morphological features of apoptosis. The predominant alterations in acutely infected cells were distended endoplasmic reticulum and abnormal mitochondria; these ultrastructural changes are consistent with necrosis, although some infected cells simultaneously displayed features of both necrosis and apoptosis. Viability of cells persistently infected by HIV-1 was only minimally reduced from that of uninfected cells. This reduction was accounted for by an increased propensity of the persistently infected cells to die by apoptosis. Alterations in [Ca2+]i and deltapsi(m) occurred in both acutely and persistently infected cells. CONCLUSION: Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells.
Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction
Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina
Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1
Sialoadhesin Expressed on IFN-Induced Monocytes Binds HIV-1 and Enhances Infectivity
Rempel, Hans; Calosing, Cyrus; Sun, Bing; Pulliam, Lynn
Background HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14+ monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1), a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases. Methodology/Principal Findings We analyzed sialoadhesin expression on CD14+ monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14+ monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-α and interferon-γ but not tumor necrosis factor-α. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte self-infection. Conclusions/Significance Increased sialoadhesin expression on CD14+ monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14+ monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesin-expressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing the distribution of HIV-1. Sialoadhesin could disseminate HIV-1 to viral reservoirs during monocyte immunosurveillance or migration to sites of inflammation and then facilitate HIV-1 infection of permissive cells. PMID:18414664
The HIV matrix protein p17 induces hepatic lipid accumulation via modulation of nuclear receptor transcriptoma
Renga, Barbara; Francisci, Daniela; Carino, Adriana; Marchianò, Silvia; Cipriani, Sabrina; Chiara Monti, Maria; Del Sordo, Rachele; Schiaroli, Elisabetta; Distrutti, Eleonora; Baldelli, Franco; Fiorucci, Stefano
Liver disease is the second most common cause of mortality in HIV-infected persons. Exactly how HIV infection per se affects liver disease progression is unknown. Here we have investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in HepG2 cells upon stimulation with the HIV matrix protein p17. This viral protein regulated mRNA expression of some NRs among which LXRα and its transcriptional co-activator MED1 were highly induced at mRNA level. Dissection of p17 downstream intracellular pathway demonstrated that p17 mediated activation of Jak/STAT signaling is responsible for the promoter dependent activation of LXR. The treatment of both HepG2 as well as primary hepatocytes with HIV p17 results in the transcriptional activation of LXR target genes (SREBP1c and FAS) and lipid accumulation. These effects are lost in HepG2 cells pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide as well as in HepG2 cells pre-incubated with the natural LXR antagonist gymnestrogenin. These results suggest that HIV p17 affects NRs and their related signal transduction thus contributing to the progression of liver disease in HIV infected patients. PMID:26469385
The HIV matrix protein p17 induces hepatic lipid accumulation via modulation of nuclear receptor transcriptoma.
Renga, Barbara; Francisci, Daniela; Carino, Adriana; Marchianò, Silvia; Cipriani, Sabrina; Chiara Monti, Maria; Del Sordo, Rachele; Schiaroli, Elisabetta; Distrutti, Eleonora; Baldelli, Franco; Fiorucci, Stefano
Liver disease is the second most common cause of mortality in HIV-infected persons. Exactly how HIV infection per se affects liver disease progression is unknown. Here we have investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in HepG2 cells upon stimulation with the HIV matrix protein p17. This viral protein regulated mRNA expression of some NRs among which LXRα and its transcriptional co-activator MED1 were highly induced at mRNA level. Dissection of p17 downstream intracellular pathway demonstrated that p17 mediated activation of Jak/STAT signaling is responsible for the promoter dependent activation of LXR. The treatment of both HepG2 as well as primary hepatocytes with HIV p17 results in the transcriptional activation of LXR target genes (SREBP1c and FAS) and lipid accumulation. These effects are lost in HepG2 cells pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide as well as in HepG2 cells pre-incubated with the natural LXR antagonist gymnestrogenin. These results suggest that HIV p17 affects NRs and their related signal transduction thus contributing to the progression of liver disease in HIV infected patients.
Exploring the relationship between induced abortion and HIV infection in Brazil.
Barbosa, Regina M; Pinho, Adriana A; Santos, Naila S; Villela, Wilza V
The impact of HIV on the decision to interrupt pregnancy remains an understudied topic in Brazil and the world. The technical means to implement HIV prevention and treatment interventions are widely available in Brazil. Although Brazil has restrictive abortion laws, induced abortion occurs frequently. This qualitative study investigates the extent to which Brazilian women are motivated to seek abortion as a consequence of having HIV disease, and the extent to which the decision is part of a larger reproductive decision-making context. Researchers interviewed 30 women who were living with HIV and had terminated pregnancies or attempted to do so. Many women identified their HIV status as an important aspect of their decision-making regarding abortion. Women also took into account issues such as the stage of life when the pregnancy occurred and the absence of support from partners and families. Contraceptive practices, pregnancy and abortion in this population are influenced by multiple factors that act on the structural, social, interpersonal and individual levels. We hypothesize that HIV infection and abortion are sometimes associated with similar contexts of vulnerability. Health services therefore should address HIV and reproductive issues together, with reproductive and sexual rights serving as the fundamental basis of health care.
Analysis of the Role of Vaccinia Virus H7 in Virion Membrane Biogenesis with an H7-Deletion Mutant
Meng, Xiangzhi; Wu, Xiang; Yan, Bo; Deng, Junpeng
Essential vaccinia virus genes are often studied with conditional-lethal inducible mutants. Here, we constructed a deletion mutant lacking the essential H7R gene (the ΔH7 mutant) with an H7-expressing cell line. Compared to an inducible H7 mutant, the ΔH7 mutant showed a defect at an earlier step of virion membrane biogenesis, before the development of short crescent-shaped precursors of the viral envelope. Our studies refine the role of H7 in virion membrane biogenesis and highlight the values of analyzing deletion mutants. PMID:23678177
Nigella sativa concoction induced sustained seroreversion in HIV patient.
Onifade, Abdulfatah Adekunle; Jewell, Andrew Paul; Adedeji, Waheed Adeola
Nigella sativa had been documented to possess many therapeutic functions in medicine but the least expected is sero-reversion in HIV infection which is very rare despite extensive therapy with highly active anti-retroviral therapy (HAART). This case presentation is to highlight the complete recovery and sero-reversion of adult HIV patient after treatment with Nigella sativa concoction for the period of six months. The patient presented to the herbal therapist with history of chronic fever, diarrhoea, weight loss and multiple papular pruritic lesions of 3 months duration. Examination revealed moderate weight loss, and the laboratory tests of ELISA (Genscreen) and western blot (new blot 1 & 2) confirmed sero-positivity to HIV infection with pre-treatment viral (HIV-RNA) load and CD4 count of 27,000 copies/ml and CD4 count of 250 cells/ mm(3) respectively. The patient was commenced on Nigella sativa concoction 10 mls twice daily for 6 months.. He was contacted daily to monitor side-effects and drug efficacy. Fever, diarrhoea and multiple pruritic lesions disappeared on 5th, 7th and 20th day respectively on Nigella sativa therapy. The CD4 count decreased to 160 cells/ mm3 despite significant reduction in viral load (≤1000 copies/ml) on 30th day on N. sativa. Repeated EIA and Western blot tests on 187th day on Nigella sativa therapy was sero-negative. The post therapy CD4 count was 650 cells/ mm(3) with undetectable viral (HIV-RNA) load. Several repeats of the HIV tests remained sero-negative, aviraemia and normal CD4 count since 24 months without herbal therapy. This case report reflects the fact that there are possible therapeutic agents in Nigella sativa that may effectively control HIV infection.
Tetraspanins CD9 and CD81 modulate HIV-1-induced membrane fusion.
Gordón-Alonso, Mónica; Yañez-Mó, María; Barreiro, Olga; Alvarez, Susana; Muñoz-Fernández, M Angeles; Valenzuela-Fernández, Agustín; Sánchez-Madrid, Francisco
Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.
Major histocompatibility complex class-II molecules promote targeting of human immunodeficiency virus type 1 virions in late endosomes by enhancing internalization of nascent particles from the plasma membrane
Finzi, Andrés; Perlman, Mira; Bourgeois-Daigneault, Marie-Claude; Thibodeau, Jacques; Cohen, Éric A.
Summary Productive assembly of human immunodeficiency virus type 1 (HIV-1) takes place, primarily, at the plasma membrane. However, depending on the cell types, a significant proportion of nascent virus particles are internalized and routed to late endosomes. We previously reported that expression of human leucocyte antigen (HLA)-DR promoted a redistribution of Gag in late endosomes and an increased detection of mature virions in these compartments in HeLa and human embryonic kidney 293T model cell lines. Although this redistribution of Gag resulted in a marked decrease of HIV-1 release, the underlying mechanism remained undefined. Here, we provide evidence that expression of HLA-DR at the cell surface induces a redistribution of mature Gag products into late endosomes by enhancing nascent HIV-1 particle internalization from the plasma membrane through a process that relies on the presence of intact HLA-DR α and β-chain cytosolic tails. These findings raise the possibility that major histocompatibility complex class-II molecules might influence endocytic events at the plasma membrane and as a result promote endocytosis of progeny HIV-1 particles. PMID:23170932
HIV infection induces morphometrical changes on the oral (buccal mucosa and tongue) epithelial cells.
Pompermayer, Adriane Bastos; Gil, Francisca Berenice Dias; França, Beatriz Helena Sottile; Machado, Maria Ângela Naval; Trevilatto, Paula Cristina; Fernandes, Angela; de Lima, Antônio Adilson Soares
The aim of this study was to assess morphological and morphometrical alterations of oral squamous epithelial cells in type 1 HIV infected individuals. Oral smears were collected from tongue and buccal mucosa of 30 HIV infected (experimental) and 30 non-infected (control) individuals by liquid-based exfoliative cytology. The cells were morphologically analyzed and the nuclear area (NA), the cytoplasmic area (CA) and the nucleus-to-cytoplasm area ratio (NA/CA) were calculated. No morphological differences were found between the groups. The mean values of CA were decreased in tongue (P=.00006) and buccal mucosa (P=.00242) in HIV infected individual, while mean values of NA were increased (P=.00308 and .00095, respectively) in the same group. NA/CA ratio for experimental group was increased in both collected places, with P=.00001 (tongue) and P=.00000 (buccal mucosa). This study revealed that HIV infection was able to induce morphometrical changes on the oral epithelial cells.
Trichomonas vaginalis-Induced Epithelial Monolayer Disruption and Human Immunodeficiency Virus Type 1 (HIV-1) Replication: Implications for the Sexual Transmission of HIV-1
Guenthner, Patricia C.; Secor, W. Evan; Dezzutti, Charlene S.
The objective of this study was to evaluate potential mechanisms of Trichomonas vaginalis involvement in human immunodeficiency virus type 1 (HIV-1) transmission. Polarized monolayer integrity of primary cervical and prostate epithelial cells or cell lines cultured with T. vaginalis was measured by monitoring transepithelium resistance. The effect of T. vaginalis isolates on HIV-1 passage through polarized epithelial cell monolayers was evaluated for HIV-1 p24gag in the basolateral supernatants. Coincubation with T. vaginalis isolates induced disruption of monolayer integrity and resulted in passage of virus to the basolateral side of the monolayer. Furthermore, there was isolate variability in which two isolates induced greater monolayer damage and increased HIV-1 passage than did the other two isolates. Coincubation of T. vaginalis isolates with acutely HIV-1-infected peripheral blood mononuclear cells enhanced HIV-1 replication. This enhancement was associated with cellular proliferation and activation, as well as with tumor necrosis factor alpha production. In contrast to the monolayer disruption, the effect of T. vaginalis on HIV-1 replication was not isolate dependent. Thus, two mechanisms have been identified that could contribute to the epidemiologic association of trichomoniasis with the sexual transmission of HIV-1. (i) T. vaginalis disruption of urogenital epithelial monolayers could facilitate passage of HIV-1 to underlying layers. (ii) Activation of local immune cells by T. vaginalis in the presence of infectious HIV-1 might lead to increased viral replication. Collectively, these data suggest the need for more vigilant efforts in the diagnosis and treatment of T. vaginalis in women and men, especially in countries with a high prevalence of HIV-1. PMID:15972505
Trichomonas vaginalis-induced epithelial monolayer disruption and human immunodeficiency virus type 1 (HIV-1) replication: implications for the sexual transmission of HIV-1.
Guenthner, Patricia C; Secor, W Evan; Dezzutti, Charlene S
The objective of this study was to evaluate potential mechanisms of Trichomonas vaginalis involvement in human immunodeficiency virus type 1 (HIV-1) transmission. Polarized monolayer integrity of primary cervical and prostate epithelial cells or cell lines cultured with T. vaginalis was measured by monitoring transepithelium resistance. The effect of T. vaginalis isolates on HIV-1 passage through polarized epithelial cell monolayers was evaluated for HIV-1 p24gag in the basolateral supernatants. Coincubation with T. vaginalis isolates induced disruption of monolayer integrity and resulted in passage of virus to the basolateral side of the monolayer. Furthermore, there was isolate variability in which two isolates induced greater monolayer damage and increased HIV-1 passage than did the other two isolates. Coincubation of T. vaginalis isolates with acutely HIV-1-infected peripheral blood mononuclear cells enhanced HIV-1 replication. This enhancement was associated with cellular proliferation and activation, as well as with tumor necrosis factor alpha production. In contrast to the monolayer disruption, the effect of T. vaginalis on HIV-1 replication was not isolate dependent. Thus, two mechanisms have been identified that could contribute to the epidemiologic association of trichomoniasis with the sexual transmission of HIV-1. (i) T. vaginalis disruption of urogenital epithelial monolayers could facilitate passage of HIV-1 to underlying layers. (ii) Activation of local immune cells by T. vaginalis in the presence of infectious HIV-1 might lead to increased viral replication. Collectively, these data suggest the need for more vigilant efforts in the diagnosis and treatment of T. vaginalis in women and men, especially in countries with a high prevalence of HIV-1.
Interacting partners of macrophage-secreted cathepsin B contribute to HIV-induced neuronal apoptosis
CANTRES-ROSARIO, Yisel M.; HERNANDEZ, Natalia; NEGRON, Karla; PEREZ-LASPIUR, Juliana; LESZYK, John; SHAFFER, Scott A.; MELENDEZ, Loyda M.
Objective HIV-1 infection of macrophages increases cathepsin B secretion and induces neuronal apoptosis, but the molecular mechanism remains unclear. Design We identified macrophage secreted cathepsin B protein interactions extracellularly and their contribution to neuronal death in vitro. Methods Cathepsin B was immunoprecipitated from monocyte-derived macrophage supernatants after 12 days post-infection. The cathepsin B interactome was quantified by label-free tandem mass spectrometry and compared to uninfected supernatants. Proteins identified were validated by western blot. Neurons were exposed to macrophage-conditioned media in presence or absence of antibodies against cathepsin B and interacting proteins. Apoptosis was measured using TUNEL labeling. Immunohistochemistry of post-mortem brain tissue samples from healthy, HIV-infected, and Alzheimer's disease patients was performed to observe the ex vivo expression of the proteins identified. Results Nine proteins co-immunoprecipitated differentially with cathepsin B between uninfected and HIV-infected macrophages. Serum amyloid p component (SAPC) -cathepsin B interaction increased in HIV-infected macrophage supernatants, while matrix metalloprotease 9 (MMP-9) -cathepsin B interaction decreased. Pre-treatment of HIV-infected macrophage-conditioned media with antibodies against cathepsin B and SAPC decreased neuronal apoptosis. The addition of MMP-9 antibodies was not protective. SAPC was over-expressed in post-mortem brain tissue from HIV-positive neurocognitive impaired patients compared to HIV positive with normal cognition and healthy controls, while MMP-9 expression was similar in all tissues. Conclusions Inhibiting SAPC-cathepsin B interaction protects against HIV–induced neuronal death and may help to find alternative treatments for HIV-associated neurocognitive disorders. PMID:26208400
Recombinant multi-epitope vaccine induce predefined epitope-specific antibodies against HIV-1.
Li, Hua; Liu, Zu-Qiang; Ding, Jian; Chen, Ying-Hua
Monoclonal antibody 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asia, American and European strains, but antibodies responses to ELDKWA-epitope in HIV-1 infected individuals were very low. Based on the epitope-vaccine strategy suggested by us, a recombinant glutathione S-transferase (GST) fusion protein (GST-MELDKWAGELDKWAGELDKWAVDIGPGRAFYGPGRAFYGPGRAFY) as vaccine antigen containing three repeats of neutralizing epitope ELDKWA on gp41 and GPGRAFY on gp120 was designed and expressed in Escherichia coli. After vaccination course, the recombinant multi-epitope vaccine could induce high levels of predefined multi-epitope-specific antibodies in mice. These antibodies in sera could bind to both neutralizing epitopes on gp41 peptide, V3 loop peptide and recombinant soluble gp41 (aa539-684) in ELISA assay (antisera dilution: 1:1,600-25,600), while normal sera did not. Moreover, these antibodies in sera could recognize the CHO-WT cells which expressed HIV-1 envelope glycoprotein on the cell surfaces, indicating that the predefined epitope-specific antibodies could recognize natural envelope protein of HIV-1 though these antibodies were induced by recombinant multi-epitope-vaccine. These experimental results suggested a possible way to develop recombinant multi-epitope vaccine inducing multi-antiviral activities against HIV-1.
RNA polymerase activity in purified virions of avian reticuloendotheliosis viruses.
Mizutani, S; Temin, H M
An RNA polymerase activity that synthesizes a U-rich RNA hydrogen bonded to a large viral RNA molecule was found in the cores of virions of avian reticuloendotheliosis viruses (REV). The RNA polymerase activity was separable from the DNA polymerase activity of REV virions. The 5'-terminus of the newly synthesized RNA was A. In addition, a tRNA nucleotidyl transferase activity, which added -CpCpA ends to tRNA, appears to be present in the REV virions. Images PMID:183017
Endotoxin-induced cytokine and chemokine expression in the HIV-1 transgenic rat
Background Repeated exposure to a low dose of a bacterial endotoxin such as lipopolysaccharide (LPS) causes immune cells to become refractory to a subsequent endotoxin challenge, a phenomenon known as endotoxin tolerance (ET). During ET, there is an imbalance in pro- and anti-inflammatory cytokine and chemokine production, leading to a dysregulated immune response. HIV-1 viral proteins are known to have an adverse effect on the immune system. However, the effects of HIV-1 viral proteins during ET have not been investigated. Methods In this study, HIV-1 transgenic (HIV-1Tg) rats and control F344 rats (n = 12 ea) were randomly treated with 2 non-pyrogenic doses of LPS (LL) to induce ET, or saline (SS), followed by a high challenge dose of LPS (LL+L, SS+L) or saline (LL+S, SS+S). The gene expression of 84 cytokines, chemokines, and their receptors in the brain and spleen was examined by relative quantitative PCR using a PCR array, and protein levels in the brain, spleen, and serum of 7 of these 84 genes was determined using an electrochemiluminescent assay. Results In the spleen, there was an increase in key pro-inflammatory (IL1α, IL-1β, IFN-γ) and anti-inflammatory (IL-10) cytokines, and inflammatory chemokines (Ccl2, Ccl7, and Ccl9,) in response to LPS in the SS+L and LL+L (ET) groups of both the HIV-1Tg and F344 rats, but was greater in the HIV-1Tg rats than in the F344. In the ET HIV-1Tg and F344 (LL+L) rats in the spleen, the LPS-induced increase in pro-inflammatory cytokines was diminished and that of the anti-inflammatory cytokine was enhanced compared to the SS+L group rats. In the brain, IL-1β, as well as the Ccl2, Ccl3, and Ccl7 chemokines were increased to a greater extent in the HIV-1Tg rats compared to the F344; whereas Cxcl1, Cxcl10, and Cxcl11 were increased to a greater extent in the F344 rats compared to the HIV-1Tg rats in the LL+L and SS+L groups. Conclusion Our data indicate that the continuous presence of HIV-1 viral proteins can have tissue
HIV-1 Vpr potently induces programmed cell death in the CNS in vivo.
Cheng, Xiaodong; Cheng, Xiandong; Mukhtar, Muhammad; Acheampong, Edward A; Srinivasan, Algarsamy; Rafi, Mohammad; Pomerantz, Roger J; Parveen, Zahida
The human immunodeficiency virus type I (HIV-1) accessory protein Vpr has been associated with the induction of programmed cell death (apoptosis) and cell-cycle arrest. Studies have shown the apoptotic effect of Vpr on primary and established cell lines and on diverse tissues including the central nervous system (CNS) in vitro. However, the relevance of the effect of Vpr observed in vitro to HIV-1 neuropathogenesis in vivo, remains unknown. Due to the narrow host range of HIV-1 infection, no animal model is currently available. This has prompted us to consider a small animal model to evaluate the effects of Vpr on CNS in vivo through surrogate viruses expressing HIV-1Vpr. A single round of replication competent viral vectors, expressing Vpr, were used to investigate the apoptosis-inducing capabilities of HIV-1Vpr in vivo. Viral particles pseudotyped with VSV-G or N2c envelopes were generated from spleen necrosis virus (SNV) and HIV-1-based vectors to transduce CNS cells. The in vitro studies have demonstrated that Vpr generated by SNV vectors had less apoptotic effects on CNS cells compared with Vpr expressed by HIV-1 vectors. The in vivo study has suggested that viral particles, expressing Vpr generated by HIV-1-based vectors, when delivered through the ventricle, caused loss of neurons and dendritic processes in the cortical region. The apoptotic effect was extended beyond the cortical region and affected the hippocampus neurons, the lining of the choroids plexus, and the cerebellum. However, the effect of Vpr, when delivered through the cortex, showed neuronal damage only around the site of injection. Interestingly, the number of apoptotic neurons were significantly higher with HIV-1 vectors expressing Vpr than by the SNV vectors. This may be due to the differences in the proteins expressed by these viral vectors. These results suggest that Vpr induces apoptosis in CNS cells in vitro and in vivo. To our knowledge, this is the first study to investigate the
The Envelope Gene of Transmitted HIV-1 Resists a Late Interferon Gamma-Induced Block
Rihn, Suzannah J.; Foster, Toshana L.; Busnadiego, Idoia; Aziz, Muhamad Afiq; Hughes, Joseph; Neil, Stuart J. D.
ABSTRACT Type I interferon (IFN) signaling engenders an antiviral state that likely plays an important role in constraining HIV-1 transmission and contributes to defining subsequent AIDS pathogenesis. Type II IFN (IFN-γ) also induces an antiviral state but is often primarily considered to be an immunomodulatory cytokine. We report that IFN-γ stimulation can induce an antiviral state that can be both distinct from that of type I interferon and can potently inhibit HIV-1 in primary CD4+ T cells and a number of human cell lines. Strikingly, we find that transmitted/founder (TF) HIV-1 viruses can resist a late block that is induced by type II IFN, and the use of chimeric IFN-γ-sensitive/resistant viruses indicates that interferon resistance maps to the env gene. Simultaneously, in vitro evolution also revealed that just a single amino acid substitution in the envelope can confer substantial resistance to IFN-mediated inhibition. Thus, the env gene of transmitted HIV-1 confers resistance to a late block that is phenotypically distinct from blocks previously described to be resisted by env and is therefore mediated by unknown IFN-γ-stimulated factor(s) in human CD4+ T cells and cell lines. This important unidentified block could play a key role in constraining HIV-1 transmission. IMPORTANCE The human immune system can hinder invading pathogens through interferon (IFN) signaling. One consequence of this signaling is that cells enter an antiviral state, increasing the levels of hundreds of defenses that can inhibit the replication and spread of viruses. The majority of HIV-1 infections result from a single virus particle (the transmitted/founder) that makes it past these defenses and colonizes the host. Thus, the founder virus is hypothesized to be a relatively interferon-resistant entity. Here, we show that certain HIV-1 envelope genes have the unanticipated ability to resist specific human defenses mediated by different types of interferons. Strikingly, the envelope
Teriflunomide and monomethylfumarate target HIV-induced neuroinflammation and neurotoxicity.
Ambrosius, Björn; Faissner, Simon; Guse, Kirsten; von Lehe, Marec; Grunwald, Thomas; Gold, Ralf; Grewe, Bastian; Chan, Andrew
HIV-associated neurocognitive disorders (HAND) affect about 50% of infected patients despite combined antiretroviral therapy (cART). Ongoing compartmentalized inflammation mediated by microglia which are activated by HIV-infected monocytes has been postulated to contribute to neurotoxicity independent from viral replication. Here, we investigated effects of teriflunomide and monomethylfumarate on monocyte/microglial activation and neurotoxicity. Human monocytoid cells (U937) transduced with a minimal HIV-Vector were co-cultured with human microglial cells (HMC3). Secretion of pro-inflammatory/neurotoxic cytokines (CXCL10, CCL5, and CCL2: p < 0.001; IL-6: p < 0.01) by co-cultures was strongly increased compared to microglia in contact with HIV-particles alone. Upon treatment with teriflunomide, cytokine secretion was decreased (CXCL10, 3-fold; CCL2, 2.5-fold; IL-6, 2.2-fold; p < 0.001) and monomethylfumarate treatment led to 2.9-fold lower CXCL10 secretion (p < 0.001). Reduced toxicity of co-culture conditioned media on human fetal neurons by teriflunomide (29%, p < 0.01) and monomethylfumarate (27%, p < 0.05) indicated functional relevance. Modulation of innate immune functions by teriflunomide and monomethylfumarate may target neurotoxic inflammation in the context of HAND.
miR-34a is a common link in both HIV- and antiretroviral therapy-induced vascular aging
Lu, Lili; Hu, Xiamin; Zhou, Jun; Sun, Yeying; Yang, Jian; Liu, Ying; Wang, Zunzhe; Tan, Ning; Chen, Jiyan; Zhang, Chunxiang
Both HIV and antiretroviral therapy could induce vascular aging with unclear mechanisms. In this study, via microarray analysis, we identified, for the first time, that miR-34a expression was significantly increased in both HIV-infected, and antiretroviral agents-treated vessels and vascular endothelial cells (ECs) from these vessels. In cultured ECs, miR-34a expression was significantly increased by HIV-Tat protein and by the antiretroviral agents, lopinavir/ritonavir. Both HIV-Tat protein and antiretroviral agents could induce EC senescence, which was inhibited by miR-34a inhibition. In contrast, EC senescence was exacerbated by miR-34a overexpression. In addition, the vascular ECs isolated from miR-34a knockout mice were resistant to HIV and antiretroviral agents-mediated senescence. In vivo, miR-34a expression in mouse vascular walls and their ECs was increased by antiretroviral therapy and by HIV-1 Tat transgenic approach. miR-34a inhibition could effectively inhibit both HIV-Tat protein and antiretroviral therapy-induced vascular aging in mice. The increased miR-34a was induced via p53, whereas Sirt1 was a downstream target gene of miR-34a in both HIV-Tat protein and antiretroviral agents-treated ECs and vessels. The study has demonstrated that miR-34a is a common link in both HIV and antiretroviral therapy-mediated vascular aging. PMID:27889708
MSM ameliorates HIV-1 Tat induced neuronal oxidative stress via rebalance of the glutathione cycle
Kim, Seol-hee; Smith, Adam J; Tan, Jun; Shytle, R Douglas; Giunta, Brian
HIV-1 Tat protein is a key neuropathological element in HIV associated neurogcognitive disorders (HAND); a type of cognitive syndrome thought to be at least partially mediated by increased levels of brain reactive oxygen species (ROS) and nitric oxide (NO). Methylsulfonylmethane (MSM) is a sulfur-containing compound known to reduce oxidative stress. This study was conducted to determine whether administration of MSM attenuates HIV-1 Tat induced oxidative stress in mouse neuronal cells. MSM treatment significantly decreased neuronal cell NO and ROS secretion. Further, MSM significantly reversed HIV-1 Tat mediated reductions in reduced glutathione (GSH) as well as HIV-1 Tat mediated increases in oxidized glutathione (GSSG). In addition, Tat reduced nuclear translocation of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), a key nuclear promoter of antioxidant activity, while MSM increased its translocation to the nucleus in the presence of Tat. These results suggest that HIV-1 Tat reduces the resiliency of neuron cells to oxidative stress which can be reversed by MSM. Given the clinical safety of MSM, future preclinical in vivo studies will be required to further confirm these results in effort to validate MSM as a neuroprotectant in patients at risk of, or who are already diagnosed with, HAND. PMID:25893035
HIV-1 Vpr Protein Induces Proteasomal Degradation of Chromatin-associated Class I HDACs to Overcome Latent Infection of Macrophages.
Romani, Bizhan; Baygloo, Nima Shaykh; Hamidi-Fard, Mojtaba; Aghasadeghi, Mohammad Reza; Allahbakhshi, Elham
Mechanisms underlying HIV-1 latency remain among the most crucial questions that need to be answered to adopt strategies for purging the latent viral reservoirs. Here we show that HIV-1 accessory protein Vpr induces depletion of class I HDACs, including HDAC1, 2, 3, and 8, to overcome latency in macrophages. We found that Vpr binds and depletes chromatin-associated class I HDACs through a VprBP-dependent mechanism, with HDAC3 as the most affected class I HDAC. De novo expression of Vpr in infected macrophages induced depletion of HDAC1 and 3 on the HIV-1 LTR that was associated with hyperacetylation of histones on the HIV-1 LTR. As a result of hyperacetylation of histones on HIV-1 promotor, the virus established an active promotor and this contributed to the acute infection of macrophages. Collectively, HIV-1 Vpr down-regulates class I HDACs on chromatin to counteract latent infections of macrophages.
A Single Amino Acid Mutation in the Carnation Ringspot Virus Capsid Protein Allows Virion Formation but Prevents Systemic Infection
Sit, Tim L.; Haikal, Patrick R.; Callaway, Anton S.; Lommel, Steven A.
A Carnation ringspot virus (CRSV) variant (1.26) was identified that accumulates virions but is incapable of forming a systemic infection. The 1.26 capsid protein gene possesses a Ser→Pro mutation at amino acid 282. Conversion of 1.26 amino acid 282 to Ser restored systemic infection, while the reciprocal mutation in wild-type CRSV abolished systemic infection. Similar mutations introduced into the related Red clover necrotic mosaic virus capsid protein gene failed to induce the packaging but nonsystemic movement phenotype. These results provide additional support for the theory that virion formation is necessary but not sufficient for systemic movement with the dianthoviruses. PMID:11533217
Structure of Hepatitis E Virion-Sized Particle Reveals an RNA-Dependent Viral Assembly Pathway
Xing, L.; Wall, J.; Li, T.-C.; Mayazaki, N.; Simon, M. N.; Moore, M.; Wang, C.-Y.; Takeda, N.; Wakita, T.; Miyamura, T.; Cheng, R. H.
Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T=1 capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T=3 icosahedral lattice and contains RNA fragment in contrast to the RNA-free T=1 capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids.
Synergistic effect of combined HIV/HCV immunogens: a combined HIV-1/HCV candidate vaccine induces a higher level of CD8+ T cell-immune responses in HLA-A2.1 mice.
Azizi, Ali; Ghorbani, Masoud; Soare, Catalina; Mojibian, Majid; Diaz-Mitoma, Francisco
Dual infections with HIV-1 and Hepatitis C virus (HCV) may proceed in concert to cause severe disease. HIV positive individuals that become infected with HCV advance more rapidly to AIDS than those that are infected with HIV-1 alone. In this study, HLA-A2.1 mice were immunized with a combination vaccine including HIV and HCV immunogens (polycistronic DNA + proteins) or vaccine containing either HIV or HCV immunogens. Mice immunized with the combined HIV/HCV regimen had similar antibody titers as the group receiving either the HIV-1 or HCV only regimen. Proliferative immune responses showed that mice receiving the combined HIV/HCV vaccine exhibited a three fold higher stimulation index (SI) to gp120 than mice immunized with the vaccine containing HIV alone. To determine whether our vaccine strategy induced Th1 or Th2 immune responses, IFN-gamma and IL-4/IL-5 were measured. The combined HIV/HCV vaccine induced a higher level of Th1 responses to HIV-1 gag protein compared with the other groups, as measured by IFN-gamma production. Interestingly, detection of IFN-gamma by ELISPOT assay demonstrated that the combined HIV/HCV vaccine group had increased numbers of spot forming cells (SFC) to HIV-gp120 peptides when compared to that of the HIV-1 only vaccine group. The combined HIV/HCV vaccine group also showed an increase in SFC to HCV-core peptides in comparison with the group receiving the HCV only vaccine. Intracellular IFN-gamma staining confirmed the ELISPOT results and demonstrated that the combined HIV/HCV group had significantly higher percentages of HIV and HCV-specific CD8+ T cells in comparison to the groups receiving the HIV or HCV vaccines. These results suggest a new approach to maximize vaccine efficacy against HIV and HCV.
CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells
Pritschet, Kathrin; Donhauser, Norbert; Schuster, Philipp; Ries, Moritz; Haupt, Sabrina; Kittan, Nicolai A.; Korn, Klaus; Poehlmann, Stefan; Holland, Gudrun; Bannert, Norbert; Bogner, Elke; Schmidt, Barbara
Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p < 0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.
BCA2/Rabring7 Interferes with HIV-1 Proviral Transcription by Enhancing the SUMOylation of IκBα
Colomer-Lluch, Marta
ABSTRACT BCA2/Rabring7 is a BST2 cofactor that promotes the lysosomal degradation of trapped HIV-1 virions but also functions as a BST2-independent anti-HIV factor by targeting Gag for lysosomal degradation. Since many antiviral factors regulate the NF-κB innate signaling pathway, we investigated whether BCA2 is also connected to this proinflammatory cascade. Here, we show for the first time that BCA2 is induced by NF-κB-activating proinflammatory cytokines and that upregulation of BCA2 provides regulatory negative feedback on NF-κB. Specifically, BCA2 serves as an E3 SUMO ligase in the SUMOylation of IκBα, which in turn enhances the sequestration of NF-κB components in the cytoplasm. Since HIV-1 utilizes NF-κB to promote proviral transcription, the BCA2-mediated inhibition of NF-κB significantly decreases the transcriptional activity of HIV-1 (up to 4.4-fold in CD4+ T cells). Therefore, our findings indicate that BCA2 poses an additional barrier to HIV-1 infection: not only does BCA2 prevent assembly and release of nascent virions, it also significantly restricts HIV-1 transcription by inhibiting the NF-κB pathway. IMPORTANCE Understanding the interactions between HIV-1 and its host cells is highly relevant to the design of new drugs aimed at eliminating HIV-1 from infected individuals. We have previously shown that BCA2, a cofactor of BST2 in the restriction of HIV-1, also prevents virion assembly in a BST2-independent manner. In this study, we found that BCA2 negatively regulates the NF-κB pathway—a signaling cascade necessary for HIV-1 replication and infectivity—which in turn detrimentally affects proviral transcription and virus propagation. Thus, our results indicate that, besides its previously described functions as an antiviral factor, BCA2 poses an additional barrier to HIV-1 replication at the transcriptional level. PMID:28122985
A Wnt5a signaling pathway in the pathogenesis of HIV-1 gp120-induced pain
Yuan, Su-Bo; Ji, Guangchen; Li, Bei; Andersson, Tommy; Neugebauer, Volker; Tang, Shao-Jun
Pathological pain is one of the most common neurological complications in HIV-1/AIDS patients. However, the pathogenic process is unclear. Our recent studies show that Wnt5a is up-regulated in the spinal cord dorsal horn of the HIV patients who develop pain and that HIV-1 gp120, a potential causal factor of the HIV-associated pain, rapidly up-regulates Wnt5a in the mouse SDH. Using a mouse model, we show here that a specific Wnt5a antagonist, Box-5, attenuated gp120-induced mechanical allodynia. Conversely, a Wnt5a agonist, Foxy5, facilitated the allodynia. To elucidate the molecular mechanism by which Wnt5a regulates gp120-induced allodynia, we tested the role of the JNK/TNF-α pathway. We observed that the JNK-specific inhibitor SP600125 blocked either gp120- or Foxy5-induced allodynia. Similarly, the TNF-α-specific antagonist Enbrel also reversed either gp120- or Foxy5-induced allodynia. These data suggest that JNK and TNF-α mediate the biological effects of Wnt5a in regulating gp120-induced allodynia. To investigate the cellular mechanism, we performed extracellular single-unit recording from SDH neurons in anesthetized mice. Both Box5 and SP600125 negated gp120-induced potentiation of SDH neuron spiking evoked by mechanical stimulation of the hindpaw. Furthermore, while Foxy5 potentiated spike frequency of SDH neurons, either SP600125 or Enbrel blocked the potentiation. The data indicate that Wnt5a potentiates the activity of SDH neurons via the JNK-TNF-α pathway. Collectively, our findings suggest that Wnt5a regulates the pathogenesis of gp120-induced pain, likely by sensitizing pain-processing SDH neurons via JNK/TNF-α signaling. PMID:25840108
A Wnt5a signaling pathway in the pathogenesis of HIV-1 gp120-induced pain.
Yuan, Su-Bo; Ji, Guangchen; Li, Bei; Andersson, Tommy; Neugebauer, Volker; Tang, Shao-Jun
Pathological pain is one of the most common neurological complications in patients with HIV-1/AIDS. However, the pathogenic process is unclear. Our recent studies show that Wnt5a is upregulated in the spinal cord dorsal horn (SDH) of the patients with HIV who develop pain and that HIV-1 gp120, a potential causal factor of the HIV-associated pain, rapidly upregulates Wnt5a in the mouse SDH. Using a mouse model, we show here that a specific Wnt5a antagonist, Box-5, attenuated gp120-induced mechanical allodynia. Conversely, a Wnt5a agonist, Foxy5, facilitated the allodynia. To elucidate the molecular mechanism by which Wnt5a regulates gp120-induced allodynia, we tested the role of the JNK/TNF-α pathway. We observed that the JNK-specific inhibitor SP600125 blocked either gp120- or Foxy5-induced allodynia. Similarly, the TNF-α-specific antagonist Enbrel also reversed either gp120- or Foxy5-induced allodynia. These data suggest that JNK and TNF-α mediate the biological effects of Wnt5a in regulating gp120-induced allodynia. To investigate the cellular mechanism, we performed extracellular single-unit recording from SDH neurons in anesthetized mice. Both Box-5 and SP600125 negated gp120-induced potentiation of SDH neuron spiking evoked by mechanical stimulation of the hind paw. Furthermore, while Foxy5 potentiated spike frequency of SDH neurons, either SP600125 or Enbrel blocked the potentiation. The data indicate that Wnt5a potentiates the activity of SDH neurons through the JNK-TNF-α pathway. Collectively, our findings suggest that Wnt5a regulates the pathogenesis of gp120-induced pain, likely by sensitizing pain-processing SDH neurons through JNK/TNF-α signaling.
Recombinant influenza virus expressing HIV-1 p24 capsid protein induces mucosal HIV-specific CD8 T-cell responses.
Tan, Hyon-Xhi; Gilbertson, Brad P; Jegaskanda, Sinthujan; Alcantara, Sheilajen; Amarasena, Thakshila; Stambas, John; McAuley, Julie L; Kent, Stephen J; De Rose, Robert
Influenza viruses are promising mucosal vaccine vectors for HIV but their use has been limited by difficulties in engineering the expression of large amounts of foreign protein. We developed recombinant influenza viruses incorporating the HIV-1 p24 gag capsid into the NS-segment of PR8 (H1N1) and X31 (H3N2) influenza viruses with the use of multiple 2A ribosomal skip sequences. Despite the insertion of a sizable HIV-1 gene into the influenza genome, recombinant viruses were readily rescued to high titers. Intracellular expression of p24 capsid was confirmed by in vitro infection assays. The recombinant influenza viruses were subsequently tested as mucosal vaccines in BALB/c mice. Recombinant viruses were attenuated and safe in immunized mice. Systemic and mucosal HIV-specific CD8 T-cell responses were elicited in mice that were immunized via intranasal route with a prime-boost regimen. Isolated HIV-specific CD8 T-cells displayed polyfunctional cytokine and degranulation profiles. Mice boosted via intravaginal route induced recall responses from the distal lung mucosa and developed heightened HIV-specific CD8 T-cell responses in the vaginal mucosa. These findings demonstrate the potential utility of recombinant influenza viruses as vaccines for mucosal immunity against HIV-1 infection.
Anti-Leu3a induces combining site-related anti-idiotypic antibody without inducing anti-HIV activity.
Reeves, J P; Buck, D; Berkower, I; Murphy, D; Epstein, S L
Development of a vaccine for acquired immunodeficiency syndrome (AIDS) has proven difficult, and so alternative approaches such as idiotypic manipulation have been suggested. As applied to AIDS, this approach could involve immunizing with an anti-CD4 antibody resembling gp120, to induce anti-idiotypic antibodies which would bind to gp120. The CD4 binding site on gp120 is conserved, and so, such an immune response should protect against all variants. Induction of anti-human immunodeficiency virus (HIV) immunity has been reported using anti-Leu3a, and this result has led to testing in humans. Negative results obtained by others have been attributed to differences in immunization protocols. Because of the importance of this question, we reinvestigated the potential of anti-Leu3a to induce anti-HIV antibodies, compared with control immunizations with OKT4A (another anti-CD4 antibody) and the irrelevant Ig MOPC-21. Responses to anti-Leu3a showed induction of high-titer anti-idiotypic activity, and included combining-site-related activity. Yet sera showed no binding to gp160 above controls and no detectable neutralizing activity in a sensitive HIV plaque assay, so the anti-idiotypes induced were not internal images of CD4. We conclude that the pronounced anti-HIV responses reported with anti-Leu3a cannot be generalized, and thus that anti-Leu3a does not offer promise as an HIV vaccine. However, these results do not negate the promise of the idiotypic approach, and a vaccine for AIDS based on idiotype manipulation remains a possibility.
Protein Primary Structure of the Vaccinia Virion at Increased Resolution
Ngo, Tuan; Mirzakhanyan, Yeva; Moussatche, Nissin; Gershon, Paul David
Here we examine the protein covalent structure of the vaccinia virus virion. Within two virion preparations, >88% of the theoretical vaccinia virus-encoded proteome was detected with high confidence, including the first detection of products from 27 open reading frames (ORFs) previously designated "predicted," "uncharacterized," "inferred," or "hypothetical" polypeptides containing as few as 39 amino acids (aa) and six proteins whose detection required nontryptic proteolysis. We also detected the expression of four short ORFs, each of which was located within an ORF ("ORF-within-ORF"), including one not previously recognized or known to be expressed. Using quantitative mass spectrometry (MS), between 58 and 74 proteins were determined to be packaged. A total of 63 host proteins were also identified as candidates for packaging. Evidence is provided that some portion of virion proteins are "nicked" via a combination of endoproteolysis and concerted exoproteolysis in a manner, and at sites, independent of virus origin or laboratory procedures. The size of the characterized virion phosphoproteome was doubled from 189 (J. Matson, W. Chou, T. Ngo, and P. D. Gershon, Virology 452-453:310-323, 2014, doi:http://dx.doi.org/10.1016/j.virol.2014.01.012) to 396 confident, unique phosphorylation sites, 268 of which were within the packaged proteome. This included the unambiguous identification of phosphorylation "hot spots" within virion proteins. Using isotopically enriched ATP, 23 sites of intravirion kinase phosphorylation were detected within nine virion proteins, all at sites already partially occupied within the virion preparations. The clear phosphorylation of proteins RAP94 and RP19 was consistent with the roles of these proteins in intravirion early gene transcription. In a blind search for protein modifications, cysteine glutathionylation and O-linked glycosylation featured prominently. We provide evidence for the phosphoglycosylation of vaccinia virus proteins.
HIV-1-infected Blood Mononuclear Cells Form an Integrin- and Agrin-dependent Viral Synapse to Induce Efficient HIV-1 Transcytosis across Epithelial Cell Monolayer
Alfsen, Annette; Yu, Huifeng; Magérus-Chatinet, Aude; Schmitt, Alain; Bomsel, Morgane
The heparan sulfate proteoglycan agrin and adhesion molecules are key players in the formation of neuronal and immune synapses that evolved for efficient communication at the sites of cell-cell contact. Transcytosis of infectious virus across epithelial cells upon contact between HIV-1-infected cells and the mucosal pole of the epithelial cells is one mechanism for HIV-1 entry at mucosal sites. In contrast, transcytosis of cell-free HIV-1 is not efficient. A synapse between HIV-1-infected cells and the mucosal epithelial surface that resembles neuronal and immune synapses is visualized by electron microscopy. We have termed this the "viral synapse." Similarities of the viral synapse also extend to the functional level. HIV-1-infected cell-induced transcytosis depends on RGD-dependent integrins and efficient cell-free virus transcytosis is inducible upon RGD-dependent integrin cross-linking. Agrin appears differentially expressed at the apical epithelial surface and acts as an HIV-1 attachment receptor. Envelope glycoprotein subunit gp41 binds specifically to agrin, reinforcing the interaction of gp41 to its epithelial receptor galactosyl ceramide. PMID:15975901
HIV-1-infected blood mononuclear cells form an integrin- and agrin-dependent viral synapse to induce efficient HIV-1 transcytosis across epithelial cell monolayer.
Alfsen, Annette; Yu, Huifeng; Magérus-Chatinet, Aude; Schmitt, Alain; Bomsel, Morgane
The heparan sulfate proteoglycan agrin and adhesion molecules are key players in the formation of neuronal and immune synapses that evolved for efficient communication at the sites of cell-cell contact. Transcytosis of infectious virus across epithelial cells upon contact between HIV-1-infected cells and the mucosal pole of the epithelial cells is one mechanism for HIV-1 entry at mucosal sites. In contrast, transcytosis of cell-free HIV-1 is not efficient. A synapse between HIV-1-infected cells and the mucosal epithelial surface that resembles neuronal and immune synapses is visualized by electron microscopy. We have termed this the "viral synapse." Similarities of the viral synapse also extend to the functional level. HIV-1-infected cell-induced transcytosis depends on RGD-dependent integrins and efficient cell-free virus transcytosis is inducible upon RGD-dependent integrin cross-linking. Agrin appears differentially expressed at the apical epithelial surface and acts as an HIV-1 attachment receptor. Envelope glycoprotein subunit gp41 binds specifically to agrin, reinforcing the interaction of gp41 to its epithelial receptor galactosyl ceramide.
Early type I Interferon response induces upregulation of human β-defensin 1 during acute HIV-1 infection
Lisanti, Antonella C.; Körner, Christian; Schiff, Abigail E.; Rosenberg, Eric S.; Allen, Todd M.; Altfeld, Marcus; Kwon, Douglas S.
HIV-1 is able to evade innate antiviral responses during acute infection to establish a chronic systemic infection which, in the absence of antiretroviral therapy (ART), typically progresses to severe immunodeficiency. Understanding these early innate immune responses against HIV-1 and their mechanisms of failure is relevant to the development of interventions to better prevent HIV-1 transmission. Human beta defensins (HBDs) are antibacterial peptides but have recently also been associated with control of viral replication. HBD1 and 2 are expressed in PBMCs as well as intestinal tissue, but their expression in vivo during HIV-1 infection has not been characterized. We demonstrate that during acute HIV-1 infection, HBD1 but not HBD2 is highly upregulated in circulating monocytes but returns to baseline levels during chronic infection. HBD1 expression in monocytes can be induced by HIV-1 in vitro, although direct infection may not entirely account for the increase in HBD1 during acute infection. We provide evidence that HIV-1 triggers antiviral IFN-α responses, which act as a potent inducer of HBD1. Our results show the first characterization of induction of an HBD during acute and chronic viral infection in humans. HBD1 has been reported to have low activity against HIV-1 compared to other defensins, suggesting that in vivo induced defensins may not significantly contribute to the robust early antiviral response against HIV-1. These data provide important insight into the in vivo kinetics of HBD expression, the mechanism of HBD1 induction by HIV-1, and the role of HBDs in the early innate response to HIV-1 during acute infection. PMID:28253319
Human Immunodeficiency Virus Type 1 Vpr Induces Apoptosis through Caspase Activation
Stewart, Sheila A.; Poon, Betty; Song, Joo Y.; Chen, Irvin S. Y.
Human immunodeficiency virus type 1 (HIV-1) Vpr is a 96-amino-acid protein that is found associated with the HIV-1 virion. Vpr induces cell cycle arrest at the G2/M phase of the cell cycle, and this arrest is followed by apoptosis. We examined the mechanism of Vpr-induced apoptosis and found that HIV-1 Vpr-induced apoptosis requires the activation of a number of cellular cysteinyl aspartate-specific proteases (caspases). We demonstrate that ectopic expression of anti-apoptotic viral proteins, which inhibit caspase activity, and addition of synthetic peptides, which represent caspase cleavage sites, can inhibit Vpr-induced apoptosis. Finally, inhibition of caspase activity and subsequent inhibition of apoptosis results in increased viral expression, suggesting that therapeutic strategies aimed at reducing Vpr-induced apoptosis in vivo require careful consideration. PMID:10708425
HIV-1-infected macrophages induce astrogliosis by SDF-1{alpha} and matrix metalloproteinases
Okamoto, Mika; Wang, Xin; Baba, Masanori . E-mail: firstname.lastname@example.com
Brain macrophages/microglia and astrocytes are known to be involved in the pathogenesis of HIV-1-associated dementia (HAD). To clarify their interaction and contribution to the pathogenesis, HIV-1-infected or uninfected macrophages were used as a model of brain macrophages/microglia, and their effects on human astrocytes in vitro were examined. The culture supernatants of HIV-1-infected or uninfected macrophages induced significant astrocyte proliferation, which was annihilated with a neutralizing antibody to stromal cell-derived factor (SDF)-1{alpha} or a matrix metalloproteinase (MMP) inhibitor. In these astrocytes, CXCR4, MMP, and tissue inhibitors of matrix metalloproteinase mRNA expression and SDF-1{alpha} production were significantly up-regulated. The supernatants of infected macrophages were always more effective than those of uninfected cells. Moreover, the enhanced production of SDF-1{alpha} was suppressed by the MMP inhibitor. These results indicate that the activated and HIV-1-infected macrophages can indirectly induce astrocyte proliferation through up-regulating SDF-1{alpha} and MMP production, which implies a mechanism of astrogliosis in HAD.
Somatic Hypermutation-Induced Changes in the Structure and Dynamics of HIV-1 Broadly Neutralizing Antibodies.
Davenport, Thaddeus M; Gorman, Jason; Joyce, M Gordon; Zhou, Tongqing; Soto, Cinque; Guttman, Miklos; Moquin, Stephanie; Yang, Yongping; Zhang, Baoshan; Doria-Rose, Nicole A; Hu, Shiu-Lok; Mascola, John R; Kwong, Peter D; Lee, Kelly K
Antibody somatic hypermutation (SHM) and affinity maturation enhance antigen recognition by modifying antibody paratope structure to improve its complementarity with the target epitope. SHM-induced changes in paratope dynamics may also contribute to antibody maturation, but direct evidence of this is limited. Here, we examine two classes of HIV-1 broadly neutralizing antibodies (bNAbs) for SHM-induced changes in structure and dynamics, and delineate the effects of these changes on interactions with the HIV-1 envelope glycoprotein (Env). In combination with new and existing structures of unmutated and affinity matured antibody Fab fragments, we used hydrogen/deuterium exchange with mass spectrometry to directly measure Fab structural dynamics. Changes in antibody structure and dynamics were positioned to improve complementarity with Env, with changes in dynamics primarily observed at the paratope peripheries. We conclude that SHM optimizes paratope complementarity to conserved HIV-1 epitopes and restricts the mobility of paratope-peripheral residues to minimize clashes with variable features on HIV-1 Env.
HIV-1 Tat modulates T-bet expression and induces Th1 type of immune response
Kulkarni, Asavari; Ravi, Dyavar S.; Singh, Kamini; Rampalli, Shravanti; Parekh, Vrajesh; Mitra, Debashis; Chattopadhyay, Samit . E-mail: email@example.com
The HIV-1 transactivator Tat performs various viral and cellular functions. Primarily, it induces processive transcription from the HIV-1 LTR promoter. However, Tat secreted from infected cells is known to activate uninfected lymphocytes through receptors. To further delineate the specific target genes, extracellular Tat was expressed on the cell membrane of stimulator cells and co-cultured with human PBMCs. Along with induced CD4{sup +} T cell proliferation and IFN-{gamma} secretion, there was strong upregulation of T-bet expression which is majorly implicated in generating T{sub H}1 type of immune response. To further delineate the effect of extracellular Tat on HIV replication, both p24 analysis and in vivo GFP expression were performed. There was a significant inhibition of HIV-1 replication in human CEM-GFP cell line and hPBMCs. Thus, for the first time we report that apart from its transactivation activity, extracellular Tat acts as a costimulatory molecule that affects viral replication by modulating host immune response through induction of T-bet expression and IFN-{gamma} secretion.
HIV infection of macrophages is enhanced in the presence of increased expression of CD163 induced by substance P.
Tuluc, Florin; Meshki, John; Spitsin, Sergei; Douglas, Steven D
Activation of NK1R by SP contributes to increased HIV-1 infection in macrophages. The scavenger receptor CD163 is expressed on cells of monocyte-macrophage origin. Our main goal was to determine if there is interplay among SP, CD163 expression, and HIV infection in macrophages. We showed that SP triggers intracellular calcium elevation and increased CD163 expression in human monocytes in a time- and concentration-dependent manner. The role of CD163 on HIV infection was examined by RT-PCR in sorted monocytes (CD163(low) and CD163(high)) and in macrophages having CD163 knocked down using siRNA. We found that the productivity of HIV infection was higher in CD163(high) cells. Additionally, in macrophages with CD163 expression knocked down, we found a significant decrease of HIV infection. Furthermore, Hb-Hp complexes, which function as an endogenous ligand for CD163, decreased HIV infection in macrophages in a dose-dependent manner. Thus, we demonstrate that SP induces higher levels of CD163 in monocytes and that high expression of CD163 is associated with increases HIV infection in macrophages. Thus, in addition to being a prognostic marker of HIV infection, the expression of CD163 on macrophages may be critical in HIV immunopathogenesis.
Incorporation of Spike and Membrane Glycoproteins into Coronavirus Virions
Ujike, Makoto; Taguchi, Fumihiro
The envelopes of coronaviruses (CoVs) contain primarily three proteins; the two major glycoproteins spike (S) and membrane (M), and envelope (E), a non-glycosylated protein. Unlike other enveloped viruses, CoVs bud and assemble at the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). For efficient virion assembly, these proteins must be targeted to the budding site and to interact with each other or the ribonucleoprotein. Thus, the efficient incorporation of viral envelope proteins into CoV virions depends on protein trafficking and protein–protein interactions near the ERGIC. The goal of this review is to summarize recent findings on the mechanism of incorporation of the M and S glycoproteins into the CoV virion, focusing on protein trafficking and protein–protein interactions. PMID:25855243
Retroviral env glycoprotein trafficking and incorporation into virions.
Murakami, Tsutomu
Together with the Gag protein, the Env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles. Env is synthesized, processed, and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins. Incorporation of Env into progeny virions is probably mediated by the interaction between Env and Gag, in some cases with the additional involvement of certain host factors. Although several general models have been proposed to explain the incorporation of retroviral Env glycoproteins into virions, the actual mechanism for this process is still unclear, partly because structural data on the Env protein cytoplasmic tail is lacking. This paper presents the current understanding of the synthesis, trafficking, and virion incorporation of retroviral Env proteins.
Inhibition of the receptor-mediated virion attachment to a lipid membrane
NASA Astrophysics Data System (ADS)
Zhdanov, Vladimir P.
The forefront of the anti-viral defence is sometimes aimed at virion attachment to a host membrane. This step or, more specifically, virion contacts with cellular membrane receptors (or, e.g., glycolipids) can be inhibited by antibodies (or specially chosen or designed compounds) via their association with virions. In this case, the full-scale attachment of virions to a host membrane occurs via a subtle interplay of the formation and rupture of multiple virion-inhibitor and virion-receptor bonds. We present a kinetic model describing this interplay and illustrating general trends in the process under consideration.
Ribonucleic Acid Polymerase Activity in Sendai Virions and Nucleocapsid
Robinson, William S.
After dissociation of purified Sendai virus with the neutral detergent Nonidet P-40 and 2-mercaptoethanol, it catalyzed the incorporation of ribonucleoside triphosphates into an acid-insoluble product. The enzyme activity was associated with viral nucleocapsid as well as whole virions. The reaction product was ribonucleic acid (RNA) which annealed specifically with virion RNA. Sedimentation of the 3H-RNA reaction product revealed two components, a 45S component with properties of double-stranded RNA and 4 to 6S component which appeared to be mostly single-stranded RNA. PMID:4328418
Vpx-containing Dendritic Cell Vaccine Vectors Induce CTLs and Reactivate Latent HIV-1 in vitro
Norton, Thomas D.; Miller, Elizabeth A.; Bhardwaj, Nina; Landau, Nathaniel R.
Eradication of HIV-1 from an infected individual requires a means of inducing production of virus from latently infected cells and stimulating an immune response against the infected cells. We report the development of lentiviral vectors that transduce dendritic cells (DCs) to both induce production of virus from latently infected cells and stimulate antigen-specific CTLs. The vectors package Vpx, a lentiviral accessory protein that counteracts the SAMHD1-mediated block to DC transduction, allowing for long-term expression of vector-encoded proteins. The vectors encode influenza or HIV-1-derived epitopes fused via a self-cleaving peptide to CD40L that releases the peptide into the endoplasmic reticulum for entry into the antigen presentation pathway. Expression of CD40L caused transduced DCs to mature and produce Th1-skewing cytokines. The DCs presented antigen to CD8 T cells, enhancing antigen-specific CTLs. Coculture of the transduced DCs with latently infected cells induced high level virus production, an effect that was mediated by TNF-α. The ability of a DC vaccine to reactivate latent HIV-1 and stimulate an adaptive immune response provides a means to reduce the size of the latent reservoir in patients. This strategy can also be applied to develop DC vaccines for other diseases. PMID:25567537
During Infection, Theiler's Virions Are Cleaved by Caspases and Disassembled into Pentamers
Arslan, Sevim Yildiz; Son, Kyung-No
spinal cord that undergo apoptosis and in turn may facilitate viral spread via infected apoptotic blebs. Infection of murine macrophages in culture results in restricted virus yields late in infection. Here it is shown that the early steps of the virus life cycle in infected macrophages in vitro do not differ from these processes in TMEV-infected BHK-21 cells, which undergo necroptosis. However, the findings late in infection suggest that caspases cleave sites in exposed capsid loops and possibly internal sites of assembled virions occurring contemporaneously with onset and progression of apoptosis. Mechanistically, this would explain the dramatic loss in virus yields during TMEV-induced apoptosis and attenuate the virus, enabling persistence. PMID:26792734
HIV-2 genomic RNA accumulates in stress granules in the absence of active translation.
Soto-Rifo, Ricardo; Valiente-Echeverria, Fernando; Rubilar, Paulina S; Garcia-de-Gracia, Francisco; Ricci, Emiliano P; Limousin, Taran; Décimo, Didier; Mouland, Andrew J; Ohlmann, Théophile
During the post-transcriptional events of the HIV-2 replication cycle, the full-length unspliced genomic RNA (gRNA) is first used as an mRNA to synthesize Gag and Gag-Pol proteins and then packaged into progeny virions. However, the mechanisms responsible for the coordinate usage of the gRNA during these two mutually exclusive events are poorly understood. Here, we present evidence showing that HIV-2 expression induces stress granule assembly in cultured cells. This contrasts with HIV-1, which interferes with stress granules assembly even upon induced cellular stress. Moreover, we observed that the RNA-binding protein and stress granules assembly factor TIAR associates with the gRNA to form a TIAR-HIV-2 ribonucleoprotein (TH2RNP) complex localizing diffuse in the cytoplasm or aggregated in stress granules. Although the assembly of TH2RNP in stress granules did not require the binding of the Gag protein to the gRNA, we observed that increased levels of Gag promoted both translational arrest and stress granule assembly. Moreover, HIV-2 Gag also localizes to stress granules in the absence of a 'packageable' gRNA. Our results indicate that the HIV-2 gRNA is compartmentalized in stress granules in the absence of active translation prior to being selected for packaging by the Gag polyprotein.
Murine Antibody Responses to Cleaved Soluble HIV-1 Envelope Trimers Are Highly Restricted in Specificity
Hu, Joyce K.; Crampton, Jordan C.; Cupo, Albert; Ketas, Thomas; van Gils, Marit J.; Sliepen, Kwinten; de Taeye, Steven W.; Sok, Devin; Ozorowski, Gabriel; Deresa, Isaiah; Stanfield, Robyn; Ward, Andrew B.; Burton, Dennis R.; Klasse, Per Johan; Sanders, Rogier W.; Moore, John P.
ABSTRACT Generating neutralizing antibodies (nAbs) is a major goal of many current HIV-1 vaccine efforts. To be of practical value, these nAbs must be both potent and cross-reactive in order to be capable of preventing the transmission of the highly diverse and generally neutralization resistant (Tier-2) HIV-1 strains that are in circulation. The HIV-1 envelope glycoprotein (Env) spike is the only target for nAbs. To explore whether Tier-2 nAbs can be induced by Env proteins, we immunized conventional mice with soluble BG505 SOSIP.664 trimers that mimic the native Env spike. Here, we report that it is extremely difficult for murine B cells to recognize the Env epitopes necessary for inducing Tier-2 nAbs. Thus, while trimer-immunized mice raised Env-binding IgG Abs and had high-quality T follicular helper (Tfh) cell and germinal center (GC) responses, they did not make BG505.T332N nAbs. Epitope mapping studies showed that Ab responses in mice were specific to areas near the base of the soluble trimer. These areas are not well shielded by glycans and likely are occluded on virions, which is consistent with the lack of BG505.T332N nAbs. These data inform immunogen design and suggest that it is useful to obscure nonneutralizing epitopes presented on the base of soluble Env trimers and that the glycan shield of well-formed HIV Env trimers is virtually impenetrable for murine B cell receptors (BCRs). IMPORTANCE Human HIV vaccine efficacy trials have not generated meaningful neutralizing antibodies to circulating HIV strains. One possible hindrance has been the lack of immunogens that properly mimic the native conformation of the HIV envelope trimer protein. Here, we tested the first generation of soluble, native-like envelope trimer immunogens in a conventional mouse model. We attempted to generate neutralizing antibodies to neutralization-resistant circulating HIV strains. Various vaccine strategies failed to induce neutralizing antibodies to a neutralization
PPARα and PPARγ attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities
Huang, Wen; Eum, Sung Yong; András, Ibolya E; Hennig, Bernhard; Toborek, Michal
The blood-brain barrier (BBB) plays an important role in HIV trafficking into the brain and the development of the central nervous system complications in HIV infection. Tight junctions are the main structural and functional elements that regulate the BBB integrity. Exposure of human brain microvascular endothelial cells (hCMEC/D3 cell line) to HIV-infected monocytes resulted in decreased expression of tight junction proteins, such as junctional adhesion molecule-A (JAM)-A, occludin, and zonula occludens (ZO)-1. Control experiments involved exposure to uninfected monocytes. Alterations of tight junction protein expression were associated with increased endothelial permeability and elevated transendothelial migration of HIV-infected monocytes across an in vitro model of the BBB. Notably, overexpression of the peroxisome proliferator-activated receptor (PPAR)α or PPARγ attenuated HIV-mediated dysregulation of tight junction proteins. With the use of exogenous PPARγ agonists and silencing of PPARα or PPARγ, these protective effects were connected to down-regulation of matrix metalloproteinase (MMP) and proteasome activities. Indeed, the HIV-induced decrease in the expression of JAM-A and occludin was restored by inhibition of MMP activity. Moreover, both MMP and proteasome inhibitors attenuated HIV-mediated altered expression of ZO-1. The present data indicate that down-regulation of MMP and proteasome activities constitutes a novel mechanism of PPAR-induced protections against HIV-induced disruption of brain endothelial cells.—Huang, W., Eum, S. Y., András, I. E., Hennig, B., Toborek, M. PPARα and PPARγ attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities. PMID:19141539
Vaccine-delivered HIV envelope inhibits CD4+ T-cell activation, a mechanism for poor HIV vaccine responses
Fernando, Kathy; Hu, Haitao; Ni, Houping; Hoxie, James A.
The human immunodeficiency virus (HIV) causes impairment of the immune system in part by targeting CD4+ T cells for infection and dysfunction. HIV envelope (Env) present on free virions and infected cells causes dysfunction of uninfected bystander CD4+ T cells via interaction with both CD4 and coreceptors. Env is commonly used as part of a cocktail of HIV antigens in current vaccines. In DNA and viral vector vaccine approaches, antigen-presenting cells (APCs) and non-APCs in the vicinity of the vaccine delivery site and draining lymph node express vaccine-derived antigens. The studies here demonstrate that cell-surface expression of Env on APCs and non-APCs as part of the vaccine action causes an inhibition of antigen-induced CD4+ T-cell activation and proliferation mediated by CD4 binding and suggests a potential mechanism for reduced activity of Env-containing HIV vaccines. Similar studies using a functional Env lacking CD4 binding circumvented suppression, suggesting an alternative and potentially superior approach to HIV vaccine design. PMID:17158230
DNA driven self-assembly of micron-sized rods using DNA-grafted bacteriophage fd virions
NASA Astrophysics Data System (ADS)
Unwin, R. R.; Cabanas, R. A.; Yanagishima, T.; Blower, T. R.; Takahashi, H.; Salmond, G. P. C.; Edwardson, J. M.; Fraden, S.; Eiser, E.
We have functionalized the sides of fd bacteriophage virions with oligonucleotides to induce DNA hybridization driven self-assembly of high aspect ratio filamentous particles. Potential impacts of this new structure range from an entirely new building block in DNA origami structures, inclusion of virions in DNA nanostructures and nanomachines, to a new means of adding thermotropic control to lyotropic liquid crystal systems. A protocol for producing the virions in bulk is reviewed. Thiolated oligonucleotides are attached to the viral capsid using a heterobifunctional chemical linker. A commonly used system is utilized, where a sticky, single-stranded DNA strand is connected to an inert double-stranded spacer to increase inter-particle connectivity. Solutions of fd virions carrying complementary strands are mixed, annealed, and their aggregation is studied using dynamic light scattering (DLS), fluorescence microscopy, and atomic force microscopy (AFM). Aggregation is clearly observed on cooling, with some degree of local order, and is reversible when temperature is cycled through the DNA hybridization transition.
Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission.
Dinh, Minh H; Anderson, Meegan R; McRaven, Michael D; Cianci, Gianguido C; McCoombe, Scott G; Kelley, Z L; Gioia, Casey J; Fought, Angela J; Rademaker, Alfred W; Veazey, Ronald S; Hope, Thomas J
To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/- 0.0154 and 0.0171 +/- 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/- 0.0079 virions/image) than glans tissue (0.0167 +/- 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/- 0.0188 vs. 0.0151 +/- 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/- 3.908 vs. 12.466 +/- 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV
Increased Sensitivity to Binge Alcohol-Induced Gut Leakiness and Inflammatory Liver Disease in HIV Transgenic Rats.
Banerjee, Atrayee; Abdelmegeed, Mohamed A; Jang, Sehwan; Song, Byoung-Joon
The mechanisms of alcohol-mediated advanced liver injury in HIV-infected individuals are poorly understood. Thus, this study was aimed to investigate the effect of binge alcohol on the inflammatory liver disease in HIV transgenic rats as a model for simulating human conditions. Female wild-type (WT) or HIV transgenic rats were treated with three consecutive doses of binge ethanol (EtOH) (3.5 g/kg/dose oral gavages at 12-h intervals) or dextrose (Control). Blood and liver tissues were collected at 1 or 6-h following the last dose of ethanol or dextrose for the measurements of serum endotoxin and liver pathology, respectively. Compared to the WT, the HIV rats showed increased sensitivity to alcohol-mediated gut leakiness, hepatic steatosis and inflammation, as evidenced with the significantly elevated levels of serum endotoxin, hepatic triglycerides, histological fat accumulation and F4/80 staining. Real-time PCR analysis revealed that hepatic levels of toll-like receptor-4 (TLR4), leptin and the downstream target monocyte chemoattractant protein-1 (MCP-1) were significantly up-regulated in the HIV-EtOH rats, compared to all other groups. Subsequent experiments with primary cultured cells showed that both hepatocytes and hepatic Kupffer cells were the sources of the elevated MCP-1 in HIV-EtOH rats. Further, TLR4 and MCP-1 were found to be upregulated by leptin. Collectively, these results show that HIV rats, similar to HIV-infected people being treated with the highly active anti-retroviral therapy (HAART), are more susceptible to binge alcohol-induced gut leakiness and inflammatory liver disease than the corresponding WT, possibly due to additive or synergistic interaction between binge alcohol exposure and HIV infection. Based on these results, HIV transgenic rats can be used as a surrogate model to study the molecular mechanisms of many disease states caused by heavy alcohol intake in HIV-infected people on HAART.
A rodent model of HIV protease inhibitor indinavir induced peripheral neuropathy.
Huang, Wenlong; Calvo, Margarita; Pheby, Tim; Bennett, David L H; Rice, Andrew S C
HIV-associated sensory neuropathy (HIV-SN) is the most frequent manifestation of HIV disease. It often presents with significant neuropathic pain and is associated with previous exposure to neurotoxic nucleoside reverse transcriptase inhibitors. However, HIV-SN prevalence remains high even in resource-rich settings where these drugs are no longer used. Previous evidence suggests that exposure to indinavir, a protease inhibitor commonly used in antiretroviral therapy, may link to elevated HIV-SN risk. Here, we investigated whether indinavir treatment was associated with the development of a "dying back" axonal neuropathy and changes in pain-relevant limb withdrawal and thigmotactic behaviours. After 2 intravenous injections of indinavir (50 mg/kg, 4 days apart), adult rats developed hind paw mechanical hypersensitivity, which peaked around 2 weeks post first injection (44% reduction from baseline). At this time, animals also had (1) significantly changed thigmotactic behaviour (62% reduction in central zone entries) comparing with the controls and (2) a significant reduction (45%) in hind paw intraepidermal nerve fibre density. Treatment with gabapentin, but not amitriptyline, was associated with a complete attenuation of hind paw mechanical hypersensitivity observed with indinavir treatment. Furthermore, we found a small but significant increase in microglia with the effector morphology in the lumbar spinal dorsal horn in indinavir-treated animals, coupled with significantly increased expression of phospho-p38 in microglia. In summary, we have reported neuropathic pain-related sensory and behavioural changes accompanied by a significant loss of hind paw skin sensory innervation in a rat model of indinavir-induced peripheral neuropathy that is suitable for further pathophysiological investigation and preclinical evaluation of novel analgesics.
Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics
Technology Transfer Automated Retrieval System (TEKTRAN)
Virion-associated peptidoglycan hydrolases (VAPGH) are phage-encoded lytic enzymes that locally degrade the peptidoglycan (PG) of the bacterial cell wall during infection. Their action usually generates a small hole through which the phage tail crosses the cell envelope to inject the phage genetic m...
HIV-associated disruption of tight and adherens junctions of oral epithelial cells facilitates HSV-1 infection and spread.
Sufiawati, Irna; Tugizov, Sharof M
Herpes simplex virus (HSV) types 1 and 2 are the most common opportunistic infections in HIV/AIDS. In these immunocompromised individuals, HSV-1 reactivates and replicates in oral epithelium, leading to oral disorders such as ulcers, gingivitis, and necrotic lesions. Although the increased risk of HSV infection may be mediated in part by HIV-induced immune dysfunction, direct or indirect interactions of HIV and HSV at the molecular level may also play a role. In this report we show that prolonged interaction of the HIV proteins tat and gp120 and cell-free HIV virions with polarized oral epithelial cells leads to disruption of tight and adherens junctions of epithelial cells through the mitogen-activated protein kinase signaling pathway. HIV-induced disruption of oral epithelial junctions facilitates HSV-1 paracellular spread between the epithelial cells. Furthermore, HIV-associated disruption of adherens junctions exposes sequestered nectin-1, an adhesion protein and critical receptor for HSV envelope glycoprotein D (gD). Exposure of nectin-1 facilitates binding of HSV-1 gD, which substantially increases HSV-1 infection of epithelial cells with disrupted junctions over that of cells with intact junctions. Exposed nectin-1 from disrupted adherens junctions also increases the cell-to-cell spread of HSV-1 from infected to uninfected oral epithelial cells. Antibodies to nectin-1 and HSV-1 gD substantially reduce HSV-1 infection and cell-to-cell spread, indicating that HIV-promoted HSV infection and spread are mediated by the interaction of HSV gD with HIV-exposed nectin-1. Our data suggest that HIV-associated disruption of oral epithelial junctions may potentiate HSV-1 infection and its paracellular and cell-to-cell spread within the oral mucosal epithelium. This could be one of the possible mechanisms of rapid development of HSV-associated oral lesions in HIV-infected individuals.
Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the Receptor-Binding Site.
Bradley, Todd; Fera, Daniela; Bhiman, Jinal; Eslamizar, Leila; Lu, Xiaozhi; Anasti, Kara; Zhang, Ruijung; Sutherland, Laura L; Scearce, Richard M; Bowman, Cindy M; Stolarchuk, Christina; Lloyd, Krissey E; Parks, Robert; Eaton, Amanda; Foulger, Andrew; Nie, Xiaoyan; Karim, Salim S Abdool; Barnett, Susan; Kelsoe, Garnett; Kepler, Thomas B; Alam, S Munir; Montefiori, David C; Moody, M Anthony; Liao, Hua-Xin; Morris, Lynn; Santra, Sampa; Harrison, Stephen C; Haynes, Barton F
Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization.
Asymptomatic Tuberculosis-Induced Ileal Perforation in an HIV- Infected Individual; A Case Report
Tahmasebi, Sedigheh; Moslemi, Sam; Tahamtan, Maryam; Taheri, Lohrasb; Davarpanah, Mohammad Ali
The co-existence of acquired immune deficiency syndrome (AIDS) and tuberculosis is a major cause of morbidity and mortality because of a widespread organ involvement. The gastrointestinal tract is a common site for localization of opportunistic microorganisms in AIDS. However, surgical abdominal emergencies such as intestinal perforation resulted from tuberculosis are uncommon in these patients. The asymptomatic occurrence of such intestinal perforation has not been reported our knowledge. We represent an HIV and HCV co-infected man with miliary tuberculosis and an incidentally detected free air under diaphragm in the chest X-ray eventually resulting in exploratory laparotomy which then revealed two tubercular-induced intestinal perforations. It seems that as the tuberculosis is increasing in incidence, mostly due to reactivation in HIV-infected patients especially in developing countries, we should not underestimate its acute abdominal emergencies such as bowel perforation. PMID:27162854
Role of incidence function in vaccine-induced backward bifurcation in some HIV models.
Sharomi, O; Podder, C N; Gumel, A B; Elbasha, E H; Watmough, James
The phenomenon of backward bifurcation in disease models, where a stable endemic equilibrium co-exists with a stable disease-free equilibrium when the associated reproduction number is less than unity, has important implications for disease control. In such a scenario, the classical requirement of the reproduction number being less than unity becomes only a necessary, but not sufficient, condition for disease elimination. This paper addresses the role of the choice of incidence function in a vaccine-induced backward bifurcation in HIV models. Several examples are given where backward bifurcations occur using standard incidence, but not with their equivalents that employ mass action incidence. Furthermore, this result is independent of the type of vaccination program adopted. These results emphasize the need for further work on the incidence functions used in HIV models.
TLR9-adjuvanted pneumococcal conjugate vaccine induces antibody-independent memory responses in HIV-infected adults.
Offersen, Rasmus; Melchjorsen, Jesper; Paludan, Søren R; Østergaard, Lars; Tolstrup, Martin; Søgaard, Ole S
HIV-patients have excess of pneumococcal infection. We immunized 40 HIV-patients twice with pneumococcal conjugate vaccine (Prevnar, Pfizer) +/- a TLR9 agonist (CPG 7909). Peripheral blood mononuclear cells were stimulated with pneumococcal polysaccharides and cytokine concentrations measured. The CPG 7909 adjuvant group had significantly higher relative cytokine responses than the placebo group for IL-1β, IL-2R, IL-6, IFN-γ and MIP-β, which, did not correlate with IgG antibody responses. These findings suggests that CPG 7909 as adjuvant to pneumococcal conjugate vaccine induces cellular memory to pneumococcal polysaccharides in HIV-patients, independently of the humoral response.
UV-induced transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and UV-induced secretion of an extracellular factor that induces HIV-1 transcription in nonirradiated cells.
Stein, B; Krämer, M; Rahmsdorf, H J; Ponta, H; Herrlich, P
UV irradiation, but not visible sunlight, induces the transcription of human immunodeficiency virus type 1 (HIV-1). Chimeric constructs carrying all or parts of the HIV-1 long terminal repeat linked to an indicator gene were transfected into HeLa cells or murine and human T-cell lines, and their response to irradiation was tested. The cis-acting element conferring UV responsiveness is identical to the sequence binding transcription factor NF kappa B. UV irradiation enhances NF kappa B binding activity as assayed by gel retardation experiments. Interestingly, the requirement for UV irradiation can be replaced by cocultivation of transfected cells with UV-irradiated nontransfected (HIV-1-negative) cells. A UV-induced extracellular protein factor is detected in the culture medium conditioned by UV-treated cells. The factor is produced upon UV irradiation by several murine and human cell lines, including HeLa, Molt-4, and Jurkat, and acts on several cells. These data suggest that the UV response of keratinocytes in human skin can be magnified and spread to deeper layers that are more shielded, including the Langerhans cells, and that this indirect UV response may contribute to the activation of HIV-1 in humans. Images PMID:2795711
[The characteristics of the interaction of the proteins comprising the virions of the human immunodeficiency virus type 1].
Sharova, N K; Bukrinskaia, A G
Treatment of virions of human immunodeficiency virus type 1 (HIV-1) with ionic and nonionic detergents (NP-40, octylglucoside, sodium deoxycholate) exerted an effect on the virus uncommon for enveloped viruses: instead of solubilization, both glycoproteins (gp120 and gp41) were found in subviral particles, whereas the core protein p24 was found in the supernatant fluid after the removal of subviral particles by centrifugation. The matrix protein p17 and unprocessed molecules of the precursor protein p55 were associated with subviral particles. The above data confirm the proposed model of the HIV-I structural organization according to which glycoproteins are incorporated into the isometric matrix formed by protein p17. Our data indicate that the core protein p24 is not incorporated into the matrix and not associated with nucleocapsid proteins.
Human β defensin-3 induces chemokines from monocytes and macrophages: diminished activity in cells from HIV-infected persons.
Petrov, Velizar; Funderburg, Nicholas; Weinberg, Aaron; Sieg, Scott
Human β defensin-3 (hBD-3) is an antimicrobial peptide with diverse functionality. We investigated the capacity of hBD-3 and, for comparison, Pam3CSK4 and LL-37 to induce co-stimulatory molecules and chemokine expression in monocytes. These stimuli differentially induced CD80 and CD86 on the surface of monocytes and each stimulant induced a variety of chemokines including monocyte chemoattractant protein 1 (MCP-1), Gro-α, macrophage-derived chemokine (MDC) and macrophage inflammatory protein 1β (MIP1β), while only hBD-3 and Pam3CSK4 significantly induced the angiogenesis factor, vascular endothelial growth factor (VEGF). Human BD-3 induced similar chemokines in monocyte-derived macrophages and additionally induced expression of Regulated upon activation normal T-cell expressed and presumably secreted (RANTES) in these cells. Comparison of monocytes from HIV(+) and HIV(-) donors indicated that monocytes from HIV(+) donors were more likely to spontaneously express certain chemokines (MIP-1α, MIP-1β and MCP-1) and less able to increase expression of other molecules in response to hBD-3 (MDC, Gro-α and VEGF). Chemokine receptor expression (CCR5, CCR2 and CXCR2) was relatively normal in monocytes from HIV(+) donors compared with cells from HIV(-) donors with the exception of diminished expression of the receptor for MDC, CCR4, which was reduced in the patrolling monocyte subset (CD14(+) CD16(++) ) of HIV(+) donors. These observations implicate chemokine induction by hBD-3 as a potentially important mechanism for orchestrating cell migration into inflamed tissues. Alterations in chemokine production or their receptors in monocytes of HIV-infected persons could influence cell migration and modify the effects of hBD-3 at sites of inflammation.
Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes
Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi; Rhim, Hyangshuk; Bae, Yong Soo; Choi, Soo Young; Park, Jinseu
HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes. - Highlights: • Celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory genes. • Celastrol inhibited HIV-1 Tat -induced activation of JNK MAPK. • Celastrol inhibited HIV-1 Tat-induced activation of both NF-κB and AP-1. • Celastrol inhibited HIV-1 Tat-induced inflammatory responses via HO-1 induction.
Human beta-defensins 2 and -3 cointernalize with human immunodeficiency virus via heparan sulfate proteoglycans and reduce infectivity of intracellular virions in tonsil epithelial cells
Herrera, Rossana; Morris, Michael; Rosbe, Kristina; Feng, Zhimin; Weinberg, Aaron; Tugizov, Sharof
We previously showed that expression of the anti-HIV innate proteins human beta-defensin 2 (hBD2) and hBD3 in adult oral epithelial cells reduces HIV transepithelial transmission by inactivation of virus. However, fetal/infant oral epithelia lack beta-defensin expression, leading to transmission of HIV. The mechanisms of hBD2- and hBD3-mediated HIV inactivation in adult oral epithelial cells are poorly understood. Here we found that heparan sulfate proteoglycans (HSPGs) on the apical surfaces of epithelial cells facilitate simultaneous binding of hBDs and HIV gp120 to the cell surface. HSPG-facilitated binding of hBDs and HIV gp120 to the cell surface did not affect viral attachment. HBD2 or -3 cointernalized with virions in endosomes, formed oligomers, and reduced infectivity of HIV. The anti-HIV effect of combining hBD2 and hBD3 was substantially higher than that of the individual peptides. These findings advance our understanding of the mechanisms of anti-HIV resistance in adult oral epithelium. PMID:26539799
An Ensemble Method to Distinguish Bacteriophage Virion from Non-Virion Proteins Based on Protein Sequence Characteristics.
Zhang, Lina; Zhang, Chengjin; Gao, Rui; Yang, Runtao
Bacteriophage virion proteins and non-virion proteins have distinct functions in biological processes, such as specificity determination for host bacteria, bacteriophage replication and transcription. Accurate identification of bacteriophage virion proteins from bacteriophage protein sequences is significant to understand the complex virulence mechanism in host bacteria and the influence of bacteriophages on the development of antibacterial drugs. In this study, an ensemble method for bacteriophage virion protein prediction from bacteriophage protein sequences is put forward with hybrid feature spaces incorporating CTD (composition, transition and distribution), bi-profile Bayes, PseAAC (pseudo-amino acid composition) and PSSM (position-specific scoring matrix). When performing on the training dataset 10-fold cross-validation, the presented method achieves a satisfactory prediction result with a sensitivity of 0.870, a specificity of 0.830, an accuracy of 0.850 and Matthew's correlation coefficient (MCC) of 0.701, respectively. To evaluate the prediction performance objectively, an independent testing dataset is used to evaluate the proposed method. Encouragingly, our proposed method performs better than previous studies with a sensitivity of 0.853, a specificity of 0.815, an accuracy of 0.831 and MCC of 0.662 on the independent testing dataset. These results suggest that the proposed method can be a potential candidate for bacteriophage virion protein prediction, which may provide a useful tool to find novel antibacterial drugs and to understand the relationship between bacteriophage and host bacteria. For the convenience of the vast majority of experimental Int. J. Mol. Sci. 2015, 16,21735 scientists, a user-friendly and publicly-accessible web-server for the proposed ensemble method is established.
An Ensemble Method to Distinguish Bacteriophage Virion from Non-Virion Proteins Based on Protein Sequence Characteristics
Zhang, Lina; Zhang, Chengjin; Gao, Rui; Yang, Runtao
Bacteriophage virion proteins and non-virion proteins have distinct functions in biological processes, such as specificity determination for host bacteria, bacteriophage replication and transcription. Accurate identification of bacteriophage virion proteins from bacteriophage protein sequences is significant to understand the complex virulence mechanism in host bacteria and the influence of bacteriophages on the development of antibacterial drugs. In this study, an ensemble method for bacteriophage virion protein prediction from bacteriophage protein sequences is put forward with hybrid feature spaces incorporating CTD (composition, transition and distribution), bi-profile Bayes, PseAAC (pseudo-amino acid composition) and PSSM (position-specific scoring matrix). When performing on the training dataset 10-fold cross-validation, the presented method achieves a satisfactory prediction result with a sensitivity of 0.870, a specificity of 0.830, an accuracy of 0.850 and Matthew's correlation coefficient (MCC) of 0.701, respectively. To evaluate the prediction performance objectively, an independent testing dataset is used to evaluate the proposed method. Encouragingly, our proposed method performs better than previous studies with a sensitivity of 0.853, a specificity of 0.815, an accuracy of 0.831 and MCC of 0.662 on the independent testing dataset. These results suggest that the proposed method can be a potential candidate for bacteriophage virion protein prediction, which may provide a useful tool to find novel antibacterial drugs and to understand the relationship between bacteriophage and host bacteria. For the convenience of the vast majority of experimental scientists, a user-friendly and publicly-accessible web-server for the proposed ensemble method is established. PMID:26370987
Syncytial apoptosis signaling network induced by the HIV-1 envelope glycoprotein complex: an overview
Nardacci, R; Perfettini, J-L; Grieco, L; Thieffry, D; Kroemer, G; Piacentini, M
Infection by human immunodeficiency virus-1 (HIV-1) is associated with a progressive decrease in CD4 T-cell numbers and the consequent collapse of host immune defenses. The major pathogenic mechanism of AIDS is the massive apoptotic destruction of the immunocompetent cells, including uninfected cells. The latter process, also known as by-stander killing, operates by various mechanisms one of which involves the formation of syncytia which undergo cell death by following a complex pathway. We present here a detailed and curated map of the syncytial apoptosis signaling network, aimed at simplifying the whole mechanism that we have characterized at the molecular level in the last 15 years. The map was created using Systems Biology Graphical Notation language with the help of CellDesigner software and encompasses 36 components (proteins/genes) and 54 interactions. The simplification of this complex network paves the way for the development of novel therapeutic strategies to eradicate HIV-1 infection. Agents that induce the selective death of HIV-1-elicited syncytia might lead to the elimination of viral reservoirs and hence constitute an important complement to current antiretroviral therapies. PMID:26247731
HIV-1 Vpu Accessory Protein Induces Caspase-mediated Cleavage of IRF3 Transcription Factor*
Park, Sang Yoon; Waheed, Abdul A.; Zhang, Zai-Rong; Freed, Eric O.; Bonifacino, Juan S.
Vpu is an accessory protein encoded by HIV-1 that interferes with multiple host-cell functions. Herein we report that expression of Vpu by transfection into 293T cells causes partial proteolytic cleavage of interferon regulatory factor 3 (IRF3), a key transcription factor in the innate anti-viral response. Vpu-induced IRF3 cleavage is mediated by caspases and occurs mainly at Asp-121. Cleavage produces a C-terminal fragment of ∼37 kDa that comprises the IRF dimerization and transactivation domains but lacks the DNA-binding domain. A similar cleavage is observed upon infection of the Jurkat T-cell line with vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1. Two other HIV-1 accessory proteins, Vif and Vpr, also contribute to the induction of IRF3 cleavage in both the transfection and the infection systems. The C-terminal IRF3 fragment interferes with the transcriptional activity of full-length IRF3. Cleavage of IRF3 under all of these conditions correlates with cleavage of poly(ADP-ribose) polymerase, an indicator of apoptosis. We conclude that Vpu contributes to the attenuation of the anti-viral response by partial inactivation of IRF3 while host cells undergo apoptosis. PMID:25352594
Gp120 binding with DC-SIGN induces reactivation of HIV-1 provirus via the NF-κB signaling pathway.
Jin, Changzhong; Li, Jie; Cheng, Linfang; Liu, Fumin; Wu, Nanping
The reactivation mechanism of latent human immunodeficiency virus type 1 (HIV-1) infection is unclear, especially in dendritic cells (DC). DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds with HIV-1 and other pathogens to activate the extracellular regulated protein kinase (ERK) and nuclear factor-kappa B (NF-κB) pathways and regulate cytokine expression. We hypothesized that DC-SIGN-induced signaling pathways may activate HIV-1 provirus. To investigate this hypothesis, we generated a model by transfecting 293T cells with a DC-SIGN expression plasmid and an HIV-1 5' long terminal repeat (LTR) reporter plasmid, and then stimulated the 293T cells with HIV-1 gp120 protein, wild-type HIV-1 or VSV-G-pNL4.3 pseudotype virus (without gp120 protein). It was found that the HIV-1 5'LTR was reactivated by HIV-1 gp120 in DC-SIGN-expressing 293T cells. Then the HIV-1 chronically infected CEM-Bru cells were transfected with DC-SIGN expression plasmid and stimulated by HIV-1 gp120 protein. It was found that early and late HIV-1 provirus replication was reactivated by the HIV-1 gp120/DC-SIGN stimulation. We then investigated the involvement of the ERK, p38 mitogen-activated protein kinases and NF-κB signaling pathways in HIV-1 gp120/DC-SIGN-induced activation of HIV-1 provirus by inhibiting the pathways specifically. Our results indicated that HIV-1 gp120/DC-SIGN stimulation reactivates latent HIV-1 provirus via the NF-κB signal pathway.
Modeling HIV-1 Induced Neuroinflammation in Mice: Role of Platelets in Mediating Blood-Brain Barrier Dysfunction
Jones, Letitia D.; Jackson, Joseph W.; Maggirwar, Sanjay B.
The number of HIV-1 positive individuals developing some form of HIV-associated neurocognitive disorder (HAND) is increasing. In these individuals, the integrity of the blood-brain barrier (BBB) is compromised due to an increase in exposure to pro-inflammatory mediators, viral proteins, and virus released from infected cells. It has been shown that soluble CD40L (sCD40L) is released upon platelet activation and is an important mediator of the pathogenesis of HAND but the underlying mechanisms are unclear, emphasizing the need of an effective animal model. Here, we have utilized a novel animal model in which wild-type (WT) mice were infected with EcoHIV; a derivative of HIV-1 that contains a substitution of envelope protein gp120 with that of gp80 derived from murine leukemia virus-1 (MuLV-1). As early as two-weeks post-infection, EcoHIV led to increased permeability of the BBB associated with decreased expression of tight junction protein claudin-5, in CD40L and platelet activation-dependent manner. Treatment with an antiplatelet drug, eptifibatide, in EcoHIV-infected mice normalized BBB function, sCD40L release and platelet activity, thus implicating platelet activation and platelet-derived CD40L in virally induced BBB dysfunction. Our results also validate and underscore the importance of EcoHIV infection mouse model as a tool to explore therapeutic targets for HAND. PMID:26986758
High Incidence of Zidovudine Induced Anaemia in HIV Infected Patients in Southern Odisha.
Dash, Kaibalya Ranjan; Meher, Lalit Kumar; Hui, P K; Behera, S K; Nayak, S N
Zidovudine (AZT), a nucleoside reverse transcriptase inhibitor was the first breakthrough in AIDS therapy in 1990.This study was conducted with an aim to determine prevalence of AZT induced anaemia in HIV infected patients initiated on AZT containing anti retroviral therapy(ART) regimen and also to find out any risk factor for causing AZT induced anaemia. Study was carried out in ART centre, M.K.C.G, MCH, Berhampur between Jan 2009 and Dec 2011. HIV infected patients registered at ART centre were treated according to National AIDS Control Organisation (NACO) guidelines. Patients (n = 1221) with Hb >8 gm/dl were prescribed AZT based ART regimen. Patients having anaemia (<8 gm/dl) were excluded from the study. Correlation of baseline characteristics (age, sex, weight, Hb level, CD4 count, World Health Organization (WHO) clinical stage) with risk of developing anaemia was also calculated. 178 (14.6 %) patients on AZT regimen developed anaemia. Patients with low CD4 count were more prone to develop severe anaemia. Age, sex, weight, WHO clinical stage had no relation with development of anaemia. Incidence of AZT induced anaemia was very high and patients having low CD4 count were more susceptible to develop anaemia.
Are Structural Changes Induced by Lithium in the HIV Brain Accompanied by Changes in Functional Connectivity?
Schmidt, Christoph; Lehmann, Thomas; Zhu, Tong; Zhong, Jianhui; Leistritz, Lutz; Schifitto, Giovanni
Lithium therapy has been shown to affect imaging measures of brain function and microstructure in human immunodeficiency virus (HIV)-infected subjects with cognitive impairment. The aim of this proof-of-concept study was to explore whether changes in brain microstructure also entail changes in functional connectivity. Functional MRI data of seven cognitively impaired HIV infected individuals enrolled in an open-label lithium study were included in the connectivity analysis. Seven regions of interest (ROI) were defined based on previously observed lithium induced microstructural changes measured by Diffusion Tensor Imaging. Generalized partial directed coherence (gPDC), based on time-variant multivariate autoregressive models, was used to quantify the degree of connectivity between the selected ROIs. Statistical analyses using a linear mixed model showed significant differences in the average node strength between pre and post lithium therapy conditions. Specifically, we found that lithium treatment in this population induced changes suggestive of increased strength in functional connectivity. Therefore, by exploiting the information about the strength of functional interactions provided by gPDC we can quantify the connectivity changes observed in relation to a given intervention. Furthermore, in conditions where the intervention is associated with clinical changes, we suggest that this methodology could enable an interpretation of such changes in the context of disease or treatment induced modulations in functional networks. PMID:26436895
Delivery of DNA HIV-1 Vaccine to the Liver Induces High and Long-lasting Humoral Immune Responses
Raska, Milan; Moldoveanu, Zina; Novak, Jan; Hel, Zdenek; Bozja, Jadranka; Compans, Richard W.; Yang, Chinglai; Mestecky, Jiri
The quality of immune responses induced by DNA vaccination depends on the site of DNA administration, the expression, and the properties of the encoded antigen. In the present study we demonstrate that intravenous hydrodynamic HIV-1 envelope DNA injection resulted in high levels of expression of HIV-1 envelope antigen in the liver. When compared to the administration of DNA by i.n., i.d., i.m., and i.splenic routes, hydrodynamic vaccination induced, upon DNA boosting, 40 times increase of HIV-1 envelope-specific antibodies over the preimmune levels. Hydrodynamic vaccination with 1 μg DNA induced higher humoral responses than 100 μg DNA given intramuscularly in the prime – boost regimen. High levels of envelope-specific IgG and IgA antibodies were induced in genital tract secretions after two doses of DNA followed by intranasal boosting with recombinant HIV-1 gp120 protein. Furthermore, two doses of 100 μg DNA generated interferon-gamma production in ~ 4.3 ± 1.7 % of CD8+ splenocytes after in vitro stimulation with HIV-1 envelope peptides. These results demonstrate that DNA vaccines targeted to tissues with high proteosynthetic activity, such as the liver, results in enhanced immune responses. PMID:18304708
Suppression of HIV-1 Nef translation by Sam68 mutant-induced stress granules and nef mRNA sequestration
Henao-Mejia, Jorge; Liu, Ying; Park, In-Woo; Zhang, Jizhong; Sanford, Jeremy; He, Johnny J.
SUMMARY HIV-1 Nef plays important roles in HIV-1 replication and pathogenesis. It is translated from completely spliced HIV-1 RNA, its expression is inherently regulated at the levels of viral DNA transcription and RNA splicing. Here we show that Sam68 cytoplasmic mutants potently suppress Nef expression. The suppression requires Sam68 domain aa269-321 and is correlated with its ability to induce stress granules. In addition, the suppression is specific to Nef, and direct binding to nef mRNA 3′UTR confers the suppression specificity. Furthermore, nef mRNA is targeted to and enriched in these induced stress granules. Importantly, Nef suppression occurs in the context of HIV-1 infection of CD4+ T lymphocytes with little MHC I and CD4 down-regulation. Taken together, these results demonstrate that stress granule induction and nef mRNA sequestration account for this translational suppression of Nef expression and offers a new strategy for development of anti-HIV therapeutics to buttress our fight against HIV/AIDS. PMID:19150430
T cells detect intracellular DNA but fail to induce type I IFN responses: implications for restriction of HIV replication.
Berg, Randi K; Rahbek, Stine H; Kofod-Olsen, Emil; Holm, Christian K; Melchjorsen, Jesper; Jensen, David G; Hansen, Anne Louise; Jørgensen, Louise B; Ostergaard, Lars; Tolstrup, Martin; Larsen, Carsten S; Paludan, Søren R; Jakobsen, Martin R; Mogensen, Trine H
HIV infects key cell types of the immune system, most notably macrophages and CD4+ T cells. Whereas macrophages represent an important viral reservoir, activated CD4+ T cells are the most permissive cell types supporting high levels of viral replication. In recent years, it has been appreciated that the innate immune system plays an important role in controlling HIV replication, e.g. via interferon (IFN)-inducible restriction factors. Moreover, innate immune responses are involved in driving chronic immune activation and the pathogenesis of progressive immunodeficiency. Several pattern recognition receptors detecting HIV have been reported, including Toll-like receptor 7 and Retinoic-inducible gene-I, which detects viral RNA. Here we report that human primary T cells fail to induce strong IFN responses, despite the fact that this cell type does express key molecules involved in DNA signaling pathways. We demonstrate that the DNA sensor IFI16 migrates to sites of foreign DNA localization in the cytoplasm and recruits the signaling molecules stimulator of IFN genes and Tank-binding kinase, but this does not result in expression of IFN and IFN-stimulated genes. Importantly, we show that cytosolic DNA fails to affect HIV replication. However, exogenous treatment of activated T cells with type I IFN has the capacity to induce expression of IFN-stimulated genes and suppress HIV replication. Our data suggest the existence of an impaired DNA signaling machinery in T cells, which may prevent this cell type from activating cell-autonomous anti-HIV responses. This phenomenon could contribute to the high permissiveness of CD4+ T cells for HIV-1.
Assembly and architecture of HIV.
Ganser-Pornillos, Barbie K; Yeager, Mark; Pornillos, Owen
HIV forms spherical, membrane-enveloped, pleomorphic virions, 1,000-1,500 Å in diameter, which contain two copies of its single-stranded, positive-sense RNA genome. Virus particles initially bud from host cells in a noninfectious or immature form, in which the genome is further encapsulated inside a spherical protein shell composed of around 2,500 copies of the virally encoded Gag polyprotein. The Gag molecules are radially arranged, adherent to the inner leaflet of the viral membrane, and closely associated as a hexagonal, paracrystalline lattice. Gag comprises three major structural domains called MA, CA, and NC. For immature virions to become infectious, they must undergo a maturation process that is initiated by proteolytic processing of Gag by the viral protease. The new Gag-derived proteins undergo dramatic rearrangements to form the mature virus. The mature MA protein forms a "matrix" layer and remains attached to the viral envelope, NC condenses with the genome, and approximately 1,500 copies of CA assemble into a new cone-shaped protein shell, called the mature capsid, which surrounds the genomic ribonucleoprotein complex. The HIV capsid conforms to the mathematical principles of a fullerene shell, in which the CA subunits form about 250 CA hexamers arrayed on a variably curved hexagonal lattice, which is closed by incorporation of exactly 12 pentamers, seven pentamers at the wide end and five at the narrow end of the cone. This chapter describes our current understanding of HIV's virion architecture and its dynamic transformations: the process of virion assembly as orchestrated by Gag, the architecture of the immature virion, the virus maturation process, and the structure of the mature capsid.
Sublingual immunization with an HIV subunit vaccine induces antibodies and cytotoxic T cells in the mouse female genital tract.
Hervouet, Catherine; Luci, Carmelo; Cuburu, Nicolas; Cremel, Magali; Bekri, Selma; Vimeux, Lene; Marañon, Concepcion; Czerkinsky, Cecil; Hosmalin, Anne; Anjuère, Fabienne
A vaccine against heterosexual transmission by human immunodeficiency virus (HIV) should generate cytotoxic and antibody responses in the female genital tract and in extra-genital organs. We report that sublingual immunization with HIV-1 gp41 and a reverse transcriptase polypeptide coupled to the cholera toxin B subunit (CTB) induced gp41-specific IgA antibodies and antibody-secreting cells, as well as reverse transcriptase-specific CD8 T cells in the genital mucosa, contrary to intradermal immunization. Conjugation of the reverse transcriptase peptide to CTB favored its cross-presentation by human dendritic cells to a T cell line from an HIV(+) patient. Sublingual vaccination could represent a promising vaccine strategy against heterosexual transmission of HIV-1.
The vaccinia virus E6 protein influences virion protein localization during virus assembly
Condit, Richard C.; Moussatche, Nissin
Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulate in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a "pre-nucleocapsid", and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. PMID:25863879
Bathophenanthroline disulfonate and soluble CD4 as probes for early events of HIV type 1 entry.
Demaria, S; Tilley, S A; Pinter, A; Bushkin, Y
We report here that a metalloprotease inhibitor, bathophenanthroline disulfonate (Bphe-ds), neutralizes both laboratory-adapted and primary strains of HIV-1. Presaturation of Bphe-ds with zinc does not alter its neutralizing activity, suggesting that the metal-chelating ability of Bphe-ds is not required for neutralization. Bphe-ds blocks infection of CD4+ cells at the stage of viral entry, not through a direct viricidal effect, but by interfering with both binding and postbinding events. This drug interacts with HIV-1 envelope, blocking almost completely the binding of three MAbs that recognize epitopes overlapping the CD4-binding site on gp120, but has no effect on the binding of MAbs directed to the cellular receptor CD4. The exposure of epitopes in the V2 and V3 but not C5 domains of gp120 is partially decreased in the presence of Bphe-ds, suggesting that the drug induces conformational changes in the envelope glycoprotein(s). Binding of both virions and soluble gp120 to CD4+ cells is inhibited by this drug in a dose-dependent manner. This contrasted with the effects of soluble CD4, which actually increased binding of virions to cells at 4 degrees C, while inhibiting the binding of soluble gp120. Bphe-ds also increases shedding of gp120 from cells infected with HIV-1IIIB. Thus, Bphe-ds appears to be an envelope-directed inhibitor of HIV-1 that neutralizes HIV-1 infectivity via multiple mechanisms.
Protein Composition of the Vaccinia Virus Mature Virion
Resch, Wolfgang; Hixson, Kim K.; Moore, Ronald J.; Lipton, Mary S.; Moss, Bernard
The protein content of vaccinia virus mature virions, purified by rate zonal and isopycnic centrifugation and solubilized by SDS or a solution of urea and thiourea, was determined by the accurate mass and time tag technology which uses both tandem mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry to detect tryptic peptides separated by high-resolution liquid chromatography. Eighty vaccinia virus-encoded proteins representing 37% of the 218 genes annotated in the complete genome sequence were detected in at least three analyses. Ten proteins accounted for approximately 80% of the mass, while the least abundant proteins made up 1% or less of the mass. Thirteen identified proteins were not previously reported as components of virions. On the other hand, 8 previously described virion proteins were not detected here, presumably due to technical reasons including small size and hydrophobicity. In addition to vaccinia virus-encoded proteins, 24 host proteins omitting isoforms were detected. The most abundant of these were cytoskeletal proteins, heat shock proteins, and proteins involved in translation.
A Growth Factor Attenuates HIV-1 Tat and Morphine Induced Damage to Human Neurons: Implication in HIV/AIDS-Drug Abuse Cases
Malik, Shaily; Khalique, Hena; Buch, Shilpa; Seth, Pankaj
The neuropathological abnormalities of human immunodeficiency virus (HIV)-1 patients abusing illicit drugs suggest extensive interactions between the two agents, thereby leading to increased rate of progression to neurodegeneration. The role of HIV-1 transactivating protein, Tat has been elucidated in mediating neuronal damage via apoptosis, a hallmark of HIV-associated dementia (HAD), however the underlying mechanisms involved in enhanced neurodegeneration by illicit drugs remain elusive. In this study, we demonstrated that morphine enhances HIV-Tat induced toxicity in human neurons and neuroblastoma cells. Enhanced toxicity by Tat and morphine was accompanied by increased numbers of TUNEL positive apoptotic neurons, elevated caspase-3 levels and decreased ratio of anti- and pro-apoptotic proteins, Bcl2/Bax. Tat and morphine together elicited high levels of reactive oxygen species that were NADPH dependent. Significant alterations in mitochondrial membrane homeostasis were also observed with co-exposure of these agents. Extensive studies of mitogen activated protein kinase (MAPK) signaling pathways revealed the involvement of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase-1/2 (ERK1/2) pathways in enhanced toxicity of Tat and morphine. In addition to this, we found that pre-treatment of cells with platelet derived growth factor (PDGF-BB) protected neurons from HIV-Tat and morphine induced damage. PDGF-BB alleviated ROS production, maintained mitochondrial membrane potential, decreased caspase-3 activation and hence protected the cells from undergoing apoptosis. PDGF-BB mediated protection against Tat and morphine involved the phosphatidylinositol–3 kinase (PI3K) pathway, as specific inhibitor of PI3K abrogated the protection conferred by PDGF-BB. This study demonstrates the mechanism of enhanced toxicity in human neurons subjected to co-exposure of HIV protein Tat and morphine, thus implying its importance in HIV positive drug abusers
HIV antiretroviral drug combination induces endothelial mitochondrial dysfunction and reactive oxygen species production, but not apoptosis
Jiang Bo; Hebert, Valeria Y.; Li, Yuchi; Mathis, J. Michael; Alexander, J. Steven; Dugas, Tammy R.
Numerous reports now indicate that HIV patients administered long-term antiretroviral therapy (ART) are at a greater risk for developing cardiovascular diseases. Endothelial dysfunction is an initiating event in atherogenesis and may contribute to HIV-associated atherosclerosis. We previously reported that ART induces direct endothelial dysfunction in rodents. In vitro treatment of human umbilical vein endothelial cells (HUVEC) with ART indicated endothelial mitochondrial dysfunction and a significant increase in the production of reactive oxygen species (ROS). In this study, we determined whether ART-induced endothelial dysfunction is mediated via mitochondria-derived ROS and whether this mitochondrial injury culminates in endothelial cell apoptosis. Two major components of ART combination therapy, a nucleoside reverse transcriptase inhibitor and a protease inhibitor, were tested, using AZT and indinavir as representatives for each. Microscopy utilizing fluorescent indicators of ROS and mitochondria demonstrated the mitochondrial localization of ART-induced ROS. MnTBAP, a cell-permeable metalloporphyrin antioxidant, abolished ART-induced ROS production. As a final step in confirming the mitochondrial origin of the ART-induced ROS, HUVEC were transduced with a cytosolic- compared to a mitochondria-targeted catalase. Transduction with the mitochondria-targeted catalase was more effective than cytoplasmic catalase in inhibiting the ROS and 8-isoprostane (8-iso-PGF{sub 2{alpha}}) produced after treatment with either AZT or indinavir. However, both mitochondrial and cytoplasmic catalase attenuated ROS and 8-iso-PGF{sub 2{alpha}} production induced by the combination treatment, suggesting that in this case, the formation of cytoplasmic ROS may also occur, and thus, that the mechanism of toxicity in the combination treatment group may be different compared to treatment with AZT or indinavir alone. Finally, to determine whether ART-induced mitochondrial dysfunction and
Altered T cell surface glycosylation in HIV-1 infection results in increased susceptibility to galectin-1-induced cell death.
Lantéri, Marion; Giordanengo, Valérie; Hiraoka, Nobuyoshi; Fuzibet, Jean-Gabriel; Auberger, Patrick; Fukuda, Minoru; Baum, Linda G; Lefebvre, Jean-Claude
The massive T cell death that occurs in HIV type 1 (HIV-1) infection contributes profoundly to the pathophysiology associated with AIDS. The mechanisms controlling cell death of both infected and uninfected T cells ("bystander" death) are not completely understood. We have shown that HIV-1 infection of T cells results in altered glycosylation of cell surface glycoproteins; specifically, it decreased sialylation and increased expression of core 2 O-glycans. Galectin-1 is an endogenous human lectin that recognizes these types of glycosylation changes and induces cell death of activated lymphocytes. Therefore we studied the possible contribution of galectin-1 in the pathophysiology of AIDS. O-glycan modifications were investigated on peripheral lymphocytes from AIDS patients. Oligosaccharides from CD43 and CD45 of CEM cells latently infected with HIV-1 were chemically analyzed. Consistent with our previous results, we show that HIV-1 infection results in accumulation of exposed lactosamine residues, oligosaccharides recognized by galectin-1 on cell surface glycoproteins. Both latently HIV-1-infected T cell lines and peripheral CD4 and CD8 T cells from AIDS patients exhibited exposed lactosamine residues and demonstrated marked susceptibility to galectin-1-induced cell death, in contrast to control cultures or cells from uninfected donors. The fraction of cells that died in response to galectin-1 exceeded the fraction of infected cells, indicating that death of uninfected cells occurred. Altered cell surface glycosylation of T cells during HIV-1 infection increases the susceptibility to galectin-1-induced cell death, and this death pathway can contribute to loss of both infected and uninfected T cells in AIDS.
A conditionally replicating HIV-1 vector interferes with wild-type HIV-1 replication and spread.
Dropulić, B; Hĕrmánková, M; Pitha, P M
Defective-interfering viruses are known to modulate virus pathogenicity. We describe conditionally replicating HIV-1 (crHIV) vectors that interfere with wild-type HIV-1 (wt-HIV) replication and spread. crHIV vectors are defective-interfering HIV genomes that do not encode viral proteins and replicate only in the presence of wt-HIV helper virus. In cells that contain both wt-HIV and crHIV genomes, the latter are shown to have a selective advantage for packaging into progeny virions because they contain ribozymes that cleave wt-HIV RNA but not crHIV RNA. A crHIV vector containing a triple anti-U5 ribozyme significantly interferes with wt-HIV replication and spread. crHIV vectors are also shown to undergo the full viral replicative cycle after complementation with wt-HIV helper-virus. The application of defective interfering crHIV vectors may result in competition with wt-HIVs and decrease pathogenic viral loads in vivo. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8855316
Astrocytes as an HIV Reservoir: Mechanism of HIV Infection.
Li, Guan-Han; Henderson, Lisa; Nath, Avindra
If we have any hope of achieving a cure for HIV infection, close attention to the cell types capable of getting infected with HIV is necessary. Of these cell types, astrocytes are the most ideal cell type for the formation of such a reservoir. These are long-lived cells with a very low turnover rate and are found in the brain and the gastrointestinal tract. Although astrocytes are evidently resistant to infection of cell-free HIV in vitro, these cells are efficiently infected via cell-tocell contact by which immature HIV virions bud off lymphocytes and have the ability to directly bind to CXCR4, triggering the process of fusion in the absence of CD4. In this review, we closely examine the evidence for HIV infection of astrocytes in the brain and the mechanisms for viral entry and regulation in this cell type, and discuss an approach for controlling this viral reservoir.
Prevalence and Gene Characteristics of Antibodies with Cofactor-induced HIV-1 Specificity*
Lecerf, Maxime; Scheel, Tobias; Pashov, Anastas D.; Jarossay, Annaelle; Ohayon, Delphine; Planchais, Cyril; Mesnage, Stephane; Berek, Claudia; Kaveri, Srinivas V.; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D.
The healthy immune repertoire contains a fraction of antibodies that bind to various biologically relevant cofactors, including heme. Interaction of heme with some antibodies results in induction of new antigen binding specificities and acquisition of binding polyreactivity. In vivo, extracellular heme is released as a result of hemolysis or tissue damage; hence the post-translational acquisition of novel antigen specificities might play an important role in the diversification of the immunoglobulin repertoire and host defense. Here, we demonstrate that seronegative immune repertoires contain antibodies that gain reactivity to HIV-1 gp120 upon exposure to heme. Furthermore, a panel of human recombinant antibodies was cloned from different B cell subpopulations, and the prevalence of antibodies with cofactor-induced specificity for gp120 was determined. Our data reveal that upon exposure to heme, ∼24% of antibodies acquired binding specificity for divergent strains of HIV-1 gp120. Sequence analyses reveal that heme-sensitive antibodies do not differ in their repertoire of variable region genes and in most of the molecular features of their antigen-binding sites from antibodies that do not change their antigen binding specificity. However, antibodies with cofactor-induced gp120 specificity possess significantly lower numbers of somatic mutations in their variable region genes. This study contributes to the understanding of the significance of cofactor-binding antibodies in immunoglobulin repertoires and of the influence that the tissue microenvironment might have in shaping adaptive immune responses. PMID:25564611
HIV-1 gp120 Mannoses Induce Immunosuppressive Responses from Dendritic Cells
Shan, Meimei; Klasse, Per Johan; Banerjee, Kaustuv; Dey, Antu K; Iyer, Sai Prasad N; Dionisio, Robert; Charles, Dustin; Campbell-Gardener, Lila; Olson, William C; Sanders, Rogier W; Moore, John P
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a vaccine immunogen that can signal via several cell surface receptors. To investigate whether receptor biology could influence immune responses to gp120, we studied its interaction with human, monocyte-derived dendritic cells (MDDCs) in vitro. Gp120 from the HIV-1 strain JR-FL induced IL-10 expression in MDDCs from 62% of donors, via a mannose C-type lectin receptor(s) (MCLR). Gp120 from the strain LAI was also an IL-10 inducer, but gp120 from the strain KNH1144 was not. The mannose-binding protein cyanovirin-N, the 2G12 mAb to a mannose-dependent gp120 epitope, and MCLR-specific mAbs inhibited IL-10 expression, as did enzymatic removal of gp120 mannose moieties, whereas inhibitors of signaling via CD4, CCR5, or CXCR4 were ineffective. Gp120-stimulated IL-10 production correlated with DC-SIGN expression on the cells, and involved the ERK signaling pathway. Gp120-treated MDDCs also responded poorly to maturation stimuli by up-regulating activation markers inefficiently and stimulating allogeneic T cell proliferation only weakly. These adverse reactions to gp120 were MCLR-dependent but independent of IL-10 production. Since such mechanisms might suppress immune responses to Env-containing vaccines, demannosylation may be a way to improve the immunogenicity of gp120 or gp140 proteins. PMID:17983270
Immunogenicity studies of bivalent inactivated virions of EV71/CVA16 formulated with submicron emulsion systems.
Lin, Chih-Wei; Liu, Chia-Chyi; Lu, Tsung-Chun; Liu, Shih-Jen; Chow, Yen-Hung; Chong, Pele; Huang, Ming-Hsi
We assessed two strategies for preparing candidate vaccines against hand, foot, and mouth disease (HFMD) caused mainly by infections of enterovirus (EV) 71 and coxsackievirus (CV) A16. We firstly design and optimize the potency of adjuvant combinations of emulsion-based delivery systems, using EV71 candidate vaccine as a model. We then perform immunogenicity studies in mice of EV71/CVA16 antigen combinations formulated with PELC/CpG. A single dose of inactivated EV71 virion (0.2 μg) emulsified in submicron particles was found (i) to induce potent antigen-specific neutralizing antibody responses and (ii) consistently to elicit broad antibody responses against EV71 neutralization epitopes. A single dose immunogenicity study of bivalent activated EV71/CVA16 virion formulated with either Alum or PELC/CpG adjuvant showed that CVA16 antigen failed to elicit CVA16 neutralizing antibody responses and did not affect EV71-specific neutralizing antibody responses. A boosting dose of emulsified EV71/CVA16 bivalent vaccine candidate was found to be necessary to achieve high seroconversion of CVA16-specific neutralizing antibody responses. The current results are important for the design and development of prophylactic vaccines against HFMD and other emerging infectious diseases.
Role of fever in infection: has induced fever any therapeutic potential in HIV infection?
Morton, R S; Rashid, S
Ancient societies had no rational understanding of fever. The Greeks were the first to recognise that it may be part of nature's method of effecting cure in some diseases. How best to assist nature went through many trials and errors. Appreciation of the prognostic value of fever and how it may be controlled was slow to appear. That there was a place in the therapeutic arsenal for induced fever came only with the 20th century. Finding a suitable, safe, and satisfactory means came slowly. The curative power of well controlled and reproducible levels of fever was proved by the arrest of one deadly and incurable complication of a sexually transmitted disease in the first half of this century. The purpose of this review is to promote discussion and, hopefully, well ordered laboratory and clinical trials aimed at learning whether or not induced fevers have a place in the care of patients with HIV/AIDS. Images PMID:9306904
Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity
NASA Astrophysics Data System (ADS)
Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy
Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.
Nature, Decay, and Spiraling of the Effects of Fear-Inducing Arguments and HIV Counseling and Testing: A Meta-Analysis of the Short- and Long-Term Outcomes of HIV-Prevention Interventions
Earl, Allison; Albarracín, Dolores
Objective To examine the long-term efficacy of both fear-inducing arguments and HIV counseling and testing at encouraging and maintaining knowledge about HIV transmission and prevention, as well as condom use. Design Analyses were conducted with a sample of 150 treatment groups and 34 controls and included measures of change at an immediate follow-up and a delayed follow-up. Main outcome measures The main outcome measures were perceived risk of HIV infection, knowledge about HIV, and condom use. Results Results indicated that receiving fear-inducing arguments increased perceptions of risk at the immediate follow-up but decreased knowledge and condom use, whereas resolving fear via HIV counseling and testing decreased perceptions of risk and increased knowledge and condom use at both the immediate and delayed follow-ups. The effects on perceived risk and knowledge decreased over time, but the effects on condom use became more pronounced. Conclusion Inducing fear is not an effective way to promote HIV-relevant learning or condom use either immediately following the intervention or later on. However, HIV counseling and testing can provide an outlet for HIV-related anxiety and, subsequently, gains in both knowledge and behavior change immediately and longitudinally. PMID:17605570
HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection
Lee, Jung-Hyun; Schierer, Stephan; Blume, Katja; Dindorf, Jochen; Wittki, Sebastian; Xiang, Wei; Ostalecki, Christian; Koliha, Nina; Wild, Stefan; Schuler, Gerold; Fackler, Oliver T.; Saksela, Kalle; Harrer, Thomas; Baur, Andreas S.
Antiretroviral therapy (ART) efficiently suppresses HIV replication but immune activation and low CD4 T cell counts often persist. The underlying mechanism of this ART-resistant pathogenesis is not clear. We observed that levels of plasma extracellular vesicles (pEV) are strongly elevated in HIV infection and do not decline during ART. Surprisingly, these vesicles contained the viral accessory proteins Nef and Vpu, which are assumed to be not expressed under efficient ART, as well as pro-inflammatory effectors, including activated ADAM17. HIV pEV were characterized by the presence of activated αvβ3 and absence of CD81 and Tsg101. Correlating with immune activation, peripheral monocytes ingested large amounts of pEV, giving rise to an increased population of CD1c+ CD14+ cells that secreted inflammatory cytokines. Importantly, the pro-inflammatory content, particularly ADAM17 activity, correlated with low T cell counts. Preliminary evidence suggested that HIV pEV derived from peripheral mononuclear cells and from an unknown myeloid cell population. In summary we propose an important role of pro-inflammatory pEV in chronic HIV infection due to ongoing viral Nef activity. PMID:27211553
HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection.
Lee, Jung-Hyun; Schierer, Stephan; Blume, Katja; Dindorf, Jochen; Wittki, Sebastian; Xiang, Wei; Ostalecki, Christian; Koliha, Nina; Wild, Stefan; Schuler, Gerold; Fackler, Oliver T; Saksela, Kalle; Harrer, Thomas; Baur, Andreas S
Antiretroviral therapy (ART) efficiently suppresses HIV replication but immune activation and low CD4 T cell counts often persist. The underlying mechanism of this ART-resistant pathogenesis is not clear. We observed that levels of plasma extracellular vesicles (pEV) are strongly elevated in HIV infection and do not decline during ART. Surprisingly, these vesicles contained the viral accessory proteins Nef and Vpu, which are assumed to be not expressed under efficient ART, as well as pro-inflammatory effectors, including activated ADAM17. HIV pEV were characterized by the presence of activated αvβ3 and absence of CD81 and Tsg101. Correlating with immune activation, peripheral monocytes ingested large amounts of pEV, giving rise to an increased population of CD1c(+) CD14(+) cells that secreted inflammatory cytokines. Importantly, the pro-inflammatory content, particularly ADAM17 activity, correlated with low T cell counts. Preliminary evidence suggested that HIV pEV derived from peripheral mononuclear cells and from an unknown myeloid cell population. In summary we propose an important role of pro-inflammatory pEV in chronic HIV infection due to ongoing viral Nef activity.
Synthetic consensus HIV-1 DNA induces potent cellular immune responses and synthesis of granzyme B, perforin in HIV infected individuals.
Morrow, Matthew P; Tebas, Pablo; Yan, Jian; Ramirez, Lorenzo; Slager, Anna; Kraynyak, Kim; Diehl, Malissa; Shah, Divya; Khan, Amir; Lee, Jessica; Boyer, Jean; Kim, J Joseph; Sardesai, Niranjan Y; Weiner, David B; Bagarazzi, Mark L
This study evaluated the safety and immunogenicity of PENNVAX-B in 12 HIV infected individuals. PENNVAX-B is a combination of three optimized synthetic plasmids encoding for multiclade HIV Gag and Pol and a consensus CladeB Env delivered by electroporation. HIV infected individuals whose virus was effectively suppressed using highly active antiretroviral therapy (HAART) received PENNVAX-B DNA followed by electroporation with CELLECTRA-5P at study weeks 0, 4, 8, and 16. Local administration site and systemic reactions to PENNVAX-B were recorded after each treatment along with any adverse events. Pain of the treatment procedure was assessed using a Visual Analog Scale. Whole PBMCs were isolated for use in IFN ELISpot and Flow Cytometric assays. PENNVAX-B was generally safe and well tolerated. Overall, the four dose regimen was not associated with any serious adverse events or severe local or systemic reactions. A rise in antigen-specific SFU was detected in the INFγ ELISpot assay in all 12 participants. T cells from 8/12 participants loaded with both granzyme B and perforin in response to HIV antigen, an immune finding characteristic of long-term nonprogressors (LTNPs) and elite controllers (ECs). Thus administration of PENNVAX-B may prove useful adjunctive therapy to ART for treatment and control of HIV infection.
Synthetic Consensus HIV-1 DNA Induces Potent Cellular Immune Responses and Synthesis of Granzyme B, Perforin in HIV Infected Individuals
Morrow, Matthew P; Tebas, Pablo; Yan, Jian; Ramirez, Lorenzo; Slager, Anna; Kraynyak, Kim; Diehl, Malissa; Shah, Divya; Khan, Amir; Lee, Jessica; Boyer, Jean; Kim, J Joseph; Sardesai, Niranjan Y; Weiner, David B; Bagarazzi, Mark L
This study evaluated the safety and immunogenicity of PENNVAX-B in 12 HIV infected individuals. PENNVAX-B is a combination of three optimized synthetic plasmids encoding for multiclade HIV Gag and Pol and a consensus CladeB Env delivered by electroporation. HIV infected individuals whose virus was effectively suppressed using highly active antiretroviral therapy (HAART) received PENNVAX-B DNA followed by electroporation with CELLECTRA-5P at study weeks 0, 4, 8, and 16. Local administration site and systemic reactions to PENNVAX-B were recorded after each treatment along with any adverse events. Pain of the treatment procedure was assessed using a Visual Analog Scale. Whole PBMCs were isolated for use in IFN ELISpot and Flow Cytometric assays. PENNVAX-B was generally safe and well tolerated. Overall, the four dose regimen was not associated with any serious adverse events or severe local or systemic reactions. A rise in antigen-specific SFU was detected in the INFγ ELISpot assay in all 12 participants. T cells from 8/12 participants loaded with both granzyme B and perforin in response to HIV antigen, an immune finding characteristic of long-term nonprogressors (LTNPs) and elite controllers (ECs). Thus administration of PENNVAX-B may prove useful adjunctive therapy to ART for treatment and control of HIV infection. PMID:25531694
Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes.
Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi; Rhim, Hyangshuk; Bae, Yong Soo; Choi, Soo Young; Park, Jinseu
HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes.
Energetics of mutation-induced changes in potency of lersivirine against HIV-1 reverse transcriptase.
Kar, Parimal; Knecht, Volker
Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy for the treatment of HIV-1. A common problem with the first generation NNRTIs is the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular, K103N and Y181C, which lead to resistance to the entire class of inhibitor. Here we have evaluated the relative affinity of the newly designed NNRTI lersivirine (LRV) against drug-resistant mutations in HIV-1 RT using the molecular mechanics generalized Born surface area (MM-GBSA) method. Eight single and one double mutant variants of RT are considered. Our results are in good agreement with experimental results and yield insights into the mechanisms underlying mutation-induced changes in the potency of LRV against RT. The strongest (54-fold) increase in the dissociation constant is found for the mutant F227C, originating from reduced electrostatic and van der Waals interactions between LRV and RT as well as a higher energetic penalty from the desolvation of polar groups. For the mutants K103N and Y181C only a moderate (2-fold) increase in the dissociation constant is found, due to a balance of opposite changes in the polar solvation as well as the electrostatic and van der Waals interactions between LRV and RT. The dissociation constant is decreased for the Y188C and G190A (2-fold), the M184V (5-fold), and the Y188C/Y188C mutant (10-fold), due to stronger electrostatic interactions between LRV and RT. Our results thus suggest that LRV is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses, which is in agreement with experimental observations.
A cytoplasmic tail determinant in HIV-1 Vpu mediates targeting of tetherin for endosomal degradation and counteracts interferon-induced restriction.
Kueck, Tonya; Neil, Stuart J D
The HIV-1 accessory protein Vpu counteracts tetherin (BST-2/CD317) by preventing its incorporation into virions, reducing its surface expression, and ultimately promoting its degradation. Here we characterize a putative trafficking motif, EXXXLV, in the second alpha helix of the subtype-B Vpu cytoplasmic tail as being required for efficient tetherin antagonism. Mutation of this motif prevents ESCRT-dependent degradation of tetherin/Vpu complexes, tetherin cell surface downregulation, but not its physical interaction with Vpu. Importantly, this motif is required for efficient cell-free virion release from CD4+ T cells, particularly after their exposure to type-1 interferon, indicating that the ability to reduce surface tetherin levels and promote its degradation is important to counteract restriction under conditions that the virus likely encounters in vivo. Vpu EXXXLV mutants accumulate with tetherin at the cell surface and in endosomal compartments, but retain the ability to bind both β-TrCP2 and HRS, indicating that this motif is required for a post-binding trafficking event that commits tetherin for ESCRT-dependent degradation and prevents its transit to the plasma membrane and viral budding zones. We further found that while Vpu function is dependent on clathrin, and the entire second alpha helix of the Vpu tail can be functionally complemented by a clathrin adaptor binding peptide derived from HIV-1 Nef, none of the canonical clathrin adaptors nor retromer are required for this process. Finally we show that residual activity of Vpu EXXXLV mutants requires an intact endocytic motif in tetherin, suggesting that physical association of Vpu with tetherin during its recycling may be sufficient to compromise tetherin activity to some degree.
A Novel Class of HIV-1 Antiviral Agents Targeting HIV via a SUMOylation-Dependent Mechanism
Madu, Ikenna G.; Li, Shirley; Li, Baozong; Li, Haitang; Chang, Tammy; Li, Yi-Jia; Vega, Ramir; Rossi, John; Yee, Jiing-Kuan; Zaia, John; Chen, Yuan
We have recently identified a chemotype of small ubiquitin-like modifier (SUMO)-specific protease (SENP) inhibitors. Prior to the discovery of their SENP inhibitory activity, these compounds were found to inhibit HIV replication, but with an unknown mechanism. In this study, we investigated the mechanism of how these compounds inhibit HIV-1. We found that they do not affect HIV-1 viral production, but significantly inhibited the infectivity of the virus. Interestingly, virions produced from cells treated with these compounds could gain entry and carry out reverse transcription, but could not efficiently integrate into the host genome. This phenotype is different from the virus produced from cells treated with the class of anti-HIV-1 agents that inhibit HIV protease. Upon removal of the SUMO modification sites in the HIV-1 integrase, the compound no longer alters viral infectivity, indicating that the effect is related to SUMOylation of the HIV integrase. This study identifies a novel mechanism for inhibiting HIV-1 integration and a new class of small molecules that inhibits HIV-1 via such mechanism that may contribute a new strategy for cure of HIV-1 by inhibiting the production of infectious virions upon activation from latency. PMID:26643614
Morphogenesis and morphology of HIV. Structure-function relations.
Gelderblom, H R; Ozel, M; Pauli, G
Fine structure and antigenic make-up analysis of HIV were combined in a 2D model, from which functional aspects can be deduced. On the envelope 72 probably trimeric surface knobs (gp120) are connected to the virion via the transmembrane protein gp41. Gp120 is shed during ageing of the virion, but host cell antigens stay firmly anchored to the envelope. Underneath the envelope, p17 forms the matrix protein layer, while the capsid of the double cone shaped core is built up of p24. The relation between biochemical findings and morphogenesis and maturation of HIV as well as aspects of pathogenesis and vaccination are discussed.
APOBEC3G ubiquitination by Nedd4-1 favors its packaging into HIV-1 particles.
Dussart, Sylvie; Douaisi, Marc; Courcoul, Marianne; Bessou, Gilles; Vigne, Robert; Decroly, Etienne
APOBEC3G is a cytidine deaminase that limits the replication of many retroviruses. This antiviral host factor is packaged into retrovirus particles, where it targets single-stranded DNA generated during reverse transcription and induces up to 2% of G-to-A mutations, which are lethal for the HIV-1 provirus. Vif protein counteracts this antiviral factor by decreasing its packaging into lentivirus particles. Here, we demonstrate that Nedd4-1, an HECT E3 ubiquitin ligase, interacts with APOBEC3G, through its WW2 and WW3 domains. As a result of this interaction, APOBEC3G undergoes post-translational modification by addition of ubiquitin moieties. Accordingly, we demonstrate that the dominant negative Nedd4-1 C/S form prevents APOBEC3G ubiquitination. Moreover, the packaging of APOBEC3G into Pr55 Gag virus-like particles and into HIV-1 virions is reduced when Nedd4-1 C/S is expressed. During HIV-1 viral production in the presence of APOBEC3G, Nedd4-1 C/S restores partially the infectivity of Deltavif HIV-1. We conclude that the ubiquitination of APOBEC3G by Nedd4-1 favors its targeting to the virus assembly site where APOBEC3G interacts with Gag and is packaged into HIV-1 particles in the absence of Vif.
Observation of HIV-1 Nucleocapsid Protein induced TAR DNA melting at the single molecule level
NASA Astrophysics Data System (ADS)
Cosa, Gonzalo; Harbron, Elizabeth; O'Connor, Donald; Musier-Forsyth, Karin; Barbara, Paul
Reverse transcription of the HIV-1 RNA genome involves several nucleic acid rearrangement steps, and the HIV-1 nucleocapsid protein (NC) plays a key role in this process. NC is a nucleic acid chaperone protein, which facilitates the formation of the most stable nucleic acid structures. Single molecule fluorescence resonance energy transfer (SM-FRET) measurements enable us to observe the NC-induced conformational fluctuations of a transactivation response region (TAR) DNA hairpin, which is part of the initial product of reverse transcription known as minus-strand strong-stop DNA. SM-FRET studies show that the majority of conformational fluctuations of the fluorescently-labeled TAR DNA hairpin in the presence of NC occur in <100 ms. A single molecule explores a wide range of confomations unpon NC binding, with fluctuations encompassing as many as 40 bases in both arms of the hairpin. No conformational fluctuations are observed with the dye-labeled TAR DNA hairpin in the absence of NC or when a labeled TAR DNA hairpin variant lacking bulges and internal loops is analyzed in the presence of NC. This study represents the first real-time observation of NC-mediated nucleic acid conformational fluctuations, revealing new insights into NC's nucleic acid chaperone activity.
Differential expression of HERV-K (HML-2) proviruses in cells and virions of the teratocarcinoma cell line Tera-1.
Bhardwaj, Neeru; Montesion, Meagan; Roy, Farrah; Coffin, John M
Human endogenous retrovirus (HERV-K (HML-2)) proviruses are among the few endogenous retroviral elements in the human genome that retain coding sequence. HML-2 expression has been widely associated with human disease states, including different types of cancers as well as with HIV-1 infection. Understanding of the potential impact of this expression requires that it be annotated at the proviral level. Here, we utilized the high throughput capabilities of next-generation sequencing to profile HML-2 expression at the level of individual proviruses and secreted virions in the teratocarcinoma cell line Tera-1. We identified well-defined expression patterns, with transcripts emanating primarily from two proviruses located on chromosome 22, only one of which was efficiently packaged. Interestingly, there was a preference for transcripts of recently integrated proviruses, over those from other highly expressed but older elements, to be packaged into virions. We also assessed the promoter competence of the 5' long terminal repeats (LTRs) of expressed proviruses via a luciferase assay following transfection of Tera-1 cells. Consistent with the RNASeq results, we found that the activity of most LTRs corresponded to their transcript levels.
The Race against Protease Activation Defines the Role of ESCRTs in HIV Budding
Bendjennat, Mourad; Saffarian, Saveez
HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding "on-time" and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release. PMID:27280284
Uukuniemi Phlebovirus assembly and secretion leave a functional imprint on the virion glycome.
Crispin, Max; Harvey, David J; Bitto, David; Halldorsson, Steinar; Bonomelli, Camille; Edgeworth, Matthew; Scrivens, James H; Huiskonen, Juha T; Bowden, Thomas A
Uukuniemi virus (UUKV) is a model system for investigating the genus Phlebovirus of the Bunyaviridae. We report the UUKV glycome, revealing differential processing of the Gn and Gc virion glycoproteins. Both glycoproteins display poly-N-acetyllactosamines, consistent with virion assembly in the medial Golgi apparatus, whereas oligomannose-type glycans required for DC-SIGN-dependent cellular attachment are predominant on Gc. Local virion structure and the route of viral egress from the cell leave a functional imprint on the phleboviral glycome.
Human Immunodeficiency Virus (HIV) Type 1 Vpu Induces the Expression of CD40 in Endothelial Cells and Regulates HIV-Induced Adhesion of B-Lymphoma Cells
Henderson, Winnie W.; Ruhl, Rebecca; Lewis, Paul; Bentley, Matthew; Nelson, Jay A.; Moses, Ashlee V.
AIDS-related B-cell non-Hodgkin's lymphoma (AIDS-NHL) is a significant cause of morbidity and mortality among individuals infected with human immunodeficiency virus type 1 (HIV-1). AIDS-NHL is clinically and histologically heterogeneous, but common features include an aggressive clinical course and frequent extranodal presentation. HIV-1 infection of nonimmune cells that interact with malignant B cells at extranodal sites may influence both the development and the clinical presentation of disease. Our previous studies have shown that coculture of B-lymphoma (BL) cells with HIV-1-infected endothelial cells (EC) leads to contact activation of EC and firm BL-cell adhesion. The key event promoting EC-BL-cell adhesion was HIV-1 upregulation of endothelial CD40, which allowed induction of vascular cell adhesion molecule 1 (VCAM-1) in a CD40-dependent manner. The present study was designed to identify the HIV-1 protein(s) that influence EC-BL-cell adhesion. When HIV-1 proteins were individually expressed in EC by using recombinant adenoviruses, cultured BL cells adhered exclusively to Vpu-transduced EC. As with HIV-infected EC, adhesive properties were linked to the capacity of Vpu to upregulate CD40, which in turn allowed efficient expression of VCAM-1. When EC were infected with an HIV-1 pseudotype lacking the Vpu gene, CD40 upregulation and BL-cell adhesive properties were lost, indicating an essential role for Vpu in EC-BL-cell interactions. Thus, these data reveal a novel function for HIV-1 Vpu and further suggest a role for Vpu in the development of AIDS-NHL at EC-rich extranodal sites. PMID:15078922
Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells.
Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah
The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4(+) T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4(+) T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4(+) T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the "Shock and Kill" strategy for latently HIV-1 infected cells.
Curcumin improves synaptic plasticity impairment induced by HIV-1gp120 V3 loop
Shen, Ling-ling; Jiang, Ming-liang; Liu, Si-si; Cai, Min-chun; Hong, Zhong-qiu; Lin, Li-qing; Xing, Yan-yan; Chen, Gui-lin; Pan, Rui; Yang, Li-juan; Xu, Ying; Dong, Jun
Curcumin has been shown to significantly improve spatial memory impairment induced by HIV-1 gp120 V3 in rats, but the electrophysiological mechanism remains unknown. Using extracellular microelectrode recording techniques, this study confirmed that the gp120 V3 loop could suppress long-term potentiation in the rat hippocampal CA1 region and synaptic plasticity, and that curcumin could antagonize these inhibitory effects. Using a Fura-2/AM calcium ion probe, we found that curcumin resisted the effects of the gp120 V3 loop on hippocampal synaptosomes and decreased Ca2+ concentration in synaptosomes. This effect of curcumin was identical to nimodipine, suggesting that curcumin improved the inhibitory effects of gp120 on synaptic plasticity, ameliorated damage caused to the central nervous system, and might be a potential neuroprotective drug. PMID:26199609
HIV-1 protease inhibitor induced oxidative stress suppresses glucose stimulated insulin release: protection with thymoquinone.
Chandra, Surabhi; Mondal, Debasis; Agrawal, Krishna C
The highly active anti-retroviral therapy (HAART) regimen has considerably reduced the mortality rate in HIV-1 positive patients. However, long-term exposure to HAART is associated with a metabolic syndrome manifesting cardiovascular dysfunction, lipodystrophy, and insulin resistance syndrome (IRS). The inclusion of HIV-1 protease inhibitors (PIs) in HAART has been linked to the induction of IRS. Although several molecular mechanisms of PI-induced effects on insulin action have been postulated, the deleterious effects of PIs on insulin production by pancreatic beta-cells have not been fully investigated and therapeutic strategies to ameliorate insulin dysregulation at this level have not been targeted. The present study showed that exposure to several different PIs, nelfinavir (5-10 microM), saquinavir (5-10 microM) and atazanavir (8-20 microM), decreases glucose stimulated insulin secretion from rat pancreatic beta-cells (INS-1). Nelfinavir significantly increased reactive oxygen species (ROS) generation and suppressed cytosolic, but not mitochondrial superoxide dismutase (SOD) levels. Nelfinvair also decreased both glutathione and ATP and increased UCP2 levels in these cells. Simultaneous treatment with thymoquinone (TQ) (2.5 microM), an active ingredient of black seed oil, significantly inhibited the effect of nelfinavir on augmented ROS production and suppressed SOD levels. Both TQ and black seed oil exposure increased glucose stimulated insulin secretion and ameliorated the suppressive effect of nelfinavir. The present findings imply a direct role of ROS in PI induced deleterious effects on pancreatic beta-cells. Our findings also suggest that TQ may be used as a potential therapeutic agent to normalize the dysregulated insulin production observed in HAART treated patients.
Inhibitors of HIV nucleocapsid protein zinc fingers as candidates for the treatment of AIDS.
Rice, W G; Supko, J G; Malspeis, L; Buckheit, R W; Clanton, D; Bu, M; Graham, L; Schaeffer, C A; Turpin, J A; Domagala, J; Gogliotti, R; Bader, J P; Halliday, S M; Coren, L; Sowder, R C; Arthur, L O; Henderson, L E
Strategies for the treatment of human immunodeficiency virus-type 1 (HIV-1) infection must contend with the obstacle of drug resistance. HIV-1 nucleocapsid protein zinc fingers are prime antiviral targets because they are mutationally intolerant and are required both for acute infection and virion assembly. Nontoxic disulfide-substituted benzamides were identified that attack the zinc fingers, inactivate cell-free virions, inhibit acute and chronic infections, and exhibit broad antiretroviral activity. The compounds were highly synergistic with other antiviral agents, and resistant mutants have not been detected. Zinc finger-reactive compounds may offer an anti-HIV strategy that restricts drug-resistance development.
HIV-specific CD4-induced Antibodies Mediate Broad and Potent Antibody-dependent Cellular Cytotoxicity Activity and are Commonly Detected in Plasma from HIV-infected Humans
Williams, Katherine L.; Cortez, Valerie; Dingens, Adam S.; Gach, Johannes S.; Rainwater, Stephanie; Weis, Julie F.; Chen, Xuemin; Spearman, Paul; Forthal, Donald N.; Overbaugh, Julie
HIV-specific antibodies (Abs) can reduce viral burden by blocking new rounds of infection or by destroying infected cells via activation of effector cells through Fc–FcR interaction. This latter process, referred to as antibody-dependent cellular cytotoxicity (ADCC), has been associated with viral control and improved clinical outcome following both HIV and SIV infections. Here we describe an HIV viral-like particle (VLP)-based sorting strategy that led to identification of HIV-specific memory B cells encoding Abs that mediate ADCC from a subtype A-infected Kenyan woman at 914 days post-infection. Using this strategy, 12 HIV-envelope-specific monoclonal antibodies (mAbs) were isolated and three mediated potent ADCC activity when compared to well-characterized ADCC mAbs. The ADCC-mediating Abs also mediated antibody-dependent cell-mediated virus inhibition (ADCVI), which provides a net measure of Fc receptor-triggered effects against replicating virus. Two of the three ADCC-mediating Abs targeted a CD4-induced (CD4i) epitope also bound by the mAb C11; the third antibody targeted the N-terminus of V3. Both CD4i Abs identified here demonstrated strong cross-clade breadth with activity against 10 of 11 envelopes tested, including those from clades A, B, C, A/D and C/D, whereas the V3-specific antibody showed more limited breadth. Variants of these CD4i, C11-like mAbs engineered to interrupt binding to FcγRs inhibited a measurable percentage of the donor's ADCC activity starting as early as 189 days post-infection. C11-like antibodies also accounted for between 18–78% of ADCC activity in 9 chronically infected individuals from the same cohort study. Further, the two CD4i Abs originated from unique B cells, suggesting that antibodies targeting this epitope can be commonly produced. Taken together, these data provide strong evidence that CD4i, C11-like antibodies develop within the first 6 months of infection and they can arise from unique B-cell lineages in the
Paucity of Intact Non-Induced Provirus with Early, Long-Term Antiretroviral Therapy of Perinatal HIV Infection
Watson, Douglas; Luzuriaga, Katherine; Siberry, George; Petru, Ann; Chen, YaHui; Uprety, Priyanka; Ho, Ya-Chi; Persaud, Deborah
The latent reservoir is a major barrier to HIV eradication. Reservoir size is emerging as an important biomarker to assess the likelihood of HIV remission in the absence of antiretroviral therapy (ART) and may be reduced by earlier initiation of ART that restricts HIV spread into CD4+ T cells. Reservoir size is traditionally measured with a quantitative viral outgrowth assay (QVOA) that induces replication-competent HIV production through in vitro stimulation of resting CD4+ T cells. However, the recent identification of replication-intact, non-induced proviral genomes (NIPG) suggests the QVOA significantly underestimates (by 62-fold) latent reservoir size in chronically-infected adults. Whether formation and persistence of Intact, NIPG is thwarted by early ART initiation and long-term virologic suppression in perinatal infection is unclear. Here, we show that the latent reservoir in 11 early treated, long-term suppressed perinatally infected children and adolescents was not inducible by QVOA and dominated by defective, NIPG. Single genome analysis of 164 NIPG from 232 million cultured resting CD4+ T cells revealed no replication-intact, near-full length sequences. Forty-three (26%) NIPG contained APOBEC3G-mediated hypermutation, 115 (70%) NIPG contained large internal deletions, one NIPG contained nonsense mutations and indels, and 5 (3%) NIPG were assigned as "Not Evaluable" due to multiple failed sequencing attempts that precluded further classification. The lack of replication competent inducible provirus and intact NIPG in this cohort indicate early, long-term ART of perinatal infection leads to marked diminution of replication-competent HIV-1 reservoirs, creating a favorable state towards interventions aimed at virologic remission. PMID:28178277
Paucity of Intact Non-Induced Provirus with Early, Long-Term Antiretroviral Therapy of Perinatal HIV Infection.
Rainwater-Lovett, Kaitlin; Ziemniak, Carrie; Watson, Douglas; Luzuriaga, Katherine; Siberry, George; Petru, Ann; Chen, YaHui; Uprety, Priyanka; McManus, Margaret; Ho, Ya-Chi; Lamers, Susanna L; Persaud, Deborah
The latent reservoir is a major barrier to HIV eradication. Reservoir size is emerging as an important biomarker to assess the likelihood of HIV remission in the absence of antiretroviral therapy (ART) and may be reduced by earlier initiation of ART that restricts HIV spread into CD4+ T cells. Reservoir size is traditionally measured with a quantitative viral outgrowth assay (QVOA) that induces replication-competent HIV production through in vitro stimulation of resting CD4+ T cells. However, the recent identification of replication-intact, non-induced proviral genomes (NIPG) suggests the QVOA significantly underestimates (by 62-fold) latent reservoir size in chronically-infected adults. Whether formation and persistence of Intact, NIPG is thwarted by early ART initiation and long-term virologic suppression in perinatal infection is unclear. Here, we show that the latent reservoir in 11 early treated, long-term suppressed perinatally infected children and adolescents was not inducible by QVOA and dominated by defective, NIPG. Single genome analysis of 164 NIPG from 232 million cultured resting CD4+ T cells revealed no replication-intact, near-full length sequences. Forty-three (26%) NIPG contained APOBEC3G-mediated hypermutation, 115 (70%) NIPG contained large internal deletions, one NIPG contained nonsense mutations and indels, and 5 (3%) NIPG were assigned as "Not Evaluable" due to multiple failed sequencing attempts that precluded further classification. The lack of replication competent inducible provirus and intact NIPG in this cohort indicate early, long-term ART of perinatal infection leads to marked diminution of replication-competent HIV-1 reservoirs, creating a favorable state towards interventions aimed at virologic remission.
A Novel Prime and Boost Regimen of HIV Virus-Like Particles with TLR4 Adjuvant MPLA Induces Th1 Oriented Immune Responses against HIV
Poteet, Ethan; Lewis, Phoebe; Li, Feng; Zhang, Sheng; Gu, Jianhua; Chen, Changyi; Ho, Sam On; Do, Thai; Chiang, SuMing; Fujii, Gary; Yao, Qizhi
HIV virus-like particles (VLPs) present the HIV envelope protein in its native conformation, providing an ideal vaccine antigen. To enhance the immunogenicity of the VLP vaccine, we sought to improve upon two components; the route of administration and the additional adjuvant. Using HIV VLPs, we evaluated sub-cheek as a novel route of vaccine administration when combined with other conventional routes of immunization. Of five combinations of distinct prime and boost sequences, which included sub-cheek, intranasal, and intradermal routes of administration, intranasal prime and sub-cheek boost (IN+SC) resulted in the highest HIV-specific IgG titers among the groups tested. Using the IN+SC regimen we tested the adjuvant VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV) + monophosphoryl lipid A (MPLA) at MPLA concentrations of 0, 7.5, 12.5, and 25 μg/dose in combination with our VLPs. Mice that received 12.5 or 25 μg/dose MPLA had the highest concentrations of Env-specific IgG2c (20.7 and 18.4 μg/ml respectively), which represents a Th1 type of immune response in C57BL/6 mice. This was in sharp contrast to mice which received 0 or 7.5 μg MPLA adjuvant (6.05 and 5.68 μg/ml of IgG2c respectively). In contrast to IgG2c, MPLA had minor effects on Env-specific IgG1; therefore, 12.5 and 25 μg/dose of MPLA induced the optimal IgG1/IgG2c ratio of 1.3. Additionally, the percentage of germinal center B cells increased significantly from 15.4% in the control group to 31.9% in the CALV + 25 μg MPLA group. These mice also had significantly more IL-2 and less IL-4 Env-specific CD8+ T cells than controls, correlating with an increased percentage of Env-specific central memory CD4+ and CD8+ T cells. Our study shows the strong potential of IN+SC as an efficacious route of administration and the effectiveness of VLPs combined with MPLA adjuvant to induce Env specific Th1-oriented HIV-specific immune responses. PMID:26312747
Interferon-α Subtypes in an Ex Vivo Model of Acute HIV-1 Infection: Expression, Potency and Effector Mechanisms
Harper, Michael S.; Guo, Kejun; Gibbert, Kathrin; Lee, Eric J.; Dillon, Stephanie M.; Barrett, Bradley S.; McCarter, Martin D.; Hasenkrug, Kim J.; Dittmer, Ulf; Wilson, Cara C.; Santiago, Mario L.
HIV-1 is transmitted primarily across mucosal surfaces and rapidly spreads within the intestinal mucosa during acute infection. The type I interferons (IFNs) likely serve as a first line of defense, but the relative expression and antiviral properties of the 12 IFNα subtypes against HIV-1 infection of mucosal tissues remain unknown. Here, we evaluated the expression of all IFNα subtypes in HIV-1-exposed plasmacytoid dendritic cells by next-generation sequencing. We then determined the relative antiviral potency of each IFNα subtype ex vivo using the human intestinal Lamina Propria Aggregate Culture model. IFNα subtype transcripts from the centromeric half of the IFNA gene complex were highly expressed in pDCs following HIV-1 exposure. There was an inverse relationship between IFNA subtype expression and potency. IFNα8, IFNα6 and IFNα14 were the most potent in restricting HIV-1 infection. IFNα2, the clinically-approved subtype, and IFNα1 were both highly expressed but exhibited relatively weak antiviral activity. The relative potencies correlated with binding affinity to the type I IFN receptor and the induction levels of HIV-1 restriction factors Mx2 and Tetherin/BST-2 but not APOBEC3G, F and D. However, despite the lack of APOBEC3 transcriptional induction, the higher relative potency of IFNα8 and IFNα14 correlated with stronger inhibition of virion infectivity, which is linked to deaminase-independent APOBEC3 restriction activity. By contrast, both potent (IFNα8) and weak (IFNα1) subtypes significantly induced HIV-1 GG-to-AG hypermutation. The results unravel non-redundant functions of the IFNα subtypes against HIV-1 infection, with strong implications for HIV-1 mucosal immunity, viral evolution and IFNα-based functional cure strategies. PMID:26529416
A virus capsid component mediates virion retention and transmission by its insect vector
Chen, Angel Y. S.; Walker, Gregory P.; Carter, David; Ng, James C. K.
Numerous pathogens of humans, animals, and plants are transmitted by specific arthropod vectors. However, understanding the mechanisms governing these pathogen–vector interactions is hampered, in part, by the lack of easy-to-use analytical tools. We investigated whitefly transmission of Lettuce infectious yellows virus (LIYV) by using a unique immunofluorescent localization approach in which we fed virions or recombinant virus capsid components to whiteflies, followed by feeding them antibodies to the virions or capsid components, respectively. Fluorescent signals, indicating the retention of virions, were localized in the anterior foregut or cibarium of a whitefly vector biotype but not within those of a whitefly nonvector biotype. Retention of virions in these locations strongly corresponded with the whitefly vector transmission of LIYV. When four recombinant LIYV capsid components were individually fed to whitefly vectors, significantly more whiteflies retained the recombinant minor coat protein (CPm). As demonstrated previously and in the present study, whitefly vectors failed to transmit virions preincubated with anti-CPm antibodies but transmitted virions preincubated with antibodies recognizing the major coat protein (CP). Correspondingly, the number of insects that specifically retained virions preincubated with anti-CPm antibodies were significantly reduced compared with those that specifically retained virions preincubated with anti-CP antibodies. Notably, a transmission-defective CPm mutant was deficient in specific virion retention, whereas the CPm-restored virus showed WT levels of specific virion retention and transmission. These data provide strong evidence that transmission of LIYV is determined by a CPm-mediated virion retention mechanism in the anterior foregut or cibarium of whitefly vectors. PMID:21930903
Architectural Insight into Inovirus-Associated Vectors (IAVs) and Development of IAV-Based Vaccines Inducing Humoral and Cellular Responses: Implications in HIV-1 Vaccines
Hassapis, Kyriakos A.; Stylianou, Dora C.; Kostrikis, Leondios G.
Inovirus-associated vectors (IAVs) are engineered, non-lytic, filamentous bacteriophages that are assembled primarily from thousands of copies of the major coat protein gp8 and just five copies of each of the four minor coat proteins gp3, gp6, gp7 and gp9. Inovirus display studies have shown that the architecture of inoviruses makes all coat proteins of the inoviral particle accessible to the outside. This particular feature of IAVs allows foreign antigenic peptides to be displayed on the outer surface of the virion fused to its coat proteins and for more than two decades has been exploited in many applications including antibody or peptide display libraries, drug design, and vaccine development against infectious and non-infectious diseases. As vaccine carriers, IAVs have been shown to elicit both a cellular and humoral response against various pathogens through the display of antibody epitopes on their coat proteins. Despite their high immunogenicity, the goal of developing an effective vaccine against HIV-1 has not yet materialized. One possible limitation of previous efforts was the use of broadly neutralizing antibodies, which exhibited autoreactivity properties. In the past five years, however, new, more potent broadly neutralizing antibodies that do not exhibit autoreactivity properties have been isolated from HIV-1 infected individuals, suggesting that vaccination strategies aimed at producing such broadly neutralizing antibodies may confer protection against infection. The utilization of these new, broadly neutralizing antibodies in combination with the architectural traits of IAVs have driven the current developments in the design of an inovirus-based vaccine against HIV-1. This article reviews the applications of IAVs in vaccine development, with particular emphasis on the design of inoviral-based vaccines against HIV-1. PMID:25525909
Architectural insight into inovirus-associated vectors (IAVs) and development of IAV-based vaccines inducing humoral and cellular responses: implications in HIV-1 vaccines.
Hassapis, Kyriakos A; Stylianou, Dora C; Kostrikis, Leondios G
Inovirus-associated vectors (IAVs) are engineered, non-lytic, filamentous bacteriophages that are assembled primarily from thousands of copies of the major coat protein gp8 and just five copies of each of the four minor coat proteins gp3, gp6, gp7 and gp9. Inovirus display studies have shown that the architecture of inoviruses makes all coat proteins of the inoviral particle accessible to the outside. This particular feature of IAVs allows foreign antigenic peptides to be displayed on the outer surface of the virion fused to its coat proteins and for more than two decades has been exploited in many applications including antibody or peptide display libraries, drug design, and vaccine development against infectious and non-infectious diseases. As vaccine carriers, IAVs have been shown to elicit both a cellular and humoral response against various pathogens through the display of antibody epitopes on their coat proteins. Despite their high immunogenicity, the goal of developing an effective vaccine against HIV-1 has not yet materialized. One possible limitation of previous efforts was the use of broadly neutralizing antibodies, which exhibited autoreactivity properties. In the past five years, however, new, more potent broadly neutralizing antibodies that do not exhibit autoreactivity properties have been isolated from HIV-1 infected individuals, suggesting that vaccination strategies aimed at producing such broadly neutralizing antibodies may confer protection against infection. The utilization of these new, broadly neutralizing antibodies in combination with the architectural traits of IAVs have driven the current developments in the design of an inovirus-based vaccine against HIV-1. This article reviews the applications of IAVs in vaccine development, with particular emphasis on the design of inoviral-based vaccines against HIV-1.
The RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid
Errando, Manel; Bieniasz, Paul D.
The APOBEC3 (A3) cytidine deaminases are antiretroviral proteins, whose targets include human immunodeficiency virus type-1 (HIV-1). Their incorporation into viral particles is critical for antiviral activity and is driven by interactions with the RNA molecules that are packaged into virions. However, it is unclear whether A3 proteins preferentially target RNA molecules that are destined to be packaged and if so, how. Using cross-linking immunoprecipitation sequencing (CLIP-seq), we determined the RNA binding preferences of the A3F, A3G and A3H proteins. We found that A3 proteins bind preferentially to RNA segments with particular properties, both in cells and in virions. Specifically, A3 proteins target RNA sequences that are G-rich and/or A-rich and are not scanned by ribosomes during translation. Comparative analyses of HIV-1 Gag, nucleocapsid (NC) and A3 RNA binding to HIV-1 RNA in cells and virions revealed the striking finding that A3 proteins partially mimic the RNA binding specificity of the HIV-1 NC protein. These findings suggest a model for A3 incorporation into HIV-1 virions in which an NC-like RNA binding specificity is determined by nucleotide composition rather than sequence. This model reconciles the promiscuity of A3 RNA binding that has been observed in previous studies with a presumed advantage that would accompany selective binding to RNAs that are destined to be packaged into virions. PMID:27541140
Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization
Moussatche, Nissin Condit, Richard C.
The vaccinia virion is a membraned, slightly flattened, barrel-shaped particle, with a complex internal structure featuring a biconcave core flanked by lateral bodies. Although the architecture of the purified mature virion has been intensely characterized by electron microscopy, the distribution of the proteins within the virion has been examined primarily using biochemical procedures. Thus, it has been shown that non-ionic and ionic detergents combined or not with a sulfhydryl reagent can be used to disrupt virions and, to a limited degree, separate the constituent proteins in different fractions. Applying a controlled degradation technique to virions adsorbed on EM grids, we were able to immuno-localize viral proteins within the virion particle. Our results show after NP40 and DTT treatment, membrane proteins are removed from the virion surface revealing proteins that are associated with the lateral bodies and the outer layer of the core wall. Combined treatment using high salt and high DTT removed lateral body proteins and exposed proteins of the internal core wall. Cores treated with proteases could be disrupted and the internal components were exposed. Cts8, a mutant in the A3 protein, produces aberrant virus that, when treated with NP-40 and DTT, releases to the exterior the virus DNA associated with other internal core proteins. With these results, we are able to propose a model for the structure the vaccinia virion.
VirD - A Virion Display Array For Profiling Functional Membrane Proteins
Hu, Shaohui; Feng, Yingzhu; Henson, Brandon; Wang, Bochu; Huang, Xiaofang; Li, Min; Desai, Prashant; Zhu, Heng
To facilitate high-throughput biochemical analyses of membrane proteins, we have developed a novel display technology in a microarray format. Both a single-pass (CD4) and a multiple-pass (GPR77) human transmembrane proteins were engineered to be displayed in the membrane envelop of herpes simplex virions. These viruses produce large spherical virions displaying multiple copies of envelop proteins. Our aim was to engineer this virus to express these human proteins during the virus productive cycle and incorporate the human proteins into the virion during the assembly process. Another strategy presented includes engineering a fusion of glycoprotein C (gC), a major constituent of herpes simplex virus type 1 (HSV-1) virions, by hijacking the cis-acting signals to direct incorporation of the chimeric protein into the virion. The expression of the human proteins in infected cells, at the cell surface and in purified virions, is in the correct transmembrane orientation and the proteins are biochemically functional. Purified virions printed on glass slides form a high-density Virion Display (VirD) Array and the displayed proteins were demonstrated to retain their native conformations and interactions on the VirD Array judging by similar assays, such as antibody staining, as well as lectin and ligand binding. This method can be readily scaled or tailored for different modalities including to a high-content, high-throughput platform for screening ligands and drugs of human membrane proteins. PMID:23941274
Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization
Moussatche, Nissin; Condit, Richard C.
The vaccinia virion is a membraned, slightly flattened, barrel-shaped particle, with a complex internal structure featuring a biconcave core flanked by lateral bodies. Although the architecture of the purified mature virion has been intensely characterized by electron microscopy, the distribution of the proteins within the virion has been examined primarily using biochemical procedures. Thus, it has been shown that non-ionic and ionic detergents combined or not with a sulfhydryl reagent can be used to disrupt virions and, to a limited degree, separate the constituent proteins in different fractions. Applying a controlled degradation technique to virions adsorbed on EM grids, we were able to immuno-localize viral proteins within the virion particle. Our results show after NP40 and DTT treatment, membrane proteins are removed from the virion surface revealing proteins that are associated with the lateral bodies and the outer layer of the core wall. Combined treatment using high salt and high DTT removed lateral body proteins and exposed proteins of the internal core wall. Cores treated with proteases could be disrupted and the internal components were exposed. Cts8, a mutant in the A3 protein, produces aberrant virus that, when treated with NP-40 and DTT, release to the exterior the virus DNA associated with other internal core proteins. With these results, we are able to propose a model for the structure the vaccinia virion. PMID:25486587
Activation of the oxidative stress pathway by HIV-1 Vpr leads to induction of hypoxia-inducible factor 1alpha expression.
Deshmane, Satish L; Mukerjee, Ruma; Fan, Shongshan; Del Valle, Luis; Michiels, Carine; Sweet, Thersa; Rom, Inna; Khalili, Kamel; Rappaport, Jay; Amini, Shohreh; Sawaya, Bassel E
The detection of biomarkers of oxidative stress in brain tissue and cerebrospinal fluid of patients with human immunodeficiency virus, type 1 (HIV)-associated dementia indicates the involvement of stress pathways in the neuropathogenesis of AIDS. Although the biological importance of oxidative stress on events involved in AIDS neuropathogenesis and the HIV-1 proteins responsible for oxidative stress remain to be elucidated, our results point to the activation of hypoxia-inducible factor 1 (HIF-1) upon HIV-1 infection and its elevation in brain cells of AIDS patients with dementia. HIF-1 is a transcription factor that is responsive to oxygen. Under hypoxic conditions, HIF-1alpha becomes stable and translocates to the nucleus where it dimerizes with aryl hydrocarbon receptor nuclear translocator and modulates gene transcription. Activation of HIF-1 can also be mediated by the HIV-1 accessory protein Vpr. In addition, cellular components, including reactive oxygen species, contribute to the induction of HIF-1alpha. Our results show that Vpr induces reactive oxygen species by increasing H(2)O(2) production, which can contribute to HIF-1alpha accumulation. Interestingly, increased levels of HIF-1alpha stimulated HIV-1 gene transcription through HIF-1 association with HIV-1 long terminal repeat. These observations point to the existence of a positive feedback interplay between HIF-1alpha and Vpr and that, by inducing oxidative stress via activation of HIF-1, Vpr can induce HIV-1 gene expression and dysregulate multiple host cellular pathways.
Residues in the gp41 Ectodomain Regulate HIV-1 Envelope Glycoprotein Conformational Transitions Induced by gp120-Directed Inhibitors.
Pacheco, Beatriz; Alsahafi, Nirmin; Debbeche, Olfa; Prévost, Jérémie; Ding, Shilei; Chapleau, Jean-Philippe; Herschhorn, Alon; Madani, Navid; Princiotto, Amy; Melillo, Bruno; Gu, Christopher; Zeng, Xin; Mao, Youdong; Smith, Amos B; Sodroski, Joseph; Finzi, Andrés
Interactions between the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer maintain the metastable unliganded form of the viral spike. Binding of gp120 to the receptor, CD4, changes the Env conformation to promote gp120 interaction with the second receptor, CCR5 or CXCR4. CD4 binding also induces the transformation of Env into the prehairpin intermediate, in which the gp41 heptad repeat 1 (HR1) coiled coil is assembled at the trimer axis. In nature, HIV-1 Envs must balance the requirements to maintain the noncovalent association of gp120 with gp41 and to evade the host antibody response with the need to respond to CD4 binding. Here we show that the gp41 HR1 region contributes to gp120 association with the unliganded Env trimer. Changes in particular amino acid residues in the gp41 HR1 region decreased the efficiency with which Env moved from the unliganded state. Thus, these gp41 changes decreased the sensitivity of HIV-1 to cold inactivation and ligands that require Env conformational changes to bind efficiently. Conversely, these gp41 changes increased HIV-1 sensitivity to small-molecule entry inhibitors that block Env conformational changes induced by CD4. Changes in particular gp41 HR1 amino acid residues can apparently affect the relative stability of the unliganded state and CD4-induced conformations. Thus, the gp41 HR1 region contributes to the association with gp120 and regulates Env transitions from the unliganded state to downstream conformations.IMPORTANCE The development of an efficient vaccine able to prevent HIV infection is a worldwide priority. Knowledge of the envelope glycoprotein structure and the conformational changes that occur after receptor engagement will help researchers to develop an immunogen able to elicit antibodies that block HIV-1 transmission. Here we identify residues in the HIV-1 transmembrane envelope glycoprotein that stabilize the unliganded state by modulating the
Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging
Wu, Weimin; Leavitt, Justin C.; Cheng, Naiqian; Gilcrease, Eddie B.; Motwani, Tina; Teschke, Carolyn M.; Casjens, Sherwood R.
ABSTRACT The P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three "ejection proteins" (E-proteins [gp7, gp16, and gp20]) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins' locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection. PMID:27507825
HIV-1-Induced Small T Cell Syncytia Can Transfer Virus Particles to Target Cells through Transient Contacts
Symeonides, Menelaos; Murooka, Thomas T.; Bellfy, Lauren N.; Roy, Nathan H.; Mempel, Thorsten R.; Thali, Markus
HIV-1 Env mediates fusion of viral and target cell membranes, but it can also mediate fusion of infected (producer) and target cells, thus triggering the formation of multinucleated cells, so-called syncytia. Large, round, immobile syncytia are readily observable in cultures of HIV-1-infected T cells, but these fast growing "fusion sinks" are largely regarded as cell culture artifacts. In contrast, small HIV-1-induced syncytia were seen in the paracortex of peripheral lymph nodes and other secondary lymphoid tissue of HIV-1-positive individuals. Further, recent intravital imaging of lymph nodes in humanized mice early after their infection with HIV-1 demonstrated that a significant fraction of infected cells were highly mobile, small syncytia, suggesting that these entities contribute to virus dissemination. Here, we report that the formation of small, migratory syncytia, for which we provide further quantification in humanized mice, can be recapitulated in vitro if HIV-1-infected T cells are placed into 3D extracellular matrix (ECM) hydrogels rather than being kept in traditional suspension culture systems. Intriguingly, live-cell imaging in hydrogels revealed that these syncytia, similar to individual infected cells, can transiently interact with uninfected cells, leading to rapid virus transfer without cell-cell fusion. Infected cells were also observed to deposit large amounts of viral particles into the extracellular space. Altogether, these observations suggest the need to further evaluate the biological significance of small, T cell-based syncytia and to consider the possibility that these entities do indeed contribute to virus spread and pathogenesis. PMID:26703714
HIV-1-Induced Small T Cell Syncytia Can Transfer Virus Particles to Target Cells through Transient Contacts.
Symeonides, Menelaos; Murooka, Thomas T; Bellfy, Lauren N; Roy, Nathan H; Mempel, Thorsten R; Thali, Markus
HIV-1 Env mediates fusion of viral and target cell membranes, but it can also mediate fusion of infected (producer) and target cells, thus triggering the formation of multinucleated cells, so-called syncytia. Large, round, immobile syncytia are readily observable in cultures of HIV-1-infected T cells, but these fast growing "fusion sinks" are largely regarded as cell culture artifacts. In contrast, small HIV-1-induced syncytia were seen in the paracortex of peripheral lymph nodes and other secondary lymphoid tissue of HIV-1-positive individuals. Further, recent intravital imaging of lymph nodes in humanized mice early after their infection with HIV-1 demonstrated that a significant fraction of infected cells were highly mobile, small syncytia, suggesting that these entities contribute to virus dissemination. Here, we report that the formation of small, migratory syncytia, for which we provide further quantification in humanized mice, can be recapitulated in vitro if HIV-1-infected T cells are placed into 3D extracellular matrix (ECM) hydrogels rather than being kept in traditional suspension culture systems. Intriguingly, live-cell imaging in hydrogels revealed that these syncytia, similar to individual infected cells, can transiently interact with uninfected cells, leading to rapid virus transfer without cell-cell fusion. Infected cells were also observed to deposit large amounts of viral particles into the extracellular space. Altogether, these observations suggest the need to further evaluate the biological significance of small, T cell-based syncytia and to consider the possibility that these entities do indeed contribute to virus spread and pathogenesis.
Cytomegalovirus UL103 controls virion and dense body egress.
Ahlqvist, Jenny; Mocarski, Edward
Human cytomegalovirus UL103 encodes a tegument protein that is conserved across herpesvirus subgroups. Mutant viruses lacking this gene product exhibit dramatically reduced accumulation of cell-free virus progeny and poor cell-to-cell spread. Given that viral proteins and viral DNA accumulate with normal kinetics in cells infected with mutant virus, UL103 appears to function during the late phase of replication, playing a critical role in egress of capsidless dense bodies and virions. Few dense bodies were observed in the extracellular space in mutant virus-infected cells in the presence or absence of the DNA encapsidation inhibitor 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole. Upon reversal of encapsidation inhibition, UL103 had a striking impact on accumulation of cell-free virus, but not on accumulation of cell-associated virus. Thus, UL103 plays a novel and important role during maturation, regulating virus particle and dense body egress from infected cells.
Sample preparation for single virion atomic force microscopy and super-resolution fluorescence imaging.
Hodges, Jeffery A; Saffarian, Saveez
Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.
HIV-induced alteration in gut microbiota: driving factors, consequences, and effects of antiretroviral therapy.
Lozupone, Catherine A; Rhodes, Matthew E; Neff, Charles P; Fontenot, Andrew P; Campbell, Thomas B; Palmer, Brent E
Consistent with an important role for adaptive immunity in modulating interactions between intestinal bacteria and host, dramatic alteration in the composition of gut microbes during chronic HIV infection was recently reported by ourselves and independently by four other research groups. Here we evaluate our results in the context of these other studies and delve into the effects of antiretroviral therapy (ART). Although gut microbiota of HIV-positive individuals on ART usually does not resemble that of HIV-negative individuals, the degree to which ART restores health-associated prevalence varies across bacterial taxa. Finally, we discuss potential drivers and health consequences of gut microbiota alterations. We propose that understanding the mechanism of HIV-associated gut microbiota changes will elucidate the role of adaptive immunity in shaping gut microbiota composition, and lay the foundation for therapeutics targeting the microbiota to attenuate HIV disease progression and reduce the risk of gut-linked disease in people with HIV.
[Mechanism Underlying Increased Expression of a Member of the Serine/Threonine Kinase Family (Citron kinase) Induced by HIV-1 Infection].
Ding, Jiwei; Mi, Zeyun; Zhao, Jianyuan; Zhou, Jinming; Li, Xiaoyu; Cen, Shan
Human immunodeficiency virus (HIV)-1 infection changes transcriptional profiles and regulates. the factors and machinery of the host that facilitate viral replication. Our previous study suggested that the serine/threonine kinase citron kinase (citK) promotes HIV-1 egress. To ascertain if HIV-1 infection affects citK expression in primary cells, peripheral blood mononuclear cells were infected with vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1 vector NL4-3-luc viruses, which resulted in remarkably increased expression of citK. citK overexpression led to a more than two-fold increase in HIV-1 production, whereas a significant decrease was observed when citK was depleted in CD4+ T cells. Infection with HIV-1 pseudoviruses induced increases in the mRNA and protein levels of citK by 2. 5- and 2. 7-fold in HEK293T cells, respectively. By cloning the 5-kb promoter of citK into a luciferase reporter system and transfecting the construct into HEK293T cells, enhanced luciferase activity was observed during HIV-1 infection. Taken together, these data demonstrate that HIV-1 infection upregulates citK expression at the transcriptional level, and thereby renders the host more susceptible to invasion by HIV-1.
CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs.
Zhang, Yonggang; Yin, Chaoran; Zhang, Ting; Li, Fang; Yang, Wensheng; Kaminski, Rafal; Fagan, Philip Regis; Putatunda, Raj; Young, Won-Bin; Khalili, Kamel; Hu, Wenhui
Current antiretroviral therapy does not eliminate the integrated and transcriptionally silent HIV-1 provirus in latently infected cells. Recently, a "shock and kill" strategy has been extensively explored to eradicate the HIV-1 latent reservoirs for a permanent cure of AIDS. The therapeutic efficacy of currently used agents remains disappointing because of low efficiency, non-specificity and cellular toxicity. Here we present a novel catalytically-deficient Cas9-synergistic activation mediator (dCas9-SAM) technology to selectively, potently and persistently reactivate the HIV-1 latent reservoirs. By screening 16 MS2-mediated single guide RNAs, we identified long terminal repeat (LTR)-L and O that surround the enhancer region (-165/-145 for L and -92/-112 for O) and induce robust reactivation of HIV-1 provirus in HIV-1 latent TZM-bI epithelial, Jurkat T lymphocytic and CHME5 microglial cells. This compulsory reactivation induced cellular suicide via toxic buildup of viral proteins within HIV-1 latent Jurkat T and CHME5 microglial cells. These results suggest that this highly effective and target-specific dCas9-SAM system can serve as a novel HIV-latency-reversing therapeutic tool for the permanent elimination of HIV-1 latent reservoirs.
CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs
Zhang, Yonggang; Yin, Chaoran; Zhang, Ting; Li, Fang; Yang, Wensheng; Kaminski, Rafal; Fagan, Philip Regis; Putatunda, Raj; Young, Won-Bin; Khalili, Kamel; Hu, Wenhui
Current antiretroviral therapy does not eliminate the integrated and transcriptionally silent HIV-1 provirus in latently infected cells. Recently, a "shock and kill" strategy has been extensively explored to eradicate the HIV-1 latent reservoirs for a permanent cure of AIDS. The therapeutic efficacy of currently used agents remains disappointing because of low efficiency, non-specificity and cellular toxicity. Here we present a novel catalytically-deficient Cas9-synergistic activation mediator (dCas9-SAM) technology to selectively, potently and persistently reactivate the HIV-1 latent reservoirs. By screening 16 MS2-mediated single guide RNAs, we identified long terminal repeat (LTR)-L and O that surround the enhancer region (-165/-145 for L and -92/-112 for O) and induce robust reactivation of HIV-1 provirus in HIV-1 latent TZM-bI epithelial, Jurkat T lymphocytic and CHME5 microglial cells. This compulsory reactivation induced cellular suicide via toxic buildup of viral proteins within HIV-1 latent Jurkat T and CHME5 microglial cells. These results suggest that this highly effective and target-specific dCas9-SAM system can serve as a novel HIV-latency-reversing therapeutic tool for the permanent elimination of HIV-1 latent reservoirs. PMID:26538064
Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder.
Kim, WooJin; Zekas, Erin; Lodge, Robert; Susan-Resiga, Delia; Marcinkiewicz, Edwidge; Essalmani, Rachid; Mihara, Koichiro; Ramachandran, Rithwik; Asahchop, Eugene; Gelman, Benjamin; Cohen, Éric A; Power, Christopher; Hollenberg, Morley D; Seidah, Nabil G
The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg41↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg41XXXXArg46↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg46↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND.
Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder
Kim, WooJin; Zekas, Erin; Lodge, Robert; Susan-Resiga, Delia; Marcinkiewicz, Edwidge; Essalmani, Rachid; Mihara, Koichiro; Ramachandran, Rithwik; Asahchop, Eugene; Gelman, Benjamin; Cohen, Éric A.; Power, Christopher; Hollenberg, Morley D.
The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg41↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg41XXXXArg46↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg46↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND. PMID:26283733
Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells
Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah
The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4+ T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4+ T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4+ T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the "Shock and Kill" strategy for latently HIV-1 infected cells. PMID:26184775
The HIV-1 Entry Process: A Stoichiometric View.
Brandenberg, Oliver F; Magnus, Carsten; Regoes, Roland R; Trkola, Alexandra
HIV-1 infection starts with fusion of the viral and the host cell membranes, a process mediated by the HIV-1 envelope glycoprotein trimer. The number of trimers required to complete membrane fusion, referred to as HIV-1 entry stoichiometry, remains under debate. A precise definition of HIV-1 entry stoichiometry is important as it reflects the efficacy of the viral entry process and steers the infectivity of HIV-1 virion populations. Initial estimates suggested a unanimous entry stoichiometry across HIV-1 strains while recent findings showed that HIV-1 strains can differ in entry stoichiometry. Here, we review current analyses of HIV-1 entry stoichiometry and point out future research directions to further define the interplay between entry stoichiometry, virus entry fitness, transmission, and susceptibility to antibody neutralization.
CD4-Induced Antibodies Promote Association of the HIV-1 Envelope Glycoprotein with CD4-Binding Site Antibodies
Fellinger, Christoph H.; Prasad, Neha R.; Zhou, Amber S.; Kondur, Hema R.; Joshi, Vinita R.; Quinlan, Brian D.; Farzan, Michael
ABSTRACT The HIV-1 envelope glycoprotein (Env) is a trimer of gp120/gp41 heterodimers that mediates viral entry. Env binds cellular CD4, an association which stabilizes a conformation favorable to its subsequent association with a coreceptor, typically CCR5 or CXCR4. The CD4- and coreceptor-binding sites serve as epitopes for two classes of HIV-1-neutralizing antibodies: CD4-binding site (CD4bs) and CD4-induced (CD4i) antibodies, respectively. Here we observed that, at a fixed total concentration, mixtures of the CD4i antibodies (E51 or 412d) and the CD4bs antibody VRC01 neutralized the HIV-1 isolates 89.6, ADA, SG3, and SA32 more efficiently than either antibody alone. We found that E51, and to a lesser extent 412d and 17b, promoted association of four CD4bs antibodies to the Env trimer but not to monomeric gp120. We further demonstrated that the binding of the sulfotyrosine-binding pocket by CCR5mim2-Ig was sufficient for promoting CD4bs antibody binding to Env. Interestingly, the relationship is not reciprocal: CD4bs antibodies were not as efficient as CD4-Ig at promoting E51 or 412d binding to Env trimer. Consistent with these observations, CD4-Ig, but none of the CD4bs antibodies tested, substantially increased HIV-1 infection of a CD4-negative, CCR5-positive cell line. We conclude that the ability of CD4i antibodies to promote VRC01 association with Env trimers accounts for the increase potency of VRC01 and CD4i antibody mixtures. Our data further suggest that potent CD4bs antibodies avoid inducing Env conformations that bind CD4i antibodies or CCR5. IMPORTANCE Potent HIV-1-neutralizing antibodies can prevent viral transmission and suppress an ongoing infection. Here we show that CD4-induced (CD4i) antibodies, which recognize the conserved coreceptor-binding site of the HIV-1 envelope glycoprotein (Env), can increase the association of Env with potent broadly neutralizing antibodies that recognize the CD4-binding site (CD4bs antibodies). We further show that
Range of CD4-Bound Conformations of HIV-1 gp120, as Defined Using Conditional CD4-Induced Antibodies
Kaplan, Gilad; Roitburd-Berman, Anna; Lewis, George K.
ABSTRACT The HIV envelope binds cellular CD4 and undergoes a range of conformational changes that lead to membrane fusion and delivery of the viral nucleocapsid into the cellular cytoplasm. This binding to CD4 reveals cryptic and highly conserved epitopes, the molecular nature of which is still not fully understood. The atomic structures of CD4 complexed with gp120 core molecules (a form of gp120 in which the V1, V2, and V3 loops and N and C termini have been truncated) have indicated that a hallmark feature of the CD4-bound conformation is the bridging sheet minidomain. Variations in the orientation of the bridging sheet hairpins have been revealed when CD4-liganded gp120 was compared to CD4-unliganded trimeric envelope structures. Hence, there appears to be a number of conformational transitions possible in HIV-1 monomeric gp120 that are affected by CD4 binding. The spectrum of CD4-bound conformations has been interrogated in this study by using a well-characterized panel of conditional, CD4-induced (CD4i) monoclonal antibodies (MAbs) that bind HIV-1 gp120 and its mutations under various conditions. Two distinct CD4i epitopes of the outer domain were studied: the first comprises the bridging sheet, while the second contains elements of the V2 loop. Furthermore, we show that the unliganded extended monomeric core of gp120 (coree) assumes an intermediate CD4i conformation in solution that further undergoes detectable rearrangements upon association with CD4. These discoveries impact both accepted paradigms concerning gp120 structure and the field of HIV immunogen design. IMPORTANCE Elucidation of the conformational transitions that the HIV-1 envelope protein undergoes during the course of entry into CD4+ cells is fundamental to our understanding of HIV biology. The binding of CD4 triggers a range of gp120 structural rearrangements that could present targets for future drug design and development of preventive vaccines. Here we have systematically interrogated and
Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR
Sahu, Geetaram; Farley, Kalamo; El-Hage, Nazira; Aiamkitsumrit, Benjamas; Fassnacht, Ryan; Kashanchi, Fatah; Ochem, Alex; Simon, Gary L.; Karn, Jonathan; Hauser, Kurt F.; Tyagi, Mudit
Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-ĸB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-ĸB at 276th serine residue. These modifications enhance the interaction of NF-ĸB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. - Highlights: • Cocaine induces the initiation phase of HIV transcription by activating NF-ĸB. • Cocaine induced NF-ĸB phosphorylation promotes its interaction with P300. • Cocaine enhances the elongation phase of HIV transcription by stimulating MSK1. • Cocaine activated MSK1 catalyzes the phosphorylation of histone H3 at its Ser10. • Cocaine induced H3S10 phosphorylation facilitates the recruitment of P-TEFb at LTR.
Acrolein enhances epigenetic modifications, FasL expression and hepatocyte toxicity induced by anti-HIV drug Zidovudine.
Ghare, Smita S; Donde, Hridgandh; Chen, Wei-Yang; Barker, David F; Gobejishvilli, Leila; McClain, Craig J; Barve, Shirish S; Joshi-Barve, Swati
Zidovudine (AZT) remains the mainstay of antiretroviral therapy against HIV in resource-poor countries; however, its use is frequently associated with hepatotoxicity. Not all HIV patients on AZT develop hepatotoxicity, and the determining factors are unclear. Alcohol consumption and cigarette smoking are known risk factors for HIV hepatotoxicity, and both are significant sources of acrolein, a highly reactive and toxic aldehyde. This study examines the potential hepatotoxic interactions between acrolein and AZT. Our data demonstrate that acrolein markedly enhanced AZT-induced transcriptionally permissive histone modifications (H3K9Ac and H3K9Me3) allowing the recruitment of transcription factor NF-kB and RNA polymerase II at the FasL gene promoter, resulting in FasL upregulation and apoptosis in hepatocytes. Notably, the acrolein scavenger, hydralazine prevented these promoter-associated epigenetic changes and inhibited FasL upregulation and apoptosis induced by the combination of AZT and acrolein, as well as AZT alone. Our data strongly suggest that acrolein enhancement of promoter histone modifications and FasL upregulation are major pathogenic mechanisms driving AZT-induced hepatotoxicity. Moreover, these data also indicate the therapeutic potential of hydralazine in mitigating AZT hepatotoxicity.
The glucocorticoid receptor type II complex is a target of the HIV-1 vpr gene product.
Refaeli, Y; Levy, D N; Weiner, D B
The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 15-kDa virion-associated protein that functions as a regulator of cellular processes linked to the HIV life cycle. We report the interaction of a 41-kDa cytosolic viral protein R interacting protein 1 (Rip-1) with Vpr in vitro. Rip-1 displays a wide tissue distribution, including relevant targets of HIV infection. Vpr protein induced nuclear translocation of Rip-1, as did glucocorticoid receptor (GR)-II-stimulating steroids. Importantly, Vpr and Rip-1 coimmunoprecipitated with the human GR as part of an activated receptor complex. Vpr complementation of a vpr mutant virus was also mimicked by GR-II-stimulating steroids. Vpr and GR-II actions were inhibited by mifepristone, a GR-II pathway inhibitor. Together these data directly link the activity of the vpr gene product to the glucocorticoid steroid pathway and provide a biochemical mechanism for the cellular and viral activity of Vpr, as well as suggest that a unique class of antivirals, which includes mifepristone (RU486), may influence HIV-1 replication. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 PMID:7724608
Envelope Conformational Changes Induced by Human Immunodeficiency Virus Type 1 Attachment Inhibitors Prevent CD4 Binding and Downstream Entry Events
Ho, Hsu-Tso; Fan, Li; Nowicka-Sans, Beata; McAuliffe, Brian; Li, Chang-Ben; Yamanaka, Gregory; Zhou, Nannan; Fang, Hua; Dicker, Ira; Dalterio, Richard; Gong, Yi-Fei; Wang, Tao; Yin, Zhiwei; Ueda, Yasutsugu; Matiskella, John; Kadow, John; Clapham, Paul; Robinson, James; Colonno, Richard; Lin, Pin-Fang
BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes. PMID:16571818
HIV-1 Capsid: The Multifaceted Key Player in HIV-1 infection
Campbell, Edward M.; Hope, Thomas J.
In a mature, infectious HIV-1 virion, the viral genome is housed within a conical capsid core comprised of the viral capsid (CA) protein. The CA protein, and the structure into which it assembles, facilitate virtually every step of infection through a series of interactions with multiple host cell factors. This review describes our understanding of the interactions between the viral capsid core and several cellular factors that enable efficient HIV-1 genome replication, timely core disassembly, nuclear import and the integration of the viral genome into the genome of the target cell. We then discuss how elucidating these interactions can reveal new targets for therapeutic interactions against HIV-1. PMID:26179359
Folded monomers and hexamers of the ectodomain of the HIV gp41 membrane fusion protein: potential roles in fusion and synergy between the fusion peptide, hairpin, and membrane-proximal external region.
Banerjee, Koyeli; Weliky, David P
HIV is an enveloped virus and fusion between the HIV and host cell membranes is catalyzed by the ectodomain of the HIV gp41 membrane protein. Both the N-terminal fusion peptide (FP) and C-terminal membrane-proximal external region (MPER) are critical for fusion and are postulated to bind to the host cell and HIV membranes, respectively. Prior to fusion, the gp41 on the virion is a trimer in noncovalent complex with larger gp120 subunits. The gp120 bind host cell receptors and move away or dissociate from gp41 which subsequently catalyzes fusion. In the present work, large gp41 ectodomain constructs were produced and biophysically and structurally characterized. One significant finding is observation of synergy between the FP, hairpin, and MPER in vesicle fusion. The ectodomain-induced fusion can be very efficient with only ∼15 gp41 per vesicle, which is comparable to the number of gp41 on a virion. Conditions are found with predominant monomer or hexamer but not trimer and these may be oligomeric states during fusion. Monomer gp41 ectodomain is hyperthermostable and has helical hairpin structure. A new HIV fusion model is presented where (1) hemifusion is catalyzed by folding of gp41 ectodomain monomers into hairpins and (2) subsequent fusion steps are catalyzed by assembly into a hexamer with FPs in an antiparallel β sheet. There is also significant interest in the gp41 MPER because it is the epitope of several broadly neutralizing antibodies. Two of these antibodies bind our gp41 ectodomain constructs and support investigation of the gp41 ectodomain as an immunogen in HIV vaccine development.
Human Ubc9 Is Involved in Intracellular HIV-1 Env Stability after Trafficking out of the Trans-Golgi Network in a Gag Dependent Manner
Bohl, Christopher R.; Abrahamyan, Levon G.; Wood, Charles
The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env) that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER) and its trafficking to the trans-Golgi network (TGN) were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions. PMID:23861967
Plasma gelsolin protects HIV-1 gp120-Induced neuronal injury via voltage-gated K(+) channel Kv2.1.
Liu, Han; Liu, Jianuo; Liang, Shangdong; Xiong, Huangui
Plasma gelsolin (pGSN), a secreted form of gelsolin, is constitutively expressed throughout the central nervous system (CNS). Neurons, astrocytes and oligodendrocytes are the major sources of pGSN in the CNS. It has been shown that levels of pGSN in cerebrospinal fluid (CSF) are decreased in several neurological conditions including HIV-1-associated neurocognitive disorders (HAND). Although there is no direct evidence that a decreased level of pGSN in CSF is causally related to the pathogenesis of neurological disorders, neural cells, if lacking pGSN, are more vulnerable to cell death. To understand how GSN levels relate to neuronal injury in HAND, we studied the effects of pGSN on HIV-1 gp120-activated outward K(+) currents in primary rat cortical neuronal cultures. Incubation of rat cortical neurons with gp120 enhanced the outward K(+) currents induced by voltage steps and resulted in neuronal apoptosis. Treatment with pGSN suppressed the gp120-induced increase of delayed rectifier current (IK) and reduced vulnerability to gp120-induced neuronal apoptosis. Application of Guangxitoxin-1E (GxTx), a Kv2.1 specific channel inhibitor, inhibited gp120 enhancement of IK and associated neuronal apoptosis, similar effects to pGSN. Western blot and PCR analysis revealed gp120 exposure to up-regulate Kv2.1 channel expression, which was also inhibited by treatment with pGSN. Taken together, these results indicate pGSN protects neurons by suppressing gp120 enhancement of IK through Kv2.1 channels and reduction of pGSN in HIV-1-infected brain may contribute to HIV-1-associated neuropathy.
Plasma gelsolin protects HIV-1 gp120-induced neuronal injury via voltage-gated K+ channel Kv2.1.
Liu, Han; Liu, Jianuo; Liang, Shangdong; Xiong, Huangui
Plasma gelsolin (pGSN), a secreted form of gelsolin, is constitutively expressed throughout the central nervous system (CNS). The neurons, astrocytes and oligodendrocytes are the major sources of pGSN in the CNS. It has been shown that levels of pGSN in the cerebrospinal fluid (CSF) are decreased in several neurological conditions including HIV-1-associated neurocognitive disorders (HAND). Although there is no direct evidence that a decreased level of pGSN in CSF is causally related to the pathogenesis of neurological disorders, neural cells, if lacking pGSN, are more vulnerable to cell death. To understand how GSN levels relate to neuronal injury in HAND, we studied the effects of pGSN on HIV-1 gp120-activated outward K+ currents in primary rat cortical neuronal cultures. Incubation of rat cortical neurons with gp120 enhanced the outward K+ currents induced by voltage steps and resulted in neuronal apoptosis. Treatment with pGSN suppressed the gp120-induced increase of delayed rectifier current (IK) and reduced vulnerability to gp120-induced neuronal apoptosis. Application of Guangxitoxin-1E (GxTx), a Kv2.1 specific channel inhibitor, inhibited gp120 enhancement of IK and associated neuronal apoptosis, similar effects to pGSN. Western blot and PCR analysis revealed gp120 exposure to up-regulate Kv2.1 channel expression, which was also inhibited by treatment with pGSN. Taken together, these results indicate pGSN protects neurons by suppressing gp120 enhancement of IK through Kv2.1 channels and reduction of pGSN in HIV-1-infected brain may contribute to HIV-1-associated neuropathy.
Romidepsin-induced HIV-1 viremia during effective antiretroviral therapy contains identical viral sequences with few deleterious mutations
Winckelmann, Anni; Barton, Kirston; Hiener, Bonnie; Schlub, Timothy E.; Shao, Wei; Rasmussen, Thomas A.; Østergaard, Lars; Søgaard, Ole S.; Tolstrup, Martin; Palmer, Sarah
Objective: To investigate the origin of the HIV-1 viremia induced by the latency-reversing agent romidepsin. Design: Six individuals on suppressive antiretroviral therapy received romidepsin administered intravenously once weekly for 3 consecutive weeks. CD4+ T cells were obtained at baseline, following the second and third romidepsin infusion, and 10 weeks after the final romidepsin treatment. Plasma samples were collected 24 and 72 h after romidepsin infusions. Methods: Single-genome sequencing of the env and p24-RT region was used to genetically characterize the virus from proviral DNA, the transcribed cell-associated RNA and the plasma RNA pool. Results: In three of six participants with available plasma samples we identified plasma HIV-1 RNA sequences that were identical to DNA and/or cell-associated RNA sequences from peripheral blood CD4+ T cells. In two participants, plasma RNA sequences contained expansions of identical sequences, corresponding to 62 and 100% of the total sequences, respectively. Plasma HIV-1 RNA had very low amounts of defective viruses compared to cell-associated RNA (odds ratio 20.85, P < 0.001) and to DNA (odds ratio 7.07, P = 0.011) during romidepsin therapy. Conclusions: Romidepsin induced transcription from proviruses in peripheral blood cells, which contributed to viremia in patients on suppressive therapy. The intermingling of these cell-associated HIV-1 RNA with DNA sequences indicates transcription from a diverse range of proviruses, but the expansions of identical viral plasma sequences with few defects indicate that the romidepsin-induced viremia arises from intact proviruses with highly similar or identical genetic backgrounds. PMID:28272134
A human recombinant Fab identifies a human immunodeficiency virus type 1-induced conformational change in cell surface-expressed CD4.
Bachelder, R E; Bilancieri, J; Lin, W; Letvin, N L
To explore the role of the CD4 molecule in human immunodeficiency virus (HIV) infection following initial virus-CD4 binding, we have characterized CD4-specific antibodies raised by immunizing an HIV-1-infected human with human recombinant soluble CD4 (rsCD4). Fabs were selected from a human recombinant Fab library constructed from the bone marrow of this immunized individual. Here, we describe a human rsCD4-specific recombinant Fab clone selected by panning the library over complexes of human rsCD4 and recombinant HIV-1 envelope protein. While this Fab does not bind to CD4-positive T-cell lines or to human T lymphocytes, it recognizes cell surface-expressed CD4 following the incubation of these cells with a recombinant form of HIV-1 gp120 or with HIV-1 virions. The Fab is not HIV-1 envelope specific, since it does not bind to recombinant gp120 or to native cell surface-expressed HIV-1 envelope proteins. As confirmation of its CD4 specificity, we show that this Fab immunoprecipitates a 55-kDa protein, corresponding to the molecular mass of cellular CD4, from an H9 cell lysate. The specificity of this human Fab provides evidence for a virus-induced conformational change in cell surface-expressed on CD4. The characterization of this altered CD4 conformation and its effects on the host cell will be important in defining postbinding events in HIV infection. PMID:7637018
Abacavir-induced fulminant hepatic failure in a HIV/HCV co-infected patient.
Haas, Christopher; Ziccardi, Mary Rodriguez; Borgman, Jody
Abacavir hypersensitivity is a rare, yet significant adverse reaction that results in a spectrum of physical and laboratory abnormalities, and has been postulated to stem from a variety of aetiological factors. The major histocompatibility complex haplotype human leucocyte antigen (HLA)-B5701 is a significant risk factor in development of hypersensitivity reactions, yet only 55% of HLA-B5701+ individuals develop such reactions, suggesting a multifactorial aetiology. Nevertheless, prospective screening and avoidance of abacavir in these patients has limited adverse events. Within this spectrum of adverse events, abacavir-induced liver toxicity is exceedingly rare and reported events have ranged from mild elevations of aminotransferases to fulminant hepatic failure. We report the case of a 50-year-old Caucasian woman with a history significant for HIV, hepatitis C virus and a HLA-B5701+ status, transferred to our emergency department in a hypotensive state and found to have acute liver failure, acute renal failure and significant rhabdomyolysis following a change of highly active antiretroviral therapy regimen.
Methods for the Analyses of Inhibitor-Induced Aberrant Multimerization of HIV-1 Integrase
Kessl, Jacques J.; Sharma, Amit; Kvaratskhelia, Mamuka
HIV-1 integrase (IN) is an important therapeutic target as its function is essential for the viral lifecycle. The discovery of multifunctional allosteric IN inhibitors or ALLINIs, which potently impair viral replication by promoting aberrant, higher order IN multimerization as well as inhibit IN interactions with its cellular cofactor, LEDGF/p75, has opened new venues to exploit IN multimerization as a therapeutic target. Furthermore, the recent discovery of multimerization selective IN inhibitors or MINIs, has provided new investigational probes to study the direct effects of aberrant IN multimerization in vitro and in infected cells. Here we describe three complementary methods designed to detect and quantify the effects of these new classes of inhibitors on IN multimerization. These methods include a homogenous time-resolved fluorescence-based assay which allows for measuring EC50 values for the inhibitor-induced aberrant IN multimerization, a dynamic light scattering-based assay which allows for monitoring the formation and sizes of oligomeric IN particles in a time-dependent manner, and a chemical cross-linking-based assay of interacting IN subunits which allows for the determination of IN oligomers in viral particles. PMID:26714710
Identification of bacteriophage virion proteins by the ANOVA feature selection and analysis.
Ding, Hui; Feng, Peng-Mian; Chen, Wei; Lin, Hao
The bacteriophage virion proteins play extremely important roles in the fate of host bacterial cells. Accurate identification of bacteriophage virion proteins is very important for understanding their functions and clarifying the lysis mechanism of bacterial cells. In this study, a new sequence-based method was developed to identify phage virion proteins. In the new method, the protein sequences were initially formulated by the g-gap dipeptide compositions. Subsequently, the analysis of variance (ANOVA) with incremental feature selection (IFS) was used to search for the optimal feature set. It was observed that, in jackknife cross-validation, the optimal feature set including 160 optimized features can produce the maximum accuracy of 85.02%. By performing feature analysis, we found that the correlation between two amino acids with one gap was more important than other correlations for phage virion protein prediction and that some of the 1-gap dipeptides were important and mainly contributed to the virion protein prediction. This analysis will provide novel insights into the function of phage virion proteins. On the basis of the proposed method, an online web-server, PVPred, was established and can be freely accessed from the website (http://lin.uestc.edu.cn/server/PVPred). We believe that the PVPred will become a powerful tool to study phage virion proteins and to guide the related experimental validations.
The vaccinia virus E6 protein influences virion protein localization during virus assembly
Condit, Richard C. Moussatche, Nissin
Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulate in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a "pre-nucleocapsid", and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. - Highlights: • Mutation of E6 disrupts association of viral membranes with viral core proteins • Mutation of E6 does not perturb viral membrane biosynthesis • Mutation of E6 does not perturb localization of viral transcription enzymes • Mutation of E6 causes mis-localization and aggregation of viral core proteins • Vaccinia assembly uses three subassemblies: membranes, viroplasm, prenucleocapsid.
HIV-induced immunosuppression is associated with colonization of the proximal gut by environmental bacteria
Yang, Liying; Poles, Michael A.; Fisch, Gene S; Ma, Yingfei; Nossa, Carlos; Phelan, Joan A; Pei, Zhiheng
Objectives To evaluate the impact of HIV infection on colonization resistance in the proximal gut. Design It was a case-control study. Methods We contrasted microbiota composition between 8 HIV-1-infected patients and 8 HIV-negative controls to characterize community alteration and detect exogenous bacteria in the esophagus, stomach, and duodenum as well as the mouth using a universal 16S rRNA gene survey and correlated the findings with HIV serostatus and peripheral blood T-cell counts. Results HIV infection was associated with an enrichment of Proteobacteria (p=0.020) and depletion of Firmicutes (p=0.005) in the proximal gut. In particular, environmental species Burkholderia fungorum and Bradyrhizobium pachyrhizi colonized the duodenum of HIV patients who had abnormal blood CD4+ T-cell counts but were absent in HIV-negative controls or HIV patients whose CD4 counts were normal. The two species coexisted and exhibited a decreasing trend proximally towards the stomach and esophagus and were virtually absent in the mouth. B. fungorum always outnumbered B. pachyrhizi in a ratio of approximately 15 to 1 regardless of the body sites (p<0.0001, r2=0.965). Their abundance was inversely correlated with CD4 counts (p=0.004) but not viral load. Overgrowth of potential opportunistic pathogens e.g. Prevotella, Fusobacterium, and Ralstonia and depletion of beneficial bacteria, e.g. Lactobacillus were also observed in HIV patients. Conclusions The colonization of the duodenum by environmental bacteria reflects loss of colonization resistance in HIV infection. Their correlation with CD4 counts suggests that compromised immunity could be responsible for the observed invasion by exogenous microbes. PMID:26731752
The Thai phase III trial (RV144) vaccine regimen induces T cell responses that preferentially target epitopes within the V2 region of HIV-1 envelope.
de Souza, Mark S; Ratto-Kim, Silvia; Chuenarom, Weerawan; Schuetz, Alexandra; Chantakulkij, Somsak; Nuntapinit, Bessara; Valencia-Micolta, Anais; Thelian, Doris; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Paris, Robert M; Kaewkungwal, Jaranit; Michael, Nelson L; Rerks-Ngarm, Supachai; Mathieson, Bonnie; Marovich, Mary; Currier, Jeffrey R; Kim, Jerome H
The Thai HIV phase III prime/boost vaccine trial (RV144) using ALVAC-HIV (vCP1521) and AIDSVAX B/E was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT was performed on PBMCs from HIV-1-uninfected vaccine (n = 61) and placebo (n = 10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4(+) T cell-mediated. Responses were targeted within the HIV Env region, with 15 of 25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α(4)β(7) integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19 of 30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4(+) T cells, with the majority of responders producing both IL-2 and IFN-γ (12 of 19; 63%). HIV Env Ab titers were higher in subjects with IL-2 compared with those without IL-2-secreting HIV Env-specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4(+), with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality, and functional cytolytic capacity. Although the RV144 T cell responses were modest in frequency compared with humoral immune responses, the CD4(+) T cell response was directed to HIV-1 Env and more particularly the V2 region.
Virion Structure of Israeli Acute Bee Paralysis Virus
Mullapudi, Edukondalu; Přidal, Antonín; Pálková, Lenka; de Miranda, Joachim R.
ABSTRACT The pollination services provided by the western honeybee (Apis mellifera) are critical for agricultural production and the diversity of wild flowering plants. However, honeybees suffer from environmental pollution, habitat loss, and pathogens, including viruses that can cause fatal diseases. Israeli acute bee paralysis virus (IAPV), from the family Dicistroviridae, has been shown to cause colony collapse disorder in the United States. Here, we present the IAPV virion structure determined to a resolution of 4.0 Å and the structure of a pentamer of capsid protein protomers at a resolution of 2.7 Å. IAPV has major capsid proteins VP1 and VP3 with noncanonical jellyroll β-barrel folds composed of only seven instead of eight β-strands, as is the rule for proteins of other viruses with the same fold. The maturation of dicistroviruses is connected to the cleavage of precursor capsid protein VP0 into subunits VP3 and VP4. We show that a putative catalytic site formed by the residues Asp-Asp-Phe of VP1 is optimally positioned to perform the cleavage. Furthermore, unlike many picornaviruses, IAPV does not contain a hydrophobic pocket in capsid protein VP1 that could be targeted by capsid-binding antiviral compounds. IMPORTANCE Honeybee pollination is required for agricultural production and to sustain the biodiversity of wild flora. However, honeybee populations in Europe and North America are under pressure from pathogens, including viruses that cause colony losses. Viruses from the family Dicistroviridae can cause honeybee infections that are lethal, not only to individual honeybees, but to whole colonies. Here, we present the virion structure of an Aparavirus, Israeli acute bee paralysis virus (IAPV), a member of a complex of closely related viruses that are distributed worldwide. IAPV exhibits unique structural features not observed in other picorna-like viruses. Capsid protein VP1 of IAPV does not contain a hydrophobic pocket, implying that capsid
Moesin is required for HIV-1-induced CD4-CXCR4 interaction, F-actin redistribution, membrane fusion and viral infection in lymphocytes.
Barrero-Villar, Marta; Cabrero, José Román; Gordón-Alonso, Mónica; Barroso-González, Jonathan; Alvarez-Losada, Susana; Muñoz-Fernández, M Angeles; Sánchez-Madrid, Francisco; Valenzuela-Fernández, Agustín
The human immunodeficiency virus 1 (HIV-1) envelope regulates the initial attachment of viral particles to target cells through its association with CD4 and either CXCR4 or CCR5. Although F-actin is required for CD4 and CXCR4 redistribution, little is known about the molecular mechanisms underlying this fundamental process in HIV infection. Using CD4(+) CXCR4(+) permissive human leukemic CEM T cells and primary lymphocytes, we have investigated whether HIV-1 Env might promote viral entry and infection by activating ERM (ezrin-radixin-moesin) proteins to regulate F-actin reorganization and CD4/CXCR4 co-clustering. The interaction of the X4-tropic protein HIV-1 gp120 with CD4 augments ezrin and moesin phosphorylation in human permissive T cells, thereby regulating ezrin-moesin activation. Moreover, the association and clustering of CD4-CXCR4 induced by HIV-1 gp120 requires moesin-mediated anchoring of actin in the plasma membrane. Suppression of moesin expression with dominant-negative N-moesin or specific moesin silencing impedes reorganization of F-actin and HIV-1 entry and infection mediated by the HIV-1 envelope protein complex. Therefore, we propose that activated moesin promotes F-actin redistribution and CD4-CXCR4 clustering and is also required for efficient X4-tropic HIV-1 infection in permissive lymphocytes.
NK cytotoxicity against CD4+ T cells during HIV-1 infection: A gp41 peptide induces the expression of an NKp44 ligand
Vieillard, Vincent; Strominger, Jack L.; Debré, Patrice
HIV infection leads to a state of chronic immune activation and progressive deterioration in immune function, manifested most recognizably by the progressive depletion of CD4+ T cells. A substantial percentage of natural killer (NK) cells from patients with HIV infection are activated and express the natural cytotoxicity receptor (NCR) NKp44. Here we show that a cellular ligand for NKp44 (NKp44L) is expressed during HIV-1 infection and is correlated with both the progression of CD4+ T cell depletion and the increase of viral load. CD4+ T cells expressing this ligand are highly sensitive to the NK lysis activity mediated by NKp44+ NK cells. The expression of NKp44L is induced by the linear motif NH2-SWSNKS-COOH of the HIV-1 envelope gp41 protein. This highly conserved motif appears critical to the sharp increase in NK lysis of CD4+ T cells from HIV-infected patients. These studies strongly suggest that induction of NKp44L plays a key role in the lysis of CD4+ T cells by activated NK cells in HIV infection and consequently provide a framework for considering how HIV-1 may use NK cell immune surveillance to trigger CD4+ T cells. Understanding this mechanism may help to develop future therapeutic strategies and vaccines against HIV-1 infection. PMID:16046540
Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry
Pham, Son; Tabarin, Thibault; Garvey, Megan; Pade, Corinna; Rossy, Jérémie; Monaghan, Paul; Hyatt, Alex; Böcking, Till; Leis, Andrew; Gaus, Katharina; Mak, Johnson
Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the 'lynchpin' for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection. - Highlights: • Cell free viruses are able to receive external trigger that leads to apparent size expansion. • Virus envelope and CD4 receptor engagement is the lynchpin of virus size expansion. • Internal capsid organisation can influence receptor mediated virus size expansion. • Pre-existing virus-associated lipid membrane in cell free virus can accommodate the receptor mediated virus size expansion.
Drug fever induced by piperacillin/tazobactam in an elderly patient with underlying human immunodeficiency virus (HIV) infection.
Swe, Thein; Ali, Mir; Naing, Akari Thein
Our search of the literature revealed no detailed case reports about drug fever induced by piperacillin/tazobactam in a patient with HIV infection although there were a few case reports about drug fever due to piperacillin/tazobactam with other comorbidities. A 63-year-old male patient with HIV positive was admitted for acute cholecystitis. He was started on piperacillin/tazobactam. For the next 8 days, he had intermittent fever up to 103°F (39.4°C) with relative bradycardia although he showed clinical improvement. There was no laboratory or imaging findings suggestive of another infectious source and drug fever was suspected. The antibiotics were stopped and after 48 hours no fever was observed until the day of discharge. Piperacillin/tazobactam can induce fever in patients with cystic fibrosis and in patients with other conditions. Drug fever may be more prevalent in patients with HIV infection. It has no characteristic pattern and may not be associated with eosinophilia.
Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis.
Murray, Catherine L; Jones, Christopher T; Rice, Charles M
Viruses of the Flaviviridae family, including hepatitis C, dengue and bovine viral diarrhoea, are responsible for considerable morbidity and mortality worldwide. Recent advances in our understanding of virion assembly have uncovered commonalities among distantly related members of this family. We discuss the emerging hypothesis that physical virion components are not alone in forming the infectious particle, but that non-structural proteins are intimately involved in orchestrating morphogenesis. Pinpointing the roles of Flaviviridae proteins in virion production could reveal new avenues for antiviral therapeutics.
Vaccine-induced Env V1-V2 IgG3 correlates with lower HIV-1 infection risk and declines soon after vaccination.
Yates, Nicole L; Liao, Hua-Xin; Fong, Youyi; deCamp, Allan; Vandergrift, Nathan A; Williams, William T; Alam, S Munir; Ferrari, Guido; Yang, Zhi-yong; Seaton, Kelly E; Berman, Phillip W; Alpert, Michael D; Evans, David T; O'Connell, Robert J; Francis, Donald; Sinangil, Faruk; Lee, Carter; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Tartaglia, James; Pinter, Abraham; Zolla-Pazner, Susan; Gilbert, Peter B; Nabel, Gary J; Michael, Nelson L; Kim, Jerome H; Montefiori, David C; Haynes, Barton F; Tomaras, Georgia D
HIV-1-specific immunoglobulin G (IgG) subclass antibodies bind to distinct cellular Fc receptors. Antibodies of the same epitope specificity but of a different subclass therefore can have different antibody effector functions. The study of IgG subclass profiles between different vaccine regimens used in clinical trials with divergent efficacy outcomes can provide information on the quality of the vaccine-induced B cell response. We show that HIV-1-specific IgG3 distinguished two HIV-1 vaccine efficacy studies (RV144 and VAX003 clinical trials) and correlated with decreased risk of HIV-1 infection in a blinded follow-up case-control study with the RV144 vaccine. HIV-1-specific IgG3 responses were not long-lived, which was consistent with the waning efficacy of the RV144 vaccine. These data suggest that specific vaccine-induced HIV-1 IgG3 should be tested in future studies of immune correlates in HIV-1 vaccine efficacy trials.
Nef enhances HIV-1 infectivity via association with the virus assembly complex
Qi Mingli; Aiken, Christopher
The HIV-1 accessory protein Nef enhances virus infectivity by facilitating an early post-entry step of infection. Nef acts in the virus producer cell, leading to a beneficial modification to HIV-1 particles. Nef itself is incorporated into HIV-1 particles, where it is cleaved by the viral protease during virion maturation. To probe the role of virion-associated Nef in HIV-1 infection, we generated a fusion protein consisting of the host protein cyclophilin A (CypA) linked to the amino terminus of Nef. The resulting CypA-Nef protein enhanced the infectivity of Nef-defective HIV-1 particles and was specifically incorporated into the virions via association with Gag during particle assembly. Pharmacologic or genetic inhibition of CypA-Nef binding to Gag prevented incorporation of CypA-Nef into virions and inhibited infectivity enhancement. Our results indicate that infectivity enhancement by Nef requires its association with a component of the assembling HIV-1 particle.
Insight into HIV of IFN-Induced Myxovirus Resistance 2 (MX2) Expressed by Traditional Chinese Medicine
Hung, Tzu-Chieh; Lee, Wen-Yuan; Chen, Kuen-Bao; Chan, Yueh-Chiu
Recently, an important topic of the acquired immunodeficiency syndrome (AIDS) had been published in 2013. In this report, the expression of the IFN-induced myxovirus resistance 2 (MX2) had been defined the function to kill the human immunodeficiency virus (HIV). The screening from the Traditional Chinese Medicine (TCM) database by simulating molecular docking and molecular dynamics could select candidate compounds, which may express MX2 against HIV. Saussureamine C, Crotalaburnine, and Precatorine are selected based on the highest docking score and other TCM compounds. The data from molecular dynamics are helpful in the analysis and detection of protein-ligand interactions. According to the docking poses, hydrophobic interactions, and hydrogen bond with structure variations, this research could assess the interaction between protein and ligand interaction. In addition to the detection of TCM compound efficacy, we suggest that Saussureamine C is better than the others in protein-ligand interaction and the structural variation to express MX2. PMID:25045710
HIV-1 Gag Blocks Selenite-Induced Stress Granule Assembly by Altering the mRNA Cap-Binding Complex
Cinti, Alessandro; Le Sage, Valerie; Ghanem, Marwan
ABSTRACT Stress granules (SGs) are dynamic accumulations of stalled preinitiation complexes and translational machinery that assemble under stressful conditions. Sodium selenite (Se) induces the assembly of noncanonical type II SGs that differ in morphology, composition, and mechanism of assembly from canonical SGs. Se inhibits translation initiation by altering the cap-binding activity of eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1). In this work, we show that human immunodeficiency virus type 1 (HIV-1) Gag is able to block the assembly of type II noncanonical SGs to facilitate continued Gag protein synthesis. We demonstrate that expression of Gag reduces the amount of hypophosphorylated 4EBP1 associated with the 5′ cap potentially through an interaction with its target, eIF4E. These results suggest that the assembly of SGs is an important host antiviral defense that HIV-1 has evolved for inhibition through several distinct mechanisms. PMID:27025252
Insight into HIV of IFN-induced myxovirus resistance 2 (MX2) expressed by traditional Chinese medicine.
Hung, Tzu-Chieh; Lee, Wen-Yuan; Chen, Kuen-Bao; Chan, Yueh-Chiu; Chen, Calvin Yu-Chian
Recently, an important topic of the acquired immunodeficiency syndrome (AIDS) had been published in 2013. In this report, the expression of the IFN-induced myxovirus resistance 2 (MX2) had been defined the function to kill the human immunodeficiency virus (HIV). The screening from the Traditional Chinese Medicine (TCM) database by simulating molecular docking and molecular dynamics could select candidate compounds, which may express MX2 against HIV. Saussureamine C, Crotalaburnine, and Precatorine are selected based on the highest docking score and other TCM compounds. The data from molecular dynamics are helpful in the analysis and detection of protein-ligand interactions. According to the docking poses, hydrophobic interactions, and hydrogen bond with structure variations, this research could assess the interaction between protein and ligand interaction. In addition to the detection of TCM compound efficacy, we suggest that Saussureamine C is better than the others in protein-ligand interaction and the structural variation to express MX2.
Quantitative real-time single particle analysis of virions
Heider, Susanne; Metzner, Christoph
Providing information about single virus particles has for a long time been mainly the domain of electron microscopy. More recently, technologies have been developed—or adapted from other fields, such as nanotechnology—to allow for the real-time quantification of physical virion particles, while supplying additional information such as particle diameter concomitantly. These technologies have progressed to the stage of commercialization increasing the speed of viral titer measurements from hours to minutes, thus providing a significant advantage for many aspects of virology research and biotechnology applications. Additional advantages lie in the broad spectrum of virus species that may be measured and the possibility to determine the ratio of infectious to total particles. A series of disadvantages remain associated with these technologies, such as a low specificity for viral particles. In this review we will discuss these technologies by comparing four systems for real-time single virus particle analysis and quantification. - Highlights: • We introduce four methods for virus particle-based quantification of viruses. • They allow for quantification of a wide range of samples in under an hour time. • The additional measurement of size and zeta potential is possible for some.
Novel High-throughput Approach for Purification of Infectious Virions
James, Kevin T.; Cooney, Brad; Agopsowicz, Kate; Trevors, Mary Ann; Mohamed, Adil; Stoltz, Don; Hitt, Mary; Shmulevitz, Maya
Viruses are extensively studied as pathogens and exploited as molecular tools and therapeutic agents. Existing methods to purify viruses such as gradient ultracentrifugation or chromatography have limitations, for example demand for technical expertise or specialized equipment, high time consumption, and restricted capacity. Our laboratory explores mutations in oncolytic reovirus that could improve oncolytic activity, and makes routine use of numerous virus variants, genome reassortants, and reverse engineered mutants. Our research pace was limited by the lack of high-throughput virus purification methods that efficiently remove confounding cellular contaminants such as cytokines and proteases. To overcome this shortcoming, we evaluated a commercially available resin (Capto Core 700) that captures molecules smaller than 700 kDa. Capto. Core 700 chromatography produced virion purity and infectivity indistinguishable from CsCl density gradient ultracentrifugation as determined by electron microscopy, gel electrophoresis analysis and plaque titration. Capto Core 700 resin was then effectively adapted to a rapid in-slurry pull-out approach for high-throughput purification of reovirus and adenovirus. The in-slurry purification approach offered substantially increased virus purity over crude cell lysates, media, or high-spin preparations and would be especially useful for high-throughput virus screening applications where density gradient ultracentrifugation is not feasible. PMID:27827454
Terminal repetitive sequences in herpesvirus saimiri virion DNA.
Bankier, A T; Dietrich, W; Baer, R; Barrell, B G; Colbère-Garapin, F; Fleckenstein, B; Bodemer, W
The H-DNA repeat unit of Herpesvirus saimiri strain 11 was cloned in plasmid vector pAGO, and the nucleotide sequence was determined by the dideoxy chain termination method. One unit of repetitive DNA has 1,444 base pairs with 70.8% G+C content. The structural features of repeat DNA sequences at the termini of intact virion M-DNA (160 kilobases) and orientation of reiterated DNA were analyzed by radioactive end labeling of M-DNA, followed by cleavage of the end fragments with restriction endonucleases. The termini appeared to be blunt ended with a 5'-phosphate group, probably generated during encapsidation by cleavage in the immediate vicinity of the single ApaI recognition site in the H-DNA repeat unit. The sequence did not reveal sizeable open reading frames, the longest hypothetical peptide from H-DNA being 85 amino acids. There was no evidence for an mRNA promoter or terminator element, and H-DNA-specific transcription could not be found in productively infected cells.
Brugia malayi Antigen (BmA) Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells
Mouser, Emily E. I. M.; Pollakis, Georgios; Yazdanbakhsh, Maria; Harnett, William
One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA) and excretory-secretory product 62 (ES-62) from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th) cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs. PMID:26808476
Tat is required for efficient HIV-1 reverse transcription.
Harrich, D; Ulich, C; García-Martínez, L F; Gaynor, R B
The ability of human immunodeficiency virus-1 (HIV-1) to undergo efficient reverse transcription is dependent on a number of parameters. These include the binding of the tRNA(3)(Lys) to the HIV-1 primer binding site and the subsequent interaction with the heterodimeric reverse transcriptase. Recently, we demonstrated that TAR RNA was also necessary for efficient HIV-1 reverse transcription. Given the fact that the Tat protein is involved in the activation of HIV-1 gene expression in conjunction with TAR, we wished to determine whether Tat might also be involved in the control of HIV-1 reverse transcription. HIV-1 virions deleted in the tat gene were unable to initiate reverse transcription efficiently upon infection of peripheral blood mononuclear cells (PBMCs). This defect was not due to decreased amounts of genomic RNA, reverse transcriptase or other HIV-1 proteins which were incorporated into the virion. Following transfection of wild-type but not mutant tat genes into cell lines producing HIV-1 lacking tat, the virions produced could be complemented for defects in reverse transcription upon subsequent infection of PBMCs. In contrast, the defect in reverse transcription seen with HIV-1 lacking the tat gene could not be complemented when the target cells rather than the producer cells contained tat. Viruses lacking tat were also defective in endogenous assays of reverse transcription, although these viruses contained similar levels of reverse transcriptase. These results indicate that the Tat protein, in addition to regulating the level of gene expression, is also important for efficient HIV-1 reverse transcription. PMID:9135139
Intranasal immunization with a formalin-inactivated human influenza A virus whole-virion vaccine alone and intranasal immunization with a split-virion vaccine with mucosal adjuvants show similar levels of cross-protection.
Okamoto, Shigefumi; Matsuoka, Sumiko; Takenaka, Nobuyuki; Haredy, Ahmad M; Tanimoto, Takeshi; Gomi, Yasuyuki; Ishikawa, Toyokazu; Akagi, Takami; Akashi, Mitsuru; Okuno, Yoshinobu; Mori, Yasuko; Yamanishi, Koichi
The antigenicity of seasonal human influenza virus changes continuously; thus, a cross-protective influenza vaccine design needs to be established. Intranasal immunization with an influenza split-virion (SV) vaccine and a mucosal adjuvant induces cross-protection; however, no mucosal adjuvant has been assessed clinically. Formalin-inactivated intact human and avian viruses alone (without adjuvant) induce cross-protection against the highly pathogenic H5N1 avian influenza virus. However, it is unknown whether seasonal human influenza formalin-inactivated whole-virion (WV) vaccine alone induces cross-protection against strains within a subtype or in a different subtype of human influenza virus. Furthermore, there are few reports comparing the cross-protective efficacy of the WV vaccine and SV vaccine-mucosal adjuvant mixtures. Here, we found that the intranasal human influenza WV vaccine alone induced both the innate immune response and acquired immune response, resulting in cross-protection against drift variants within a subtype of human influenza virus. The cross-protective efficacy conferred by the WV vaccine in intranasally immunized mice was almost the same as that conferred by a mixture of SV vaccine and adjuvants. The level of cross-protective efficacy was correlated with the cross-reactive neutralizing antibody titer in the nasal wash and bronchoalveolar fluids. However, neither the SV vaccine with adjuvant nor the WV vaccine induced cross-reactive virus-specific cytotoxic T-lymphocyte activity. These results suggest that the intranasal human WV vaccine injection alone is effective against variants within a virus subtype, mainly through a humoral immune response, and that the cross-protection elicited by the WV vaccine and the SV vaccine plus mucosal adjuvants is similar.
Cyclophilin A is required for the replication of group M human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus SIV(CPZ)GAB but not group O HIV-1 or other primate immunodeficiency viruses.
Braaten, D; Franke, E K; Luban, J
The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein binds to cyclophilin A and incorporates this cellular peptidyl prolyl-isomerase into virions. Disruption of cyclophilin A incorporation, either by gag mutations or by cyclosporine A, inhibits virion infectivity, indicating that cyclophilin A plays an essential role in the HIV-1 life cycle. Using assays for packaging of cyclophilin A into virions and for viral replication sensitivity to cyclosporine A, as well as information gleaned from the alignment of Gag residues encoded by representative viral isolates, we demonstrate that of the five lineages of primate immunodeficiency viruses, only HIV-1 requires cyclophilin A for replication. Cloned viral isolates from clades A, B, and D of HIV-1 group M, as well as a phylogenetically related isolate from chimpanzee, all require cyclophilin A for replication. In contrast, the replication of two outlier (group O) HIV-1 isolates is unaffected by concentrations of cyclosporine A which disrupt cyclophilin A incorporation into virions, indicating that these viruses are capable of replicating independently of cyclophilin A. These studies identify the first phenotypic difference between HIV-1 group M and group O and are consistent with phylogenetic studies suggesting that the two HIV-1 groups were introduced into human populations via separate zoonotic transmission events. PMID:8676442
Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C.
Gómez, Carmen Elena; Perdiguero, Beatriz; Jiménez, Victoria; Filali-Mouhim, Abdelali; Ghneim, Khader; Haddad, Elias K; Quakkelaar, Esther D; Quakkerlaar, Esther D; Delaloye, Julie; Harari, Alexandre; Roger, Thierry; Duhen, Thomas; Dunhen, Thomas; Sékaly, Rafick P; Melief, Cornelis J M; Calandra, Thierry; Sallusto, Federica; Lanzavecchia, Antonio; Wagner, Ralf; Pantaleo, Giuseppe; Esteban, Mariano
Based on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world.
HMBA Enhances Prostratin-Induced Activation of Latent HIV-1 via Suppressing the Expression of Negative Feedback Regulator A20/TNFAIP3 in NF-κB Signaling
Chen, Duchu; Wang, Huiping; Aweya, Jude Juventus; Chen, Yanheng; Chen, Meihua; Wu, Xiaomeng; Chen, Xiaonan; Lu, Jing
In the past decade, much emphasis has been put on the transcriptional activation of HIV-1, which is proposed as a promised strategy for eradicating latent HIV-1 provirus. Two drugs, prostratin and hexamethylene bisacetamide (HMBA), have shown potent effects as inducers for releasing HIV-1 latency when used alone or in combination, although their cellular target(s) are currently not well understood, especially under drug combination. Here, we have shown that HMBA and prostratin synergistically release HIV-1 latency via different mechanisms. While prostratin strongly stimulates HMBA-induced HIV-1 transcription via improved P-TEFb activation, HMBA is capable of boosting NF-κB-dependent transcription initiation by suppressing prostratin-induced expression of the deubiquitinase A20, a negative feedback regulator in the NF-κB signaling pathway. In addition, HMBA was able to increase prostratin-induced phosphorylation and degradation of NF-κB inhibitor IκBα, thereby enhancing and prolonging prostratin-induced nuclear translocation of NF-κB, a prerequisite for stimulation of transcription initiation. Thus, by blocking the negative feedback circuit, HMBA functions as a signaling enhancer of the NF-κB signaling pathway. PMID:27529070
Isolation and preliminary characterization of herpes simplex virus 1 primary enveloped virions from the perinuclear space.
Padula, Maryn E; Sydnor, Mariam L; Wilson, Duncan W
Herpes simplex virus 1 (HSV-1) nucleocapsids exit the nucleus by budding into the inner nuclear membrane, where they exist briefly as primary enveloped virions. These virus particles subsequently fuse their envelopes with the outer nuclear membrane, permitting nucleocapsids to then enter the cytoplasm and complete assembly. We have developed a method to isolate primary enveloped virions from HSV-1-infected cells and subjected the primary enveloped virion preparation to MALDI-MS/MS (matrix-assisted laser desorption ionization-tandem mass spectrometry) analyses. We identified most capsid proteins, a tegument protein (VP22), a glycoprotein (gD), and a cellular protein (annexin A2) in the primary enveloped virion preparation. We determined that annexin A2 does not play an essential role in infection under our experimental conditions. Elucidating the structure and biochemical properties of this unique virus assembly intermediate will provide new insights into HSV-1 biology.
Translational regulation of APOBEC3G mRNA by Vif requires its 5'UTR and contributes to restoring HIV-1 infectivity.
Guerrero, Santiago; Libre, Camille; Batisse, Julien; Mercenne, Gaëlle; Richer, Delphine; Laumond, Géraldine; Decoville, Thomas; Moog, Christiane; Marquet, Roland; Paillart, Jean-Christophe
The essential HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells expressing cytidine deaminases APOBEC3G (A3G) and A3F by decreasing their cellular level, and preventing their incorporation into virions. Unlike the Vif-induced degradation of A3G, the functional role of the inhibition of A3G translation by Vif remained unclear. Here, we show that two stem-loop structures within the 5'-untranslated region of A3G mRNA are crucial for translation inhibition by Vif in cells, and most Vif alleles neutralize A3G translation efficiently. Interestingly, K26R mutation in Vif abolishes degradation of A3G by the proteasome but has no effect at the translational level, indicating these two pathways are independent. These two mechanisms, proteasomal degradation and translational inhibition, similarly contribute to decrease the cellular level of A3G by Vif and to prevent its incorporation into virions. Importantly, inhibition of A3G translation is sufficient to partially restore viral infectivity in the absence of proteosomal degradation. These findings demonstrate that HIV-1 has evolved redundant mechanisms to specifically inhibit the potent antiviral activity of A3G.
Translational regulation of APOBEC3G mRNA by Vif requires its 5′UTR and contributes to restoring HIV-1 infectivity
Guerrero, Santiago; Libre, Camille; Batisse, Julien; Mercenne, Gaëlle; Richer, Delphine; Laumond, Géraldine; Decoville, Thomas; Moog, Christiane; Marquet, Roland; Paillart, Jean-Christophe
The essential HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells expressing cytidine deaminases APOBEC3G (A3G) and A3F by decreasing their cellular level, and preventing their incorporation into virions. Unlike the Vif-induced degradation of A3G, the functional role of the inhibition of A3G translation by Vif remained unclear. Here, we show that two stem-loop structures within the 5′-untranslated region of A3G mRNA are crucial for translation inhibition by Vif in cells, and most Vif alleles neutralize A3G translation efficiently. Interestingly, K26R mutation in Vif abolishes degradation of A3G by the proteasome but has no effect at the translational level, indicating these two pathways are independent. These two mechanisms, proteasomal degradation and translational inhibition, similarly contribute to decrease the cellular level of A3G by Vif and to prevent its incorporation into virions. Importantly, inhibition of A3G translation is sufficient to partially restore viral infectivity in the absence of proteosomal degradation. These findings demonstrate that HIV-1 has evolved redundant mechanisms to specifically inhibit the potent antiviral activity of A3G. PMID:27996044
HIV-1 Vpu targets cell surface markers CD4 and BST-2 through distinct mechanisms
Andrew, Amy; Strebel, Klaus
Vpu is a small integral membrane protein encoded by HIV-1 and some SIV isolates. The protein is known to induce degradation of the viral receptor molecule CD4 and to enhance the release of newly formed virions from the cell surface. Vpu accomplishes these two functions through two distinct mechanisms. In the case of CD4, Vpu acts as a molecular adaptor to connect CD4 to an E3 ubiquitin ligase complex resulting in CD4 degradation by cellular proteasomes. This requires signals located in Vpu's cytoplasmic domain. Enhancement of virus release on the other hand involves the neutralization of a cellular host factor, BST-2 (also known as CD317, HM1.24, or tetherin) and requires Vpu's TM domain. The current review discusses recent advances on the role of Vpu in controlling degradation of CD4 and in regulating virus release. PMID:20858517
Triterpene Constituents of Euphorbia Erythradenia Bioss. and their Anti-HIV Activity
Ayatollahi, Abdul Majid; Zarei, Seyed Mohammad; Memarnejadian, Arash; Ghanadian, Mustafa; Heydarian Moghadam, Mohammad; Kobarfard, Farzad
Phytochemical investigation of the aerial parts of Euphorbia erythradenia Bioss. (Euphorbiaceae), one of Iranian endemic Euphorbias, with particular attention to triterpene constituents, using methanol solvent extraction was carried out. Five known triterpenes, including four cycloartanes and oleanolic acid, were isolated for the first time and identified using NMR and Mass techniques. Anti HIV activity of the isolated triterpenes and ingenoid diterpenes was evaluated using single cycle replicable HIV-1 (SCR HIV-1) virions. Molecular features of the most active compound (IC50 = 0.008 μM, CC50 = 3.264 μM, TI = 380.64), which showed higher therapeutic index than nevirapine, was assessed using molecular docking. Docking studies demonstrated three hydrogen bonds between HIV-1 virion protease active site and this compound with a distance less than 3 A° which can be responsible for the observed anti HIV-1 activity. PMID:28228800
All eyes on the next generation of HIV vaccines: strategies for inducing a broadly neutralizing antibody response.
Ahlers, Jeffrey D
HIV-1 broadly neutralizing antibodies (BNAbs) develop after several years of infection through a recursive process of memory B cell adaptation and maturation against co-evolving virus quasispecies. Advances in single-cell sorting and memory B cell antibody cloning methods have identified many new HIV BNAbs targeting conserved epitopes on the HIV envelope (env) protein. 3D crystal structures and biophysical analyses of BNAbs bound to invariant virus structures expressed on monomeric gp120, epitope scaffolds, core structures, and native trimers have helped us to visualize unique binding interactions and paratope orientations that have been instrumental in guiding vaccine design. A paradigm shift in the approach to structure-based design of HIV-1 envelope immunogens came recently after several laboratories discovered that native viral envelopes or "env-structures" reverse-engineered to bind with high affinity to a handful of broadly neutralizing antibodies did not in fact bind the predicted germline precursors of these broadly neutralizing antibodies. A major challenge for HIV-1 B cell vaccine development moving forward is the design of new envelope immunogens that can trigger the selection and expansion of germline precursor and intermediate memory B cells to recapitulate B cell ontogenies associated with the maturation of a broadly neutralizing antibody response. Equally important for vaccine development is the identification of delivery systems, prime-boost strategies, and synergistic adjuvant combinations that can induce the magnitude and quality of antigen-specific T follicular helper (TFH) cell responses needed to drive somatic hypermutation (SHM) and B cell maturation against heterologous primary virus envelopes. Finding the combination of multi-protein envelope immunogens and immunization strategies that can evolve a potent broadly neutralizing antibody response portends to require a complex vaccine regimen that might be difficult to implement on any scale
Nelfinavir, an HIV-1 Protease Inhibitor, Induces Oxidative Stress–Mediated, Caspase-Independent Apoptosis in Leishmania Amastigotes
Kumar, Pranav; Lodge, Robert; Trudel, Nathalie; Ouellet, Michel; Ouellette, Marc; Tremblay, Michel J.
Background Visceral leishmaniasis has now emerged as an important opportunistic disease in patients coinfected with human immunodeficiency virus type-1 (HIV-1). Although the effectiveness of HIV-1 protease inhibitors, such as nelfinavir, in antiretroviral therapies is well documented, little is known of the impact of these drugs on Leishmania in coinfected individuals. Methodology and Principal Findings Here, we show that nelfinavir generates oxidative stress in the parasite, leading to altered physiological parameters such as an increase in the sub-G1 DNA content, nuclear DNA fragmentation and loss of mitochondrial potential, which are all characteristics of apoptosis. Pretreatment of axenic amastigotes with the caspase inhibitor z-VAD-fmk did not inhibit the increase in sub-G1 DNA content in nelfinavir-treated parasites, suggesting therefore that this antiviral agent does not kill Leishmania amastigotes in a caspase-dependent manner. Furthermore, we observed that the mitochondrial resident protein endonuclease G is involved. We also demonstrate that parasites overexpressing GSH1 (the rate limiting enzyme of glutathione biosynthesis) were more resistant to nelfinavir when compared to untransfected controls. Conclusions and Significance These data suggest that nelfinavir induces oxidative stress in Leishmania amastigotes, culminating in caspase-independent apoptosis, in which DNA is degraded by endonuclease G. This study provides a rationale for future, long-term design of new therapeutic strategies to test nelfinavir as a potential antileishmanial agent as well as for possible future use in Leishmania/HIV-1 coinfections. PMID:20361030
An inducible packaging cell system for safe, efficient lentiviral vector production in the absence of HIV-1 accessory proteins.
Pacchia, A L; Adelson, M E; Kaul, M; Ron, Y; Dougherty, J P
Lentiviral vectors based on human immunodeficiency virus type 1 (HIV-1) possess the ability to deliver exogenous genes to both dividing and nondividing cells and to subsequently establish a stable provirus in these target cells, which can allow long-term expression of the transferred gene. Herein we describe a stable packaging cell line that is devoid of HIV-1 tat, vif, vpr, vpu, and nef. In order to avoid any risk of cytotoxicity associated with constitutive expression of HIV-1 protease or the VSV-G envelope protein, transcription of the packaging and envelope constructs was tightly controlled by employing the ecdysone-inducible system. Using this cell line, we have been able to consistently generate concentrated pseudotyped vector virus stocks with titers in the range of 10(8) IU/ml, which can efficiently transduce actively dividing and growth-arrested cells in vitro. This novel packaging cell line for lentiviral vectors facilitates the production of high-titer virus stocks in the absence of replication-competent virus and provides us with an important tool for use in future gene transfer studies.
Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly
Background The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6. Results Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site. Conclusions Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and
Inhibition of HIV-1 Maturation via Small Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein.
Wang, Weifeng; Zhou, Jing; Halambage, Upul D; Jurado, Kellie A; Jamin, Augusta V; Wang, Yujie; Engelman, Alan N; Aiken, Christopher
The HIV-1 capsid protein is an attractive therapeutic target owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsid to HIV-1 infectivity. To date, small molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, BI compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor Bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant crosslinks in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle.IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here we show that one such compound, Compound 1, interferes with assembly of the conical viral capsid during virion maturation, and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a
Ribonucleases in HIV type 1 inhibition: effect of recombinant RNases on infection of primary T cells and immune activation-induced RNase gene and protein expression.
Bedoya, Victoria I; Boasso, Adriano; Hardy, Andrew W; Rybak, Susanna; Shearer, Gene M; Rugeles, Maria T
Ribonucleases (RNases) have therapeutic potential against cancer and viral diseases and have been reported to inhibit replication of the human immunodeficiency virus type 1 (HIV-1) in chronically infected cell lines. The ribonuclease eosinophil-derived neurotoxin (EDN) is responsible for the anti-HIV-1 activity of a soluble factor produced in response to human alloantigens (ASF). Four recombinant RNases (EDN; a four amino acid extension of the N-terminus EDN, -4EDN; RNase A; and angiogenin) were tested for inhibition of HIV-1 replication in PHA blasts. All RNases showed anti-HIV-1 activity, irrespective of whether the RNases were added before, during, or 2 h after infection. Polyclonal antibodies against the four RNases blocked the antiviral activity. ASF inhibited HIV-1 replication in vitro if added up to 4 h after infection. We demonstrated that allostimulation induced EDN, RNase A, and angiogenin mRNA expression in peripheral blood mononuclear cells (PBMCs), although only EDN protein was detected. We identified monocytes and dendritic cells, but not macrophages or T cells, as EDN-producing cells. These findings raise the possibilities that multiple naturally occurring RNases may contribute to protection against HIV-1 infection and could be considered for utilization in HIV-1 therapy.
Three-Year Durability of Immune Responses Induced by HIV-DNA and HIV-Modified Vaccinia Virus Ankara and Effect of a Late HIV-Modified Vaccinia Virus Ankara Boost in Tanzanian Volunteers.
Joachim, Agricola; Munseri, Patricia J; Nilsson, Charlotta; Bakari, Muhammad; Aboud, Said; Lyamuya, Eligius F; Tecleab, Teghesti; Liakina, Valentina; Scarlatti, Gabriella; Robb, Merlin L; Earl, Patricia L; Moss, Bernard; Wahren, Britta; Mhalu, Fred; Ferrari, Guido; Sandstrom, Eric; Biberfeld, Gunnel
We explored the duration of immune responses and the effect of a late third HIV-modified vaccinia virus Ankara (MVA) boost in HIV-DNA primed and HIV-MVA boosted Tanzanian volunteers. Twenty volunteers who had previously received three HIV-DNA and two HIV-MVA immunizations were given a third HIV-MVA immunization 3 years after the second HIV-MVA boost. At the time of the third HIV-MVA, 90% of the vaccinees had antibodies to HIV-1 subtype C gp140 (median titer 200) and 85% to subtype B gp160 (median titer 100). The majority of vaccinees had detectable antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, 70% against CRF01_AE virus-infected cells (median titer 239) and 84% against CRF01_AE gp120-coated cells (median titer 499). A high proportion (74%) of vaccinees had IFN-γ ELISpot responses, 63% to Gag and 42% to Env, 3 years after the second HIV-MVA boost. After the third HIV-MVA, there was an increase in Env-binding antibodies and ADCC-mediating antibodies relative to the response seen at the time of the third HIV-MVA vaccination, p < .0001 and p < .05, respectively. The frequency of IFN-γ ELISpot responses increased to 95% against Gag or Env and 90% to both Gag and Env, p = .064 and p = .002, respectively. In conclusion, the HIV-DNA prime/HIV-MVA boost regimen elicited potent antibody and cellular immune responses with remarkable durability, and a third HIV-MVA immunization significantly boosted both antibody and cellular immune responses relative to the levels detected at the time of the third HIV-MVA, but not to higher levels than after the second HIV-MVA.
Herpes simplex virus 1 counteracts tetherin restriction via its virion host shutoff activity.
Zenner, Helen L; Mauricio, Rui; Banting, George; Crump, Colin M
The interferon-inducible membrane protein tetherin (Bst-2, or CD317) is an antiviral factor that inhibits enveloped virus release by cross-linking newly formed virus particles to the producing cell. The majority of viruses that are sensitive to tetherin restriction appear to be those that acquire their envelopes at the plasma membrane, although many viruses, including herpesviruses, envelope at intracellular membranes, and the effect of tetherin on such viruses has been less well studied. We investigated the tetherin sensitivity and possible countermeasures of herpes simplex virus 1 (HSV-1). We found that overexpression of tetherin inhibits HSV-1 release and that HSV-1 efficiently depletes tetherin from infected cells. We further show that the virion host shutoff protein (Vhs) is important for depletion of tetherin mRNA and protein and that removal of tetherin compensates for defects in replication and release of a Vhs-null virus. Vhs is known to be important for HSV-1 to evade the innate immune response in vivo. Taken together, our data suggest that tetherin has antiviral activity toward HSV-1 and that the removal of tetherin by Vhs is important for the efficient replication and dissemination of HSV-1.
Are Basophils and Mast Cells Masters in HIV Infection?
Marone, Gianni; Varricchi, Gilda; Loffredo, Stefania; Galdiero, Maria Rosaria; Rivellese, Felice; de Paulis, Amato
The World Health Organization AIDS epidemic update estimates that more than 37 million people are living with HIV infection. Despite the unprecedented success of antiretroviral treatments, significant challenges remain in the fight against HIV. In particular, how uninfected cells capture HIV and transmit virions to target cells remains an unanswered question. Tissue mast cells and peripheral blood basophils can be exposed to virions or HIV products during infection. Several HIV proteins (i.e., envelope glycoproteins gp120 and gp41, Tat, and Nef) can interact with distinct surface receptors expressed by human basophils and mast cells and modulate their functional responses at different levels. Additionally, several groups have provided evidence that human mast cells can be infected in vitro, as well as in vivo, by certain strains of HIV. Recently, it has been demonstrated that basophils purified from healthy donors and intestinal mast cells can efficiently capture HIV on their cell surface and, cocultured with CD4+ T cells, they can transfer the virus to the cocultured cells leading to infection. Direct contact between human basophils or intestinal mast cells and CD4+ T cells can mediate viral trans-infection of T cells through the formation of viral synapses. Thus, basophils and mast cells can provide a cellular basis for capturing and then spreading viruses throughout the body. Collectively, these findings suggest that human basophils and mast cells play a complex and possibly distinct role in HIV infection, warranting further investigations.
3D visualization of HIV transfer at the virological synapse between dendritic cells and T cells
Felts, Richard L.; Narayan, Kedar; Estes, Jacob D.; Shi, Dan; Trubey, Charles M.; Fu, Jing; Hartnell, Lisa M.; Ruthel, Gordon T.; Schneider, Douglas K.; Nagashima, Kunio; Bess, Julian W.; Bavari, Sina; Lowekamp, Bradley C.; Bliss, Donald; Lifson, Jeffrey D.; Subramaniam, Sriram
The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4+ T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4+ T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer by T cells highlight unique aspects of cell-cell HIV transmission. PMID:20624966
Multiplex RT-PCR Amplification of HIV Genes to Create a Completely Autologous DC-Based Immunotherapy for the Treatment of HIV Infection
Tcherepanova, Irina; Harris, Jason; Starr, Aijing; Cleveland, Jaclyn; Ketteringham, Helen; Calderhead, David; Horvatinovich, Joe; Healey, Don; Nicolette, Charles A.
Background Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC)-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. Methodology/Principal Findings The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. To resolve this problem we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. Conclusion/Significance This study describes a strategy to overcome patient to patient viral diversity enabling strain-independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce effective control of viral
Human cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone BiP/GRP78, which is required for virion assembly.
Buchkovich, Nicholas J; Maguire, Tobi G; Yu, Yongjun; Paton, Adrienne W; Paton, James C; Alwine, James C
The endoplasmic reticulum (ER) chaperone BiP/GRP78 regulates ER function and the unfolded protein response (UPR). Human cytomegalovirus infection of human fibroblasts induces the UPR but modifies it to benefit viral replication. BiP/GRP78 protein levels are tightly regulated during infection, rising after 36 h postinfection (hpi), peaking at 60 hpi, and decreasing thereafter. To determine the effects of this regulation on viral replication, BiP/GRP78 was depleted using the SubAB subtilase cytotoxin, which rapidly and specifically cleaves BiP/GRP78. Toxin treatment of infected cells for 12-h periods beginning at 36, 48, 60, and 84 hpi caused complete loss of BiP but had little effect on viral protein synthesis. However, progeny virion formation was significantly inhibited, suggesting that BiP/GRP78 is important for virion formation. Electron microscopic analysis showed that infected cells were resistant to the toxin and showed none of the cytotoxic effects seen in uninfected cells. However, all viral activity in the cytoplasm ceased, with nucleocapsids remaining in the nucleus or concentrated in the cytoplasmic space just outside of the outer nuclear membrane. These data suggest that one effect of the controlled expression of BiP/GRP78 in infected cells is to aid in cytoplasmic virion assembly and egress.
[A novel immunization strategy to induce strong humoral responses against HIV-1 using combined DNA, recombinant vaccinia virus and protein vaccines].
Liu, Chang; Wang, Shu-hui; Ren, Li; Hao, Yan-ling; Zhang, Qi-cheng; Liu, Ying
To optimize the immunization strategy against HIV-1, a DNA vaccine was combined with a recombinant vaccinia virus (rTV) vaccine and a protein vaccine. Immune responses against HIV-1 were detected in 30 female guinea pigs divided into six groups. Three groups of guinea pigs were primed with HIV-1 DNA vaccine three times, boosted with rTV at week 14, and then boosted with gp140 protein at intervals of 4, 8 or 12 weeks. Simultaneously, the other three groups of animals were primed with rTV vaccine once, and then boosted with gp140 after 4, 8 or 12 weeks. The HIV-1 specific binding antibody and neutralizing antibody, in addition to the relative affinity of these antibodies, were detected at different time points after the final administration of vaccine in each group. The DNA-rTV-gp140 immune regimen induced higher titers and affinity levels of HIV-1 gp120/gp140 antibodies and stronger V1V2-gp70 antibodies than the rTV-gp140 regimen. In the guinea pigs that underwent the DNA-rTV-gp140 regimen, the highest V1V2-gp70 antibody was induced in the 12-week-interval group. However, the avidity of antibodies was improved in the 4-week-interval group. Using the rTV-gp140 immunization strategy, guinea pigs boosted at 8 or 12 weeks after rTV priming elicited stronger humoral responses than those boosted at 4 weeks after priming. In conclusion, this study shows that the immunization strategy of HIV-1 DNA vaccine priming, followed by rTV and protein vaccine boosting, could strengthen the humoral response against HIV-1. Longer intervals were better to induce V1V2-gp70-specific antibodies, while shorter intervals were more beneficial to enhance the avidity of antibodies.
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Piscine reovirus encodes a cytotoxic, non-fusogenic, integral membrane protein and previously unrecognized virion outer-capsid proteins.
Key, Tim; Read, Jolene; Nibert, Max L; Duncan, Roy
Piscine reovirus (PRV) is a tentative new member of the family Reoviridae and has been linked to heart and skeletal muscle inflammation in farmed Atlantic salmon (Salmo salar L.). Recent sequence-based evidence suggests that PRV is about equally related to members of the genera Orthoreovirus and Aquareovirus. Sequence similarities have also suggested that PRV might encode a fusion-associated small transmembrane (FAST) protein, which in turn suggests that PRV might be the prototype of a new genus with syncytium-inducing potential. In previous support of this designation has been the absence of identifiable PRV-encoded homologues of either the virion outer-clamp protein of ortho- and aquareoviruses or the virion outer-fibre protein of most orthoreoviruses. In the current report, we have provided experimental evidence that the putative p13 FAST protein of PRV lacks the defining feature of the FAST protein family - the ability to induce syncytium formation. Instead, p13 is the first example of a cytosolic, integral membrane protein encoded by ortho- or aquareoviruses, and induces cytotoxicity in the absence of cell-cell fusion. Sequence analysis also identified signature motifs of the outer-clamp and outer-fibre proteins of other reoviruses in two of the predicted PRV gene products. Based on these findings, we conclude that PRV does not encode a FAST protein and is therefore unlikely to be a new fusogenic reovirus. The presence of a novel integral membrane protein and two previously unrecognized, essential outer-capsid proteins has important implications for the biology, evolution and taxonomic classification of this virus.
HIV-1 Reservoir Dynamics after Vaccination and Antiretroviral Therapy Interruption Are Associated with Dendritic Cell Vaccine-Induced T Cell Responses
Andrés, Cristina; Plana, Montserrat; Guardo, Alberto C.; Alvarez-Fernández, Carmen; Climent, Nuria; Gallart, Teresa; León, Agathe; Clotet, Bonaventura; Autran, Brigitte; Chomont, Nicolas; Gatell, Josep M.; Sánchez-Palomino, Sonsoles
ABSTRACT HIV-1-specific immune responses induced by a dendritic cell (DC)-based therapeutic vaccine might have some effect on the viral reservoir. Patients on combination antiretroviral therapy (cART) were randomized to receive DCs pulsed with autologous HIV-1 (n = 24) (DC-HIV-1) or nonpulsed DCs (n = 12) (DC-control). We measured the levels of total and integrated HIV-1 DNA in CD4 T cells isolated from these patients at 6 time points: before any cART; before the first cART interruption, which was at 56 weeks before the first immunization to isolate virus for pulsing DCs; before and after vaccinations (VAC1 and VAC2); and at weeks 12 and 48 after the second cART interruption. The vaccinations did not influence HIV-1 DNA levels in vaccinated subjects. After the cART interruption at week 12 postvaccination, while total HIV-1 DNA increased significantly in both arms, integrated HIV-1 DNA did not change in vaccinees (mean of 1.8 log10 to 1.9 copies/106 CD4 T cells, P = 0.22) and did increase in controls (mean of 1.8 log10 to 2.1 copies/106 CD4 T cells, P = 0.02) (P = 0.03 for the difference between groups). However, this lack of increase of integrated HIV-1 DNA observed in the DC-HIV-1 group was transient, and at week 48 after cART interruption, no differences were observed between the groups. The HIV-1-specific T cell responses at the VAC2 time point were inversely correlated with the total and integrated HIV-1 DNA levels after cART interruption in vaccinees (r [Pearson's correlation coefficient] = −0.69, P = 0.002, and r = −0.82, P < 0.0001, respectively). No correlations were found in controls. HIV-1-specific T cell immune responses elicited by DC therapeutic vaccines drive changes in HIV-1 DNA after vaccination and cART interruption. (This study has been registered at ClinicalTrials.gov under registration no. NCT00402142.) IMPORTANCE There is an intense interest in developing strategies to target HIV-1 reservoirs as they create barriers to curing the disease
Enzymatic treatment of duck hepatitis B virus: Topology of the surface proteins for virions and noninfectious subviral particles
Franke, Claudia; Matschl, Urte; Bruns, Michael . E-mail: email@example.com
The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles' interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: 1-preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was accessible, 2-phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions, 3-iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions.
Fullerene Derivatives Strongly Inhibit HIV-1 Replication by Affecting Virus Maturation without Impairing Protease Activity
Martinez, Zachary S.; Castro, Edison; Seong, Chang-Soo; Cerón, Maira R.
Three compounds (1, 2, and 3) previously reported to inhibit HIV-1 replication and/or in vitro activity of reverse transcriptase were studied, but only fullerene derivatives 1 and 2 showed strong antiviral activity on the replication of HIV-1 in human CD4+ T cells. However, these compounds did not inhibit infection by single-round infection vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped viruses, indicating no effect on the early steps of the viral life cycle. In contrast, analysis of single-round infection VSV-G-pseudotyped HIV-1 produced in the presence of compound 1 or 2 showed a complete lack of infectivity in human CD4+ T cells, suggesting that the late stages of the HIV-1 life cycle were affected. Quantification of virion-associated viral RNA and p24 indicates that RNA packaging and viral production were unremarkable in these viruses. However, Gag and Gag-Pol processing was affected, as evidenced by immunoblot analysis with an anti-p24 antibody and the measurement of virion-associated reverse transcriptase activity, ratifying the effect of the fullerene derivatives on virion maturation of the HIV-1 life cycle. Surprisingly, fullerenes 1 and 2 did not inhibit HIV-1 protease in an in vitro assay at the doses that potently blocked viral infectivity, suggesting a protease-independent mechanism of action. Highlighting the potential therapeutic relevance of fullerene derivatives, these compounds block infection by HIV-1 resistant to protease and maturation inhibitors. PMID:27431232
Intranasal immunization of young mice with a multigene HIV-1 vaccine in combination with the N3 adjuvant induces mucosal and systemic immune responses.
Bråve, Andreas; Hallengärd, David; Schröder, Ulf; Blomberg, Pontus; Wahren, Britta; Hinkula, Jorma
One of the major challenges for the development of an HIV vaccine is to induce potent virus-specific immune responses at the mucosal surfaces where transmission of virus occurs. Intranasal delivery of classical vaccines has been shown to induce good mucosal antibody responses, but so far for genetic vaccines the success has been limited. This study shows that young individuals are sensitive to nasal immunization with a genetic vaccine delivered in a formulation of a lipid adjuvant, the Eurocine N3. Intranasal delivery of a multiclade/multigene HIV-1 genetic vaccine gave rise to vaginal and rectal IgA responses as well as systemic humoral and cellular responses. As electroporation might become the preferred means of delivering genetic vaccines for systemic HIV immunity, nasal delivery by droplet formulation in a lipid adjuvant might become a means of priming or boosting the mucosal immunity.
RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions
Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K.; James, John; Prescott, Alan; Haga, Ismar R.; Beard, Philippa M.
Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.
Virion endocytosis is a major target for murid herpesvirus-4 neutralization
Glauser, Daniel L.; Gillet, Laurent
Herpesviruses consistently transmit from immunocompetent carriers, implying that their neutralization is hard to achieve. Murid herpesvirus-4 (MuHV-4) exploits host IgG Fc receptors to bypass blocks to cell binding, and pH-dependent protein conformation changes to unveil its fusion machinery only after endocytosis. Nevertheless, neutralization remains possible by targeting the virion glycoprotein H (gH)–gL heterodimer, and the neutralizing antibody responses of MuHV-4 carriers are improved by boosting with recombinant gH–gL. We analysed here how gH–gL-directed neutralization works. The MuHV-4 gH–gL binds to heparan sulfate. However, most gH–gL-specific neutralizing antibodies did not block this interaction; neither did they act directly on fusion. Instead, they blocked virion endocytosis and transport to the late endosomes, where membrane fusion normally occurs. The poor endocytosis of gH–gL-neutralized virions was recapitulated precisely by virions genetically lacking gL. Therefore, driving virion uptake appears to be an important function of gH–gL that provides a major target for antibody-mediated neutralization. PMID:22377583
Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis
Lussignol, Marion; Kopp, Martina; Molloy, Kelly; Vizcay-Barrena, Gema; Fleck, Roland A.; Bell, Kierstin L.; Chait, Brian T.; Rice, Charles M.; Catanese, Maria Teresa
Hepatitis C virus (HCV) is a unique enveloped virus that assembles as a hybrid lipoviral particle by tightly interacting with host lipoproteins. As a result, HCV virions display a characteristic low buoyant density and a deceiving coat, with host-derived apolipoproteins masking viral epitopes. We previously described methods to produce high-titer preparations of HCV particles with tagged envelope glycoproteins that enabled ultrastructural analysis of affinity-purified virions. Here, we performed proteomics studies of HCV isolated from culture media of infected hepatoma cells to define viral and host-encoded proteins associated with mature virions. Using two different affinity purification protocols, we detected four viral and 46 human cellular proteins specifically copurifying with extracellular HCV virions. We determined the C terminus of the mature capsid protein and reproducibly detected low levels of the viral nonstructural protein, NS3. Functional characterization of virion-associated host factors by RNAi identified cellular proteins with either proviral or antiviral roles. In particular, we discovered a novel interaction between HCV capsid protein and the nucleoporin Nup98 at cytosolic lipid droplets that is important for HCV propagation. These results provide the first comprehensive view to our knowledge of the protein composition of HCV and new insights into the complex virus–host interactions underlying HCV infection. PMID:26884193
Adenovirus vector vaccination induces expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1
Benlahrech, Adel; Harris, Julian; Meiser, Andrea; Papagatsias, Timos; Hornig, Julia; Hayes, Peter; Lieber, Andre; Athanasopoulos, Takis; Bachy, Veronique; Csomor, Eszter; Daniels, Rod; Fisher, Kerry; Gotch, Frances; Seymour, Len; Logan, Karen; Barbagallo, Romina; Klavinskis, Linda; Dickson, George; Patterson, Steven
In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memory CD4 T cells. To test this hypothesis, Ad5 and Ad11 antibody titers were measured in 20 healthy volunteers. Dendritic cells (DCs) from these individuals were pulsed with replication defective Ad5 or Ad11 and co-cultured with autologous lymphocytes. Cytokine profiles, proliferative capacity, mucosal migration potential, and susceptibility to HIV infection of the adenovirus-stimulated memory CD4 T cells were measured. Stimulation of T cells from healthy Ad5-seropositive but Ad11-seronegative individuals with Ad5, or serologically distinct Ad11 vectors induced preferential expansion of adenovirus memory CD4 T cells expressing α4β7 integrins and CCR9, indicating a mucosal-homing phenotype. CD4 T-cell proliferation and IFN-γ production in response to Ad stimulation correlated with Ad5 antibody titers. However, Ad5 serostatus did not correlate with total cytokine production upon challenge with Ad5 or Ad11. Expanded Ad5 and Ad11 memory CD4 T cells showed an increase in CCR5 expression and higher susceptibility to infection by R5 tropic HIV-1. This suggests that adenoviral-based vaccination against HIV-1 in individuals with preexisting immunity against Ad5 results in preferential expansion of HIV-susceptible activated CD4 T cells that home to mucosal tissues, increases the number of virus targets, and leads to a higher susceptibility to HIV acquisition. PMID:19918060
HSV vector-mediated GAD67 suppresses neuropathic pain induced by perineural HIV gp120 in rats through inhibition of ROS and Wnt5a
Kanda, Hirotsugu; Kanao, Megumi; Liu, Shue; Yi, Hyun; Iida, Takafumi; Levitt, Roy C; Candiotti, Keith A; Lubarsky, David A; Hao, Shuanglin
Human immunodeficiency virus (HIV)-related neuropathic pain is a debilitating chronic condition that is severe and unrelenting. Despite the extensive research, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments. Loss of GABAergic tone may play an important role in the neuropathic pain state. Glutamic acid decarboxylase 67 (GAD67) is one of isoforms that catalyze GABA synthesis. Here, we used recombinant herpes simplex virus (HSV-1) vectors that encode gad1 gene to evaluate the therapeutic potential of GAD67 in peripheral HIV gp120-induced neuropathic pain in rats. We found that 1) subcutaneous inoculation of the HSV vectors expressing GAD67 attenuated mechanical allodynia in the model of HIV gp120-induced neuropathic pain, 2) the anti-allodynic effect of GAD67 was reduced by GABA-A and-B receptors antagonists, 3) HSV vectors expressing GAD67 reversed the lowered GABA-IR expression, and 4) the HSV vectors expressing GAD67 suppressed the upregulated mitochondrial superoxide and Wnt5a in the spinal dorsal horn. Taken together, our studies support the concept that recovering GABAergic tone by the HSV vectors may reverse HIV-associated neuropathic pain through suppressing mitochondrial superoxide and Wnt5a. Our studies provide validation of HSV-mediated GAD67 gene therapy in the treatment of HIV-related neuropathic pain. PMID:26752351
HSV vector-mediated GAD67 suppresses neuropathic pain induced by perineural HIV gp120 in rats through inhibition of ROS and Wnt5a.
Kanda, H; Kanao, M; Liu, S; Yi, H; Iida, T; Levitt, R C; Candiotti, K A; Lubarsky, D A; Hao, S
Human immunodeficiency virus (HIV)-related neuropathic pain is a debilitating chronic condition that is severe and unrelenting. Despite the extensive research, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments. Loss of GABAergic tone may have an important role in the neuropathic pain state. Glutamic acid decarboxylase 67 (GAD67) is one of the isoforms that catalyze GABA synthesis. Here, we used recombinant herpes simplex virus (HSV-1) vectors that encode gad1 gene to evaluate the therapeutic potential of GAD67 in peripheral HIV gp120-induced neuropathic pain in rats. We found that (1) subcutaneous inoculation of the HSV vectors expressing GAD67 attenuated mechanical allodynia in the model of HIV gp120-induced neuropathic pain, (2) the anti-allodynic effect of GAD67 was reduced by GABA-A and-B receptors antagonists, (3) HSV vectors expressing GAD67 reversed the lowered GABA-IR expression and (4) the HSV vectors expressing GAD67 suppressed the upregulated mitochondrial superoxide and Wnt5a in the spinal dorsal horn. Taken together, our studies support the concept that recovering GABAergic tone by the HSV vectors may reverse HIV-associated neuropathic pain through suppressing mitochondrial superoxide and Wnt5a. Our studies provide validation of HSV-mediated GAD67 gene therapy in the treatment of HIV-related neuropathic pain.
HIV-1 Tat Induces Unfolded Protein Response and Endoplasmic Reticulum Stress in Astrocytes and Causes Neurotoxicity through Glial Fibrillary Acidic Protein (GFAP) Activation and Aggregation.
Fan, Yan; He, Johnny J
HIV-1 Tat is a major culprit for HIV/neuroAIDS. One of the consistent hallmarks of HIV/neuroAIDS is reactive astrocytes or astrocytosis, characterized by increased cytoplasmic accumulation of the intermediate filament glial fibrillary acidic protein (GFAP). We have shown that that Tat induces GFAP expression in astrocytes and that GFAP activation is indispensable for astrocyte-mediated Tat neurotoxicity. However, the underlying molecular mechanisms are not known. In this study, we showed that Tat expression or GFAP expression led to formation of GFAP aggregates and induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in astrocytes. In addition, we demonstrated that GFAP up-regulation and aggregation in astrocytes were necessary but also sufficient for UPR/ER stress induction in Tat-expressing astrocytes and for astrocyte-mediated Tat neurotoxicity. Importantly, we demonstrated that inhibition of Tat- or GFAP-induced UPR/ER stress by the chemical chaperone 4-phenylbutyrate significantly alleviated astrocyte-mediated Tat neurotoxicity in vitro and in the brain of Tat-expressing mice. Taken together, these results show that HIV-1 Tat expression leads to UPR/ER stress in astrocytes, which in turn contributes to astrocyte-mediated Tat neurotoxicity, and raise the possibility of developing HIV/neuroAIDS therapeutics targeted at UPR/ER stress.
Release of simian virus 40 virions from epithelial cells is polarized and occurs without cell lysis.
Clayson, E T; Brando, L V; Compans, R W
We have investigated the process of release of simian virus 40 (SV40) virions from several monkey kidney cell lines. High levels of virus release were observed prior to any significantly cytopathic effects in all cell lines examined, indicating that SV40 utilizes a mechanism for escape from the host cell which does not involve cell lysis. We demonstrate that SV40 release was polarized in two epithelial cell types (Vero C1008 and primary African green monkey kidney cells) grown on permeable supports; release of virus occurs almost exclusively at apical surfaces. In contrast, equivalent amounts of SV40 virions were recovered from apical and basal culture fluids of nonpolarized CV-1 cells. SV40 virions were observed in large numbers on apical surfaces of epithelial cells and in cytoplasmic smooth membrane vesicles. The sodium ionophore monensin, an inhibitor of vesicular transport, was found to inhibit SV40 release without altering viral protein synthesis or infectious virus production. Images PMID:2539518
Putative site for the acquisition of human herpesvirus 6 virion tegument.
Roffman, E; Albert, J P; Goff, J P; Frenkel, N
The virion of human herpesvirus 6 (HHV-6) contains a very distinct tegument layer, occupying the space between the nucleocapsid and the virion envelope. Ultrastructural analyses of thymocytes infected with HHV-6 revealed the presence of intranuclear spherical compartments, approximately 1.5 microns in diameter, in which tegumentation seems to take place. These compartments, termed tegusomes, were bounded by two membranes and contained ribosomes, consistent with their derivation by cytoplasmic invagination into the nucleus. Capsids located within the nucleus outside the tegusomes were all naked, while those located in the cytoplasm were uniformly tegumented. In contrast, capsids present inside the tegusomes contains teguments of variable thicknesses. In addition, nucleocapsids were documented in the process of budding into the tegusomes. We thus suggest that the tegusomes represent a cellular site in which HHV-6 virions acquire their tegument. Images PMID:2173796
RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions
Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K.; James, John; Prescott, Alan; Haga, Ismar R.; Beard, Philippa M.
Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. PMID:25462347
5' termini of poliovirus RNA: difference between virion and nonencapsidated 35S RNA.
Fernandez-Muñoz, R; Lavi, U
Poliovirus cytoplasmic, nonencapsidated 35S RNA yields approximately one pUp per molecule upon T2 RNase digestion, indicating that this RNA has the same 5' end as the polyribosome-associated viral RNA fraction. Double-stranded, replicative form RNA after the same treatment yielded approximately four pNp structures per molecule, 65% of which was pUp. In contrast, the 35S RNA from mature virions contained no detectable pNp, indicating that the 5' end of the virion RNA is different from that of the nonencapsidated RNA. None of the above molecules contained pppNp, ppNp, or GpppNp structures present in host mRNA. The virion RNA molecules, as we have shown previously for thenonencapsidated 35S viral RNA (Fernandez-Muñoz and Darnell, 1976), is not labeled with [methyl-3H]methionine. PMID:189096
Binding of soluble CD4 proteins to human immunodeficiency virus type 1 and infected cells induces release of envelope glycoprotein gp120.
Hart, T K; Kirsh, R; Ellens, H; Sweet, R W; Lambert, D M; Petteway, S R; Leary, J; Bugelski, P J
Human immunodeficiency virus (HIV) infects cells after binding of the viral envelope glycoprotein gp120 to the cell surface recognition marker CD4. gp120 is noncovalently associated with the HIV transmembrane envelope glycoprotein gp41, and this complex is believed responsible for the initial stages of HIV infection and cytopathic events in infected cells. Soluble constructs of CD4 that contain the gp120 binding site inhibit HIV infection in vitro. This is believed to occur by competitive inhibition of viral binding to cellular CD4. Here we suggest an alternative mechanism of viral inhibition by soluble CD4 proteins. We demonstrate biochemically and morphologically that following binding, the soluble CD4 proteins sT4, V1V2,DT, and V1[106] (amino acids 1-369, 1-183, and -2 to 106 of mature CD4) induced the release of gp120 from HIV-1 and HIV-1-infected cells. gp120 release was concentration-, time-, and temperature-dependent. The reaction was biphasic at 37 degrees C and did not take place at 4 degrees C, indicating that binding of soluble CD4 was not sufficient to release gp120. The appearance of free gp120 in the medium after incubation with sT4 correlated with a decrease in envelope glycoprotein spikes on virions and exposure of a previously cryptic epitope near the amino terminus of gp41 on virions and infected cells. The concentration of soluble CD4 proteins needed to induce the release of gp120 from virally infected cells also correlated with those required to inhibit HIV-mediated syncytium formation. These results suggest that soluble CD4 constructs may inactivate HIV by inducing the release of gp120. We propose that HIV envelope-mediated fusion is initiated following rearrangement and/or dissociation of gp120 from the gp120-gp41 complex upon binding to cellular CD4, thus exposing the fusion domain of gp41. Images PMID:2006155
Development of a novel self micro-emulsifying drug delivery system (SMEDDS) for reducing HIV protease inhibitor-induced intestinal epithelial barrier dysfunction
Lei, Bokai; Zha, Weibin; Wang, Yun; Wen, Cong; Stude, Elaine J; Wang, Xuan; Jin, Fang; Wang, Guangji; Zhang, Luyong; Zhou, Huiping
The development of HIV protease inhibitors (PIs) has been one of the most significant advances of the past decade in controlling HIV infection. Unfortunately, the benefits of HIV PIs are compromised by serious side effects. One of the most frequent and deleterious side effects of HIV PIs is severe gastrointestinal (GI) disorders including mucosal erosions, epithelial barrier dysfunction, and leak-flux diarrhea, which occurs in 16–62% of patients on HIV PIs. Although the underlying mechanisms behind HIV PI-associated serious adverse side effects remain to be identified, our recent studies have shown that activation of endoplasmic reticulum (ER) stress response plays a critical role in HIV PI-induced GI complications. The objective of this study was to develop a novel self micro-emulsifying drug delivery system (SMEDDS) using various antioxidants as surfactants and co-surfactants to reduce the GI side effects of the most commonly used HIV PI, ritonavir. The biological activities of this SMSDDS of ritonavir were compared with that of Norvir®, which is currently used in the clinic. Rat normal intestinal epithelial cells (IEC-6) and mouse Raw 264.7 macrophages were used to examine the effect of new SMEDDS of ritonavir on activation of ER stress and oxidative stress. The Sprague-Dawley rats and C57/BL6 mice were used for pharmacokinetic studies and in vivo studies. The intracellular and plasma drug concentrations were determined by HPLC analysis. Activation of ER stress was detected by western blot analysis and secreted alkaline phosphatase (SEAP) reporter assay. Reactive oxygen species (ROS) was measured using dichlorodihydrofluorescein diacetate as a probe. Cell viability was determined by Roche's cell proliferation reagent WST-1. Protein levels of inflammatory cytokines (TNF-α and IL-6) were determined by Enzyme-linked immunosorbent assays (ELISA). The intestinal permeability was assessed by luminal enteral administration of fluorescein isothiocyanate conjugated
Development of a novel self-microemulsifying drug delivery system for reducing HIV protease inhibitor-induced intestinal epithelial barrier dysfunction.
Lei, Bokai; Zha, Weibin; Wang, Yun; Wen, Cong; Studer, Elaine J; Wang, Xuan; Jin, Fang; Wang, Guangji; Zhang, Luyong; Zhou, Huiping
The development of HIV protease inhibitors (PIs) has been one of the most significant advances of the past decade in controlling HIV infection. Unfortunately, the benefits of HIV PIs are compromised by serious side effects. One of the most frequent and deleterious side effects of HIV PIs is severe gastrointestinal (GI) disorders including mucosal erosions, epithelial barrier dysfunction, and leak-flux diarrhea, which occurs in 16-62% of patients on HIV PIs. Although the underlying mechanisms behind HIV PI-associated serious adverse side effects remain to be identified, our recent studies have shown that activation of endoplasmic reticulum (ER) stress response plays a critical role in HIV PI-induced GI complications. The objective of this study was to develop a novel self-microemulsifying drug delivery system (SMEDDS) using various antioxidants as surfactants and cosurfactants to reduce the GI side effects of the most commonly used HIV PI, ritonavir. The biological activities of this SMSDDS of ritonavir were compared with that of Norvir, which is currently used in the clinic. Rat normal intestinal epithelial cells (IEC-6) and mouse Raw 264.7 macrophages were used to examine the effect of new SMEDDS of ritonavir on activation of ER stress and oxidative stress. Sprague-Dawley rats and C57/BL6 mice were used for pharmacokinetic studies and in vivo studies. The intracellular and plasma drug concentrations were determined by HPLC analysis. Activation of ER stress was detected by Western blot analysis and secreted alkaline phosphatase (SEAP) reporter assay. Reactive oxygen species (ROS) was measured using dichlorodihydrofluorescein diacetate as a probe. Cell viability was determined by Roche's cell proliferation reagent WST-1. Protein levels of inflammatory cytokines (TNF-alpha and IL-6) were determined by enzyme-linked immunosorbent assays (ELISA). The intestinal permeability was assessed by luminal enteral administration of fluorescein isothiocyanate conjugated dextran
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Trimethoprim-sulfamethoxazole-induced Steven Johnson syndrome in an HIV-infected patient.
Taqi, Syed Ahmed; Zaki, Syed Ahmed; Nilofer, Angadi Rajasab; Sami, Lateef Begum
Trimethoprim-sulfamethoxazole (TMP/SMX) is a widely prescribed antimicrobial for the management of several uncomplicated infections. It is commonly used for the treatment and prophylaxis of Pneumocystis jirovecii pneumonia (PCP) in the HIV-infected population. The adverse reaction to TMP/SMX is more frequent and severe in HIV-infected patients as compared to the general population. Here, we report a case of Stevens-Johnson syndrome (SJS) secondary to TMP/SMX. The patient had a generalized cutaneous reaction with involvement of the eyes, oral cavity, and genitals. He had elevated hepatic alanine aminotransferase and aspartate aminotransferase enzyme. TMP/SMX therapy was stopped and supportive treatment was started. His condition improved after eight days of stopping TMP/SMX therapy.
HIV-2 and SIVmac accessory virulence factor Vpx down-regulates SAMHD1 enzyme catalysis prior to proteasome-dependent degradation.
DeLucia, Maria; Mehrens, Jennifer; Wu, Ying; Ahn, Jinwoo
SAMHD1, a dGTP-regulated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase, down-regulates dNTP pools in terminally differentiated and quiescent cells, thereby inhibiting HIV-1 infection at the reverse transcription step. HIV-2 and simian immunodeficiency virus (SIV) counteract this restriction via a virion-associated virulence accessory factor, Vpx (Vpr in some SIVs), which loads SAMHD1 onto CRL4-DCAF1 E3 ubiquitin ligase for polyubiquitination, programming it for proteasome-dependent degradation. However, the detailed molecular mechanisms of SAMHD1 recruitment to the E3 ligase have not been defined. Further, whether divergent, orthologous Vpx proteins, encoded by distinct HIV/SIV strains, bind SAMHD1 in a similar manner, at a molecular level, is not known. We applied surface plasmon resonance analysis to assess the requirements for and kinetics of binding between various primate SAMHD1 proteins and Vpx proteins from SIV or HIV-2 strains. Our data indicate that Vpx proteins, bound to DCAF1, interface with the C terminus of primate SAMHD1 proteins with nanomolar affinity, manifested by rapid association and slow dissociation. Further, we provide evidence that Vpx binding to SAMHD1 inhibits its catalytic activity and induces disassembly of a dGTP-dependent oligomer. Our studies reveal a previously unrecognized biochemical mechanism of Vpx-mediated SAMHD1 inhibition: direct down-modulation of its catalytic activity, mediated by the same binding event that leads to SAMHD1 recruitment to the E3 ubiquitin ligase for proteasome-dependent degradation.
FBI-1, a factor that binds to the HIV-1 inducer of short transcripts (IST), is a POZ domain protein.
Morrison, D J; Pendergrast, P S; Stavropoulos, P; Colmenares, S U; Kobayashi, R; Hernandez, N
The HIV-1 promoter directs the synthesis of two classes of transcripts, short, non-polyadenylated transcripts and full-length, polyadenylated transcripts. The synthesis of short transcripts is activated by a bipartite DNA element, the inducer of short transcripts or IST, located downstream of the HIV-1 transcriptional start site, while the synthesis of full-length transcripts is activated by the viral activator Tat. Tat binds to the RNA element TAR, which is encoded largely between the two IST half-elements. Upon activation by Tat, the synthesis of short RNAs is repressed. We have previously purified a factor called FBI-1 (for factor that binds to IST) whose binding to wild-type and mutated ISTs correlated well with the abilities of these ISTs to direct the synthesis of short transcripts. Here, we report the cloning of cDNAs encoding FBI-1. FBI-1 contains a POZ domain at its N-terminus and four Krüppel-type zinc fingers at its C-terminus. The C-terminus is sufficient for specific binding, and FBI-1 can form homomers through its POZ domain and, in vivo, through its zinc finger domain as well. In addition, FBI-1 associates with Tat, suggesting that repression of the short transcripts by Tat may be mediated through interactions between the two factors.
Modeling global changes induced by local perturbations to the HIV-1 capsid.
Bergman, Shana; Lezon, Timothy R
The HIV-1 capsid is a conical protein shell made up of hexamers and pentamers of the capsid protein. The capsid houses the viral genome and replication machinery, and its opening, or uncoating, within the host cell marks a critical step in the HIV-1 lifecycle. Binding of host factors such as TRIM5α and cyclophilin A (CypA) can alter the capsid's stability, accelerating or delaying the onset of uncoating and disrupting infectivity. We employ coarse-grained computational modeling to investigate the effects of point mutations and host factor binding on HIV-1 capsid stability. We find that the largest fluctuations occur in the low-curvature regions of the capsid, and that its structural dynamics are affected by perturbations at the inter-hexamer interfaces and near the CypA binding loop, suggesting roles for these features in capsid stability. Our models show that linking capsid proteins across hexamers attenuates vibration in the low-curvature regions of the capsid, but that linking within hexamers does not. These results indicate a possible mechanism through which CypA binding alters capsid stability and highlight the utility of coarse-grained network modeling for understanding capsid mechanics.
HLA class II genes modulate vaccine-induced antibody responses to affect HIV-1 acquisition.
Prentice, Heather A; Tomaras, Georgia D; Geraghty, Daniel E; Apps, Richard; Fong, Youyi; Ehrenberg, Philip K; Rolland, Morgane; Kijak, Gustavo H; Krebs, Shelly J; Nelson, Wyatt; DeCamp, Allan; Shen, Xiaoying; Yates, Nicole L; Zolla-Pazner, Susan; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Ferrari, Guido; McElrath, M Juliana; Montefiori, David C; Bailer, Robert T; Koup, Richard A; O'Connell, Robert J; Robb, Merlin L; Michael, Nelson L; Gilbert, Peter B; Kim, Jerome H; Thomas, Rasmi
In the RV144 vaccine trial, two antibody responses were found to correlate with HIV-1 acquisition. Because human leukocyte antigen (HLA) class II-restricted CD4(+) T cells are involved in antibody production, we tested whether HLA class II genotypes affected HIV-1-specific antibody levels and HIV-1 acquisition in 760 individuals. Indeed, antibody responses correlated with acquisition only in the presence of single host HLA alleles. Envelope (Env)-specific immunoglobulin A (IgA) antibodies were associated with increased risk of acquisition specifically in individuals with DQB1*06. IgG antibody responses to Env amino acid positions 120 to 204 were higher and were associated with decreased risk of acquisition and increased vaccine efficacy only in the presence of DPB1*13. Screening IgG responses to overlapping peptides spanning Env 120-204 and viral sequence analysis of infected individuals defined differences in vaccine response that were associated with the presence of DPB1*13 and could be responsible for the protection observed. Overall, the underlying genetic findings indicate that HLA class II modulated the quantity, quality, and efficacy of antibody responses in the RV144 trial.
HLA class II genes modulate vaccine-induced antibody responses to affect HIV-1 acquisition
Prentice, Heather A.; Tomaras, Georgia D.; Geraghty, Daniel E.; Apps, Richard; Fong, Youyi; Ehrenberg, Philip K.; Rolland, Morgane; Kijak, Gustavo H.; Krebs, Shelly J.; Nelson, Wyatt; DeCamp, Allan; Shen, Xiaoying; Yates, Nicole L.; Zolla-Pazner, Susan; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Ferrari, Guido; Juliana McElrath, M.; Montefiori, David C.; Bailer, Robert T.; Koup, Richard A.; O'Connell, Robert J.; Robb, Merlin L.; Michael, Nelson L.; Gilbert, Peter B.; Kim, Jerome H.; Thomas, Rasmi
In the RV144 vaccine trial, two antibody responses were found to correlate with HIV-1 acquisition. Because human leukocyte antigen (HLA) class II–restricted CD4+ T cells are involved in antibody production, we tested whether HLA class II genotypes affected HIV-1–specific antibody levels and HIV-1 acquisition in 760 individuals. Indeed, antibody responses correlated with acquisition only in the presence of single host HLA alleles. Envelope (Env)–specific immunoglobulin A (IgA) antibodies were associated with increased risk of acquisition specifically in individuals with DQB1*06. IgG antibody responses to Env amino acid positions 120 to 204 were higher and were associated with decreased risk of acquisition and increased vaccine efficacy only in the presence of DPB1*13. Screening IgG responses to overlapping peptides spanning Env 120–204 and viral sequence analysis of infected individuals defined differences in vaccine response that were associated with the presence of DPB1*13 and could be responsible for the protection observed. Overall, the underlying genetic findings indicate that HLA class II modulated the quantity, quality, and efficacy of antibody responses in the RV144 trial. PMID:26180102
Myristoylation of the Arenavirus Envelope Glycoprotein Stable Signal Peptide Is Critical for Membrane Fusion but Dispensable for Virion Morphogenesis
York, Joanne
ABSTRACT Arenaviruses are responsible for severe and often fatal hemorrhagic disease. In the absence of effective antiviral therapies and vaccines, these viruses pose serious threats to public health and biodefense. Arenaviruses enter the host cell by fusion of the viral and endosomal membranes, a process mediated by the virus envelope glycoprotein GPC. Unlike other class I viral fusion proteins, GPC retains its stable signal peptide (SSP) as an essential third subunit in the mature complex. SSP spans the membrane twice and is myristoylated at its cytoplasmic N terminus. Mutations that abolish SSP myristoylation have been shown to reduce pH-induced cell-cell fusion activity of ectopically expressed GPC to ∼20% of wild-type levels. In order to examine the role of SSP myristoylation in the context of the intact virus, we used reverse genetics to generate Junín viruses (Candid #1 isolate) in which the critical glycine-2 residue in SSP was either replaced by alanine (G2A) or deleted (ΔG2). These mutant viruses produced smaller foci of infection in Vero cells and showed an ∼5-fold reduction in specific infectivity, commensurate with the defect in cell-cell fusion. However, virus assembly and GPC incorporation into budded virions were unaffected. Our findings suggest that the myristate moiety is cryptically disposed in the prefusion GPC complex and may function late in the fusion process to promote merging of the viral and cellular membranes. IMPORTANCE Hemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Arenavirus entry into the host cell is promoted by the virus envelope glycoprotein GPC. Unlike other viral envelope glycoproteins, GPC contains a myristoylated stable signal peptide (SSP) as an essential third subunit. Myristoylation has been shown to be important for the membrane fusion activity of recombinantly expressed GPC. Here, we use reverse genetics to study the role of SSP myristoylation in the context of the intact
Targeting xCT, a cystine-glutamate transporter induces apoptosis and tumor regression for KSHV/HIV-associated lymphoma
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of primary effusion lymphoma (PEL), which represents a rapidly progressing malignancy arising in HIV-infected patients. Conventional chemotherapy for PEL treatment induces unwanted toxicity and is ineffective — PEL continues to portend nearly 100% mortality within a period of months, which requires novel therapeutic strategies. The amino acid transporter, xCT, is essential for the uptake of cystine required for intracellular glutathione (GSH) synthesis and for maintaining the intracellular redox balance. Inhibition of xCT induces growth arrest in a variety of cancer cells, although its role in virus-associated malignancies including PEL remains unclear. In the current study, we identify that xCT is expressed on the surface of patient-derived KSHV+ PEL cells, and targeting xCT induces caspase-dependent cell apoptosis. Further experiments demonstrate the underlying mechanisms including host and viral factors: reducing intracellular GSH while increasing reactive oxygen species (ROS), repressing cell-proliferation-related signaling, and inducing viral lytic genes. Using an immune-deficient xenograft model, we demonstrate that an xCT selective inhibitor, Sulfasalazine (SASP), prevents PEL tumor progression in vivo. Together, our data provide innovative and mechanistic insights into the role of xCT in PEL pathogenesis, and the framework for xCT-focused therapies for AIDS-related lymphoma in future. PMID:24708874
In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype.
Roden, R B; Greenstone, H L; Kirnbauer, R; Booy, F P; Jessie, J; Lowy, D R; Schiller, J T
We report a system for generating infectious papillomaviruses in vitro that facilitates the analysis of papillomavirus assembly, infectivity, and serologic relatedness. Cultured hamster BPHE-1 cells harboring autonomously replicating bovine papillomavirus type 1 (BPV1) genomes were infected with recombinant Semliki Forest viruses that express the structural proteins of BPV1. When plated on C127 cells, extracts from cells expressing L1 and L2 together induced numerous transformed foci that could be specifically prevented by BPV neutralizing antibodies, demonstrating that BPV infection was responsible for the focal transformation. Extracts from BPHE-1 cells expressing L1 or L2 separately were not infectious. Although Semliki Forest virus-expressed L1 self-assembled into virus-like particles (VLPs), viral DNA was detected in particles only when L2 was coexpressed with L1, indicating that genome encapsidation requires L2. Expression of human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1 cells also yielded infectious virus. These pseudotyped virions were neutralized by antiserum to HPV16 VLPs derived from European (114/K) or African (Z-1194) HPV16 variants but not by antisera to BPV VLPs, to a poorly assembling mutant HPV16 L1 protein, or to VLPs of closely related genital HPV types. Extracts from BPHE-1 cells coexpressing BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were not infectious. We conclude that (i) mouse C127 cells express the cell surface receptor for HPV16 and are able to uncoat HPV16 capsids; (ii) if a papillomavirus DNA packaging signal exists, then it is conserved between the BPV and HPV16 genomes; (iii) functional L1-L2 interaction exhibits type specificity; and (iv) protection by HPV virus-like particle vaccines is likely to be type specific. PMID:8709207
Determination of molecular structures of HIV envelope glycoproteins using cryo-electron tomography and automated sub-tomogram averaging.
Meyerson, Joel R; White, Tommi A; Bliss, Donald; Moran, Amy; Bartesaghi, Alberto; Borgnia, Mario J; de la Cruz, M Jason V; Schauder, David; Hartnell, Lisa M; Nandwani, Rachna; Dawood, Moez; Kim, Brianna; Kim, Jun Hong; Sununu, John; Yang, Lisa; Bhatia, Siddhant; Subramaniam, Carolyn; Hurt, Darrell E; Gaudreault, Laurent; Subramaniam, Sriram
Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) (2). Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor (5,9). The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine. The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at ˜20 Å resolution (9,14). This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH (http://biowulf.nih.gov). This remote access, combined with low-cost computer hardware and
Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging
Meyerson, Joel R.; White, Tommi A.; Bliss, Donald; Moran, Amy; Bartesaghi, Alberto; Borgnia, Mario J.; de la Cruz, M. Jason V.; Schauder, David; Hartnell, Lisa M.; Nandwani, Rachna; Dawood, Moez; Kim, Brianna; Kim, Jun Hong; Sununu, John; Yang, Lisa; Bhatia, Siddhant; Subramaniam, Carolyn; Hurt, Darrell E.; Gaudreault, Laurent; Subramaniam, Sriram
Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine. The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at ˜20 Å resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH (http://biowulf.nih.gov). This remote access, combined with low-cost computer hardware and high
HIV-1 Vpr Abrogates the Effect of TSG101 Overexpression to Support Virus Release
Chutiwitoonchai, Nopporn; Siarot, Lowela; Takeda, Eri; Shioda, Tatsuo; Ueda, Motoki; Aida, Yoko
HIV-1 budding requires interaction between Gag and cellular TSG101 to initiate viral particle assembly and release via the endosomal sorting complexes required for transport (ESCRT) pathway. However, some reports show that overexpression of TSG101 inhibits virus release by disruption of Gag targeting process. Since a HIV-1 accessory protein, Vpr binds to Gag p6 domain at the position close to the binding site for TSG101, whether Vpr implicates TSG101 overexpression effect has not been investigated. Here, we found that Vpr abrogates TSG101 overexpression effect to rescue viral production. Co-transfection of TSG101 and Gag with Vpr prevented TSG101-induced Gag accumulation in endosomes and lysosomes. In addition, Vpr rescued virus-like particle (VLP) production in a similar manner as a lysosomal inhibitor, Bafilomycin A1 indicating that Vpr inhibits TSG101-induced Gag downregulation via lysosomal pathway. Vpr and Gag interaction is required to counteract TSG101 overexpression effect since Vpr A30F mutant which is unable to interact with Gag and incorporate into virions, reduced ability to prevent Gag accumulation and to rescue VLP production. In addition, GST pull-down assays and Biacore analysis revealed that Vpr competed with TSG101 for Gag binding. These results indicate that Vpr overcomes the effects of TSG101 overexpression to support viral production by competing with TSG101 to bind Gag. PMID:27648839
Detection and tracking of dual-labeled HIV particles using wide-field live cell imaging to follow viral core integrity
Mamede, Joao I.; Hope, Thomas J.
Summary Live cell imaging is a valuable technique that allows the characterization of the dynamic processes of the HIV-1 life-cycle. Here, we present a method of production and imaging of dual-labeled HIV viral particles that allows the visualization of two events. Varying release of the intravirion fluid phase marker reveals virion fusion and the loss of the integrity of HIV viral cores with the use of live wide-field fluorescent microscopy. PMID:26714704
Non-puerperal induced lactation: an infant feeding option in paediatric HIV/AIDS in tropical Africa.
Ogunlesi, Tinuade A; Adekanmbi, Folasade A; Fetuga, Bolanle M; Ogundeyi, Mojisola M
A major problem in the management of infants exposed to HIV is the issue of feeding, which stems from the need to avoid transmission of the virus via breast milk. Other important issues in the nutrition of infants exposed to the virus include severe maternal illness, which makes suckling extremely difficult, and feeding orphans. Wet nursing is one of the recommended steps in addressing the feeding problems of such infants but for reasons of sociocultural disapproval, it appears not to be popular in traditional African settings. Non-puerperal induced lactation or re-lactation of a close relation, usually a grandmother, which hitherto has been used to rehabilitate severely malnourished motherless infants, may be equally useful. The procedure of re-lactation and the limitations of the method are highlighted. Also, the need to employ information, education and communication in improving the sociocultural acceptability of this veritable infant feeding method in tropical Africa is discussed.
Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation
Novikova, Mariia; Ablan, Sherimay D.; Freed, Eric O.
The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals. PMID:26711999
Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies
Chen, Weizao; Feng, Yang; Wang, Yanping; Zhu, Zhongyu; Dimitrov, Dimiter S.
Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.
Porphyromonas gingivalis induces CCR5-dependent transfer of infectious HIV-1 from oral keratinocytes to permissive cells
Giacaman, Rodrigo A; Asrani, Anil C; Gebhard, Kristin H; Dietrich, Elizabeth A; Vacharaksa, Anjalee; Ross, Karen F; Herzberg, Mark C
Background Systemic infection with HIV occurs infrequently through the oral route. The frequency of occurrence may be increased by concomitant bacterial infection of the oral tissues, since co-infection and inflammation of some cell types increases HIV-1 replication. A putative periodontal pathogen, Porphyromonas gingivalis selectively up-regulates expression of the HIV-1 coreceptor CCR5 on oral keratinocytes. We, therefore, hypothesized that P. gingivalis modulates the outcome of HIV infection in oral epithelial cells. Results Oral and tonsil epithelial cells were pre-incubated with P. gingivalis, and inoculated with either an X4- or R5-type HIV-1. Between 6 and 48 hours post-inoculation, P. gingivalis selectively increased the infectivity of R5-tropic HIV-1 from oral and tonsil keratinocytes; infectivity of X4-tropic HIV-1 remained unchanged. Oral keratinocytes appeared to harbor infectious HIV-1, with no evidence of productive infection. HIV-1 was harbored at highest levels during the first 6 hours after HIV exposure and decreased to barely detectable levels at 48 hours. HIV did not appear to co-localize with P. gingivalis, which increased selective R5-tropic HIV-1 trans infection from keratinocytes to permissive cells. When CCR5 was selectively blocked, HIV-1 trans infection was reduced. Conclusion P. gingivalis up-regulation of CCR5 increases trans infection of harbored R5-tropic HIV-1 from oral keratinocytes to permissive cells. Oral infections such as periodontitis may, therefore, increase risk for oral infection and dissemination of R5-tropic HIV-1. PMID:18371227
Constitutive gene expression in monocytes from chronic HIV-1 infection overlaps with acute Toll-like receptor induced monocyte activation profiles.
Gekonge, Bethsebah; Giri, Malavika S; Kossenkov, Andrew V; Nebozyhn, Michael; Yousef, Malik; Mounzer, Karam; Showe, Louise; Montaner, Luis J
Elevated TLR expression/signalling in monocyte/macrophages has been shown to mediate systemic immune activation, a hallmark of progressive HIV-1 infection. Here we show, via differential gene expression comparisons, the presence of a constitutive in vivo TLR-like gene activation signature in steady-state circulating monocytes from chronically HIV-1 infected subjects. The TLR2-like gene signature was defined as an 82 gene subset of the 376 genes constitutively modulated in in vivo HIV-1 monocytes, based on their overlap with de novo TLR2-induced genes in uninfected subjects' monocytes following acute ex vivo stimulation with Staphylococcus Aureus Cowan (SAC). Additional comparison of in vivo gene networks with available datasets from acute TLR activations in M/M expanded the overlap to 151-gene concordance among the 376 differential genes with emphasis on ERK/MAPK, TNF/IL6 (NFκB) and p53 gene networks. TLR2 stimulation of monocytes from HIV-1 infected subjects resulted in further upregulation of inflammatory genes indicative of a sustained transcriptional potential upon stimulation. In summary, our data support the presence of a sustained TLR-like gene activation profile in circulating monocyte from steady-state viremia in HIV-1 infected subjects.
Binding Characteristics of Small Molecules that Mimic Nucleocapsid Protein-induced Maturation of Stem-loop-1 of HIV-1 RNA†
Chung, Janet; Ulyanov, Nikolai B.; Guilbert, Christophe; Mujeeb, Anwer; James, Thomas L.
As a retrovirus, the human immunodeficiency virus (HIV-1) packages two copies of the RNA genome as a dimer in the infectious virion. Dimerization is initiated at the dimer initiation site (DIS) which encompasses stem-loop 1 (SL1) in the 5'-UTR of the genome. Study of genomic dimerization has been facilitated by the discovery that short RNA fragments containing SL1 can dimerize spontaneously without any protein factors. Based on the palindromic nature of SL1, a kissing loop model has been proposed. First, a metastable kissing dimer is formed via standard Watson-Crick base pairs and then converted into a more stable extended dimer by the viral nucleocapsid protein (NCp7). This dimer maturation in vitro is believed to mimic initial steps in the RNA maturation in vivo, which is correlated with viral infectivity. We previously discovered a small molecule activator, Lys-Ala-7-amido-4-methylcoumarin (KA-AMC), which facilitates dimer maturation in vitro, and determined aspects of its structure-activity relationship. In this report, we present measurements of the binding affinity of the activators and characterization of their interactions with the SL1 RNA. Guanidinium groups and increasing positive charge on the side chain enhance affinity and activity, but features in the aromatic ring at least partially decouple affinity from activity. Although KA-AMC can bind to multiple structural motifs, NMR study showed KA-AMC preferentially binds to unique structural motifs, such as the palindromic loop and the G-rich internal loop in the SL1 RNA. NCp7 binds to SL1 only an order of magnitude tighter than the best small molecule ligand tested. The study presented here provides guidelines for design of superior small molecule binders to the SL1 RNA that have the potential of being developed as an antiviral by either interfering with SL1-NCp7 interaction at the packaging and/or maturation stages. PMID:20565056
A nucleolar localizing Rev binding element inhibits HIV replication
Michienzi, Alessandro; De Angelis, Fernanda G; Bozzoni, Irene; Rossi, John J
The Rev protein of the human immunodeficiency virus (HIV) facilitates the nuclear export of intron containing viral mRNAs allowing formation of infectious virions. Rev traffics through the nucleolus and shuttles between the nucleus and cytoplasm. Rev multimerization and interaction with the export protein CRM1 takes place in the nucleolus. To test the importance of Rev nucleolar trafficking in the HIV-1 replication cycle, we created a nucleolar localizing Rev Response Element (RRE) decoy and tested this for its anti-HIV activity. The RRE decoy provided marked inhibition of HIV-1 replication in both the CEM T-cell line and in primary CD34+ derived monocytes. These results demonstrate that titration of Rev in the nucleolus impairs HIV-1 replication and supports a functional role for Rev trafficking in this sub-cellular compartment. PMID:16712721
Sulphadiazine-induced obstructive renal failure complicating treatment of HIV-associated toxoplasmosis.
Allinson, J; Topping, W; Edwards, S G; Miller, R F
A patient with newly-diagnosed HIV infection and biopsy-proven cerebral toxoplasmosis was treated with sulphadiazine and pyrimethamine. Despite adequate hydration and daily examination of urine for sulphadiazine crystals obstructive uropathy due to bilateral ureteric stones with hydronephrosis occurred, resulting in rapid onset renal failure. Sulphadiazine was discontinued and clindamycin was substituted. With intravenous fluid hydration and bilateral nephrostomies the urolithiasis resolved. This case serves to remind clinicians of the need for vigilance when treating cerebral toxoplasmosis with sulphadiazine, in order to avoid this potentially serious complication of treatment.
Predicting First Traversal Times for Virions and Nanoparticles in Mucus with Slowed Diffusion
Erickson, Austen M.; Henry, Bruce I.; Murray, John M.; Klasse, Per Johan; Angstmann, Christopher N.
Particle-tracking experiments focusing on virions or nanoparticles in mucus have measured mean-square displacements and reported diffusion coefficients that are orders of magnitude smaller than the diffusion coefficients of such particles in water. Accurate description of this subdiffusion is important to properly estimate the likelihood of virions traversing the mucus boundary layer and infecting cells in the epithelium. However, there are several candidate models for diffusion that can fit experimental measurements of mean-square displacements. We show that these models yield very different estimates for the time taken for subdiffusive virions to traverse through a mucus layer. We explain why fits of subdiffusive mean-square displacements to standard diffusion models may be misleading. Relevant to human immunodeficiency virus infection, using computational methods for fractional subdiffusion, we show that subdiffusion in normal acidic mucus provides a more effective barrier against infection than previously thought. By contrast, the neutralization of the mucus by alkaline semen, after sexual intercourse, allows virions to cross the mucus layer and reach the epithelium in a short timeframe. The computed barrier protection from fractional subdiffusion is some orders of magnitude greater than that derived by fitting standard models of diffusion to subdiffusive data. PMID:26153713
Virion Structure of Black Queen Cell Virus, a Common Honeybee Pathogen
Spurny, Radovan; Přidal, Antonín; Pálková, Lenka; Kiem, Hoa Khanh Tran; de Miranda, Joachim R.
ABSTRACT Viral diseases are a major threat to honeybee (Apis mellifera) populations worldwide and therefore an important factor in reliable crop pollination and food security. Black queen cell virus (BQCV) is the etiological agent of a fatal disease of honeybee queen larvae and pupae. The virus belongs to the genus Triatovirus from the family Dicistroviridae, which is part of the order Picornavirales. Here we present a crystal structure of BQCV determined to a resolution of 3.4 Å. The virion is formed by 60 copies of each of the major capsid proteins VP1, VP2, and VP3; however, there is no density corresponding to a 75-residue-long minor capsid protein VP4 encoded by the BQCV genome. We show that the VP4 subunits are present in the crystallized virions that are infectious. This aspect of the BQCV virion is similar to that of the previously characterized triatoma virus and supports the recent establishment of the separate genus Triatovirus within the family Dicistroviridae. The C terminus of VP1 and CD loops of capsid proteins VP1 and VP3 of BQCV form 34-Å-tall finger-like protrusions at the virion surface. The protrusions are larger than those of related dicistroviruses. IMPORTANCE The western honeybee is the most important pollinator of all, and it is required to sustain the agricultural production and biodiversity of wild flowering plants. However, honeybee populations worldwide are suffering from virus infections that cause colony losses. One of the most common, and least known, honeybee pathogens is black queen cell virus (BQCV), which at high titers causes queen larvae and pupae to turn black and die. Here we present the three-dimensional virion structure of BQCV, determined by X-ray crystallography. The structure of BQCV reveals large protrusions on the virion surface. Capsid protein VP1 of BQCV does not contain a hydrophobic pocket. Therefore, the BQCV virion structure provides evidence that capsid-binding antiviral compounds that can prevent the
Conductive silver paste smeared glass substrates for label-free Raman spectroscopic detection of HIV-1 and HIV-1 p24 antigen in blood plasma.
Otange, Ben O; Birech, Zephania; Okonda, Justus; Rop, Ronald
We report on application of conductive silver paste smeared glass slides as Raman spectroscopy sample substrates for label-free detection of HIV-1 p24 antigen in blood plasma. We also show that the same substrates can be applied in Raman spectroscopic screening of blood plasma for presence of HIV. The characteristic Raman spectrum of HIV-1 p24 antigen displayed prominent bands that were assigned to ribonucleic acids (RNA) and proteins that constitute the antigen. This spectrum can be used as reference during Raman spectroscopic screening for HIV in plasma within the first few days after exposure (<7 days). The Raman spectra obtained from HIV+ plasma displayed unique peaks centered at wavenumbers 928, 990, 1270, 1397, and 1446 cm(-1) attributed to the Raman active vibrations in the virion carbohydrates, lipids, and proteins. Other bands similar to those reported in literature were also seen and assignments made. The attachment of the HIV virions to silver nanoparticles via gp120 glycoprotein knobs was thought to be responsible for the enhanced Raman signals of proteins associated with the virus. The principal component analysis (PCA) applied on the combined spectral data showed that HIV- and HIV+ spectra had differing spectral patterns. This indicated the great power of Raman spectroscopy in HIV detection when plasma samples are deposited onto silver paste smeared glass substrates. The Raman peaks responsible for the segregation of the spectral data in PCA were mainly those assigned to the viral proteins (645, 725, 813, 1270, and 1658 cm(-1)). Excellent results were obtained from Artificial Neural Network (ANN) applied on the HIV+ Raman spectral data around the prominent peak centered at 1270 cm(-1) with R (coefficient of correlation) and R (2) (coefficient of determination) values of 0.9958 and 0.9895, respectively. The method has the potential of being used as quick blood screening for HIV before blood transfusion with the Raman peaks assigned to the virion
Recombinant measles viruses expressing single or multiple antigens of human immunodeficiency virus (HIV-1) induce cellular and humoral immune responses.
Liniger, Matthias; Zuniga, Armando; Morin, Teldja Neige Azzouz; Combardiere, Behazine; Marty, Rene; Wiegand, Marian; Ilter, Orhan; Knuchel, Marlyse; Naim, Hussein Y
Recombinant measles viruses (rMV) based on the live attenuated measles vaccine strain (MVb) expressing antigens of HIV-1 clade B were generated by reverse genetics. Recombinants expressing single or double antigens of HIV-1 (rMV-HIV) were genetically highly stable on human diploid cells. The production process of these viruses was essentially similar to the parental MV strain, yielding comparative end titers. Immunization of tg-mice by different regimens and formulations showed potent humoral and cellular immune responses against MV and HIV antigens. Recombinant MV-HIV expressing Gag protein conferred protective immunity in tg-mice after a high-dose pseudochallenge with recombinant vaccinia virus. In addition, rMV-HIV boosted anti-HIV antibodies, in the presence of pre-existing anti-vector antibodies.
Peptide ligands selected with CD4-induced epitopes on native dualtropic HIV-1 envelope proteins mimic extracellular coreceptor domains and bind to HIV-1 gp120 independently of coreceptor usage.
Dervillez, Xavier; Klaukien, Volker; Dürr, Ralf; Koch, Joachim; Kreutz, Alexandra; Haarmann, Thomas; Stoll, Michaela; Lee, Donghan; Carlomagno, Teresa; Schnierle, Barbara; Möbius, Kalle; Königs, Christoph; Griesinger, Christian; Dietrich, Ursula
During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.
Alopecia induced by lopinavir plus ritonavir therapy in an HIV patient.
Chrysos, George; Mikros, Sotirios; Kokkoris, Stelios; Pastelli, Androula; Kontochristopoulos, George
The most commonly reported side effects related to lopinavir/ritonavir are diarrhea, vomiting, headache, nausea, and increased serum triglycerides and cholesterol levels. About 4% of the patients prescribed lopinavir/ritonavir stop taking it because of side effects. Alopecia, generally involving the scalp, has been reported in patients with HIV infection treated with indinavir but not with lopinavir/ritonavir. We present a 62-year-old man with HIV infection, stage B2, who experienced alopecia totalis of his scalp, eyebrows, and eyelashes beginning 18 months after initiating antiretroviral treatment including lopinavir/ritonavir. No hair loss on the arms, legs, and pubic area was observed. Our patient's drug regimen consisted of lopinavir/ritonavir, efavirenz, and stavudine; in addition, the patient was receiving treatment for diabetes with glivenclamide and metformin for the last 3 years. These drugs have not been shown to cause alopecia. Alopecia reversed completely 2 months after substituting nelfinavir for lopinavir/ritonavir without any other change of treatment and his eyelashes and eyebrows grew back as well. To our knowledge, this is the second case of lopinavir/ritonavir-associated alopecia totalis reported in the international literature.
[Cytomegalovirus-induced colitis in HIV infection. Considerations on its diagnosis, treatment and complications].
Sousa, A E; Lucas, M; Palhano, M J; de Deus, J; Damião, J; Victorino, R M
The diagnosis of cytomegalovirus intestinal disease in patients with HIV (human immunodeficiency virus) infection frequently raises diagnostic problems in view of the absence of definite pathological, serological or virological markers of active CMV infection. We describe the case of a 47-year-old man with a CMV colitis which illustrates several diagnostic and therapeutic problems and that was complicated by an intestinal perforation. We emphasize that in HIV+ patients with chronic diarrhea, the presence of abdominal pain should suggest the possibility of a CMV colitis and that in such cases a colonoscopy with biopsies of the right colon should be performed, in view of the higher frequency of the typical histopathological changes at this level. On the other hand, this case presented a marked thickening of the colon wall, simulating pseudotumoral images on CAT scans, as recently described in literature. The therapeutic possibilities as well as the complications of CMV colitis are discussed in the context of the occurrence of an ileal perforation, which represents the first report of this complication in Portuguese literature and which had the particularity of having a long survival after surgery in comparison with the previous cases described in international literature.
Amelioration of radiation-induced skin injury by HIV-TAT-mediated protein transduction of RP-1 from Rana pleurade.
Zhang, Shuyu; Wang, Wenjie; Peng, Ying; Gu, Qing; Luo, Judong; Zhou, Jundong; Wu, Jinchang; Hou, Yinglong; Cao, Jianping
Radiation-induced reactive oxygen species (ROS) can damage DNA and most other biological macromolecules in skin and radiation-induced skin injury is a serious concern for radiation therapy. Skin possesses an extremely efficient antioxidant system, which is conferred by two systems: antioxidant enzymes and small molecules that can scavenge ROS by donating electrons. Amphibian skin is a multifunctional organ, which protects against dangers of various oxidative stresses. Recently, a small peptide called RP-1 was isolated from the skin secretions of Rana pleurade, which shows strong antioxidant activity. However, this RP-1 peptide is limited because its inability to across the cell membrane. Protein transduction domains (PTDs) have demonstrated high efficiency for facilitating the internalization of both homologous and heterogeneous proteins into cells. This study aims to elucidate the protective effects of a HIV-TAT (TAT) PTD-coupled RP-1 fusion protein (TAT-RP1) on radiation-induced skin injury in vitro and in vivo. The synthesized fusion TAT-RP1 peptide can be incorporated into human keratinocyte HaCaT cells in a dose- and time-dependent manner without cytotoxicity. We then evaluated the protective role of TAT-RP1 against ionizing radiation. TAT-RP1 supplementation increased anti-superoxide anion ability of HaCaT cells and decreased HaCaT cell radiosensitivity to irradiation. Moreover, TAT-RP1 was able to penetrate the skin of rats, entering epidermis as well as the dermis of the subcutaneous layer in skin tissue. Topical spread of TAT-RP1 promoted the amelioration of radiation-induced skin damage in rats. These results suggest that TAT-RP1 has potential as a protein therapy for radiation-induced skin injury.
[Analysis of genetic recombination between human immunodeficiency virus type 1 (HIV-1) and HIV-2].
Motomura, Kazushi
It is estimated that one million people are dually infected with Human Immunodeficiency Virus type-I (HIV-1) and type-II (HIV-2) in West Africa and parts of India. HIV-1 and HIV-2 use the same receptor and coreceptors for entry into cells, and thus target the same cell populations in the host. Additionally, we first examined whether RNAs from HIV-1 and HIV-2 can be copackaged into the same virion. Therefore these properties suggest that in the dually infected population, it is likely that some cells can be infected by both HIV-1 and HIV-2, thereby providing opportunities for these two viruses to interact with each other. We constructed recombination assay system for measurement recombination frequencies and analyzed recombination rate between HIV-1 and HIV-2. We used modified near-full-length viruses that each contained a green fluorescent protein gene (gfp) with a different inactivating mutation. Thus, a functional gfp could be reconstituted via recombination, which was used to detect copackaging of HIV-1 and HIV-2 RNAs. In this study, approximately 0.2% of infection events generated the GFP phenotype. Therefore, the appearance of the GFP+ phenotype in the current system is approximately 35-fold lower than that between two homologous HIV-1 or HIV-2 viruses. We then mapped the general structures of the recombinant viruses and characterized the recombination junctions by DNA sequencing. We observed several different recombination patterns including those only had crossovers in gfp. The most common hybrid genomes had heterologous LTRs. Although infrequent, crossovers were also identified in the viral sequences. Such chimeric HIV-1 and HIV-2 viruses have yet to be observed in the infected population. It is unclear whether the lack of observed chimeras is due to the divergence between HIV-1 and HIV-2 being too great for such an event to occur, or whether such events could occur but have not yet been observed. Given the number of coinfected people, the potential for
Knockdown of ERM family member moesin in host cells increases HIV type 1 replication.
Capalbo, Gianni; Mueller-Kuller, Thea; Markovic, Sandra; Klein, Stefan A; Dietrich, Ursula; Hoelzer, Dieter; Ottmann, Oliver G; Scheuring, Urban J
Moesin is a member of the ERM (ezrin, radixin, moesin) family of cytoskeleton/membrane structure organizing and signal transduction proteins. Previously, we found an increased expression of moesin during HIV-1 infection. Moesin was also reported to be incorporated into HIV-1 virions. To analyze whether moesin is a host factor affecting the replication cycle of human immunodeficiency virus type 1 (HIV-1), we used small interfering RNAs (siRNAs) to evaluate the effect of moesin knockdown on HIV-1 replication in P4-CCR5 cells. Moesin's knockdown did not affect the cell viability or cell phenotype. Interestingly, we observed a marked increase in viral replication, as demonstrated by enhanced HIV-1 RNA, p24 antigen, and ß-galactosidase reporter expression. Moesin-dependent enhancement of HIV-1 replication was confirmed in lymphocytic host cells (Jurkat). These results suggest an overall rather restrictive role of moesin for HIV-1 replication in host cells in vitro.
NMR structure of the (1-51) N-terminal domain of the HIV-1 regulatory protein Vpr.
Wecker, K; Roques, B P
The human immunodeficiency virus type 1 (HIV-1) genome encodes a highly conserved 16 kDa regulatory gene product, Vpr (viral protein of regulation, 96 amino acid residues), which is incorporated into virions, in quantities equivalent to those of the viral Gag proteins. In the infected cells, Vpr is believed to function in the early phase of HIV-1 replication, including nuclear migration of preintegration complex, transcription of the provirus genome and viral multiplication by blocking cells in the G2 phase. Vpr has a critical role in long-term AIDS disease by inducing infection in nondividing cells such as monocytes and macrophages. Mutations have suggested that the N-terminal domain of Vpr encompassing the first 40 residues could be required for nuclear localization, packaging into virions and binding of transcription factor (TFIIB, Sp1), viral proteins (p6) and cellular proteins (RIP1, UNG, karyopherins). To gain insight into the structure-function relationship of Vpr, (1-51)Vpr was synthesized and its structure analyzed by circular dichroism and two-dimensional 1H NMR in aqueous trifluoroethanol (30%) solution and refined by restrained molecular dynamics. The structure is characterized by three turns around the first three prolines, Pro5, Pro10, Pro14, followed by a long amphipathic alpha helix-turn-alpha helix (Asp17-Ile46) motif ended by a turn extending from Tyr47 to Thr49. The alpha helix-turn-alpha helix motif and the amphipathic helix are well known for being implicated in protein-protein or protein-nucleic acid interaction. Therefore structural characteristics of the (1-51) N-terminal fragment of Vpr could explain why this region of Vpr plays a role in several biological functions of this protein.
RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study.
Bernacchi, Serena; Henriet, Simon; Dumas, Philippe; Paillart, Jean-Christophe; Marquet, Roland
The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors.
Clustering and Mobility of HIV-1 Env at Viral Assembly Sites Predict Its Propensity To Induce Cell-Cell Fusion
Roy, Nathan H.; Chan, Jany; Lambelé, Marie
HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env's fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag's ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells. PMID:23637402
Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope
Zolla-Pazner, Susan; Cohen, Sandra; Pinter, Abraham; Krachmarov, Chavdar; Wrin, Terri; Wang Shixia; Lu Shan
Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3{sub B}-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.
Pressure-induced structural transition of mature HIV-1 Protease from a combined NMR/MD simulation approach
Roche, Julien; Louis, John M.; Bax, Ad; Best, Robert B.
We investigate the pressure-induced structural changes in the mature human immunodeficiency virus type 1 protease dimer (HIV-1 PR), using residual dipolar coupling (RDC) measurements in a weakly oriented solution. 1DNH RDCs were measured under high-pressure conditions for an inhibitor-free PR and an inhibitor-bound complex, as well as for an inhibitor-free multidrug resistant protease bearing 20 mutations (PR20). While PR20 and the inhibitor-bound PR were little affected by pressure, inhibitor-free PR showed significant differences in the RDCs measured at 600 bar compared to 1 bar. The structural basis of such changes was investigated by MD simulations using the experimental RDC restraints, revealing substantial conformational perturbations, specifically a partial opening of the flaps and the penetration of water molecules into the hydrophobic core of the subunits at high-pressure. This study highlights the exquisite sensitivity of RDCs to pressure-induced conformational changes and illustrates how RDCs combined with MD simulations can be used to determine the structural properties of metastable intermediate states on the folding energy landscape. PMID:26385843
Different Mutagenic Potential of HIV-1 Restriction Factors APOBEC3G and APOBEC3F Is Determined by Distinct Single-Stranded DNA Scanning Mechanisms
Ara, Anjuman; Love, Robin P.; Chelico, Linda
The APOBEC3 deoxycytidine deaminase family functions as host restriction factors that can block replication of Vif (virus infectivity factor) deficient HIV-1 virions to differing degrees by deaminating cytosines to uracils in single-stranded (−)HIV-1 DNA. Upon replication of the (−)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite the uracils, thereby inducing C/G→T/A mutations that can functionally inactivate HIV-1. Although both APOBEC3F and APOBEC3G are expressed in cell types HIV-1 infects and are suppressed by Vif, there has been no prior biochemical analysis of APOBEC3F, in contrast to APOBEC3G. Using synthetic DNA substrates, we characterized APOBEC3F and found that similar to APOBEC3G; it is a processive enzyme and can deaminate at least two cytosines in a single enzyme-substrate encounter. However, APOBEC3F scanning movement is distinct from APOBEC3G, and relies on jumping rather than both jumping and sliding. APOBEC3F jumping movements were also different from APOBEC3G. The lack of sliding movement from APOBEC3F is due to an 190NPM192 motif, since insertion of this motif into APOBEC3G decreases its sliding movements. The APOBEC3G NPM mutant induced significantly less mutations in comparison to wild-type APOBEC3G in an in vitro model HIV-1 replication assay and single-cycle infectivity assay, indicating that differences in DNA scanning were relevant to restriction of HIV-1. Conversely, mutation of the APOBEC3F 191Pro to 191Gly enables APOBEC3F sliding movements to occur. Although APOBEC3F 190NGM192 could slide, the enzyme did not induce more mutagenesis than wild-type APOBEC3F, demonstrating that the unique jumping mechanism of APOBEC3F abrogates the influence of sliding on mutagenesis. Overall, we demonstrate key differences in the impact of APOBEC3F- and APOBEC3G-induced mutagenesis on HIV-1 that supports a model in which both the processive DNA scanning mechanism and preferred deamination motif (APOBEC3F, 5�
Combined HIV-1 Envelope Systemic and Mucosal Immunization of Lactating Rhesus Monkeys Induces a Robust Immunoglobulin A Isotype B Cell Response in Breast Milk
Nelson, Cody S.; Pollara, Justin; Kunz, Erika L.; Jeffries, Thomas L.; Duffy, Ryan; Beck, Charles; Stamper, Lisa; Wang, Minyue; Shen, Xiaoying; Pickup, David J.; Hudgens, Michael G.; Kepler, Thomas B.; Montefiori, David C.; Moody, M. Anthony; Tomaras, Georgia D.; Liao, Hua-Xin; Haynes, Barton F.; Ferrari, Guido; Fouda, Genevieve G. A.
ABSTRACT Maternal vaccination to induce anti-HIV immune factors in breast milk is a potential intervention to prevent postnatal HIV-1 mother-to-child transmission (MTCT). We previously demonstrated that immunization of lactating rhesus monkeys with a modified vaccinia Ankara (MVA) prime/intramuscular (i.m.) protein boost regimen induced functional IgG responses in milk, while MVA prime/intranasal (i.n.) boost induced robust milk Env-specific IgA responses. Yet, recent studies have suggested that prevention of postnatal MTCT may require both Env-specific IgA and functional IgG responses in milk. Thus, to investigate whether both responses could be elicited by a combined systemic/mucosal immunization strategy, animals previously immunized with the MVA prime/i.n. boost regimen received an i.n./i.m. combined C.1086 gp120 boost. Remarkably, high-magnitude Env-specific IgA responses were observed in milk, surpassing those in plasma. Furthermore, 29% of vaccine-elicited Env-specific B cells isolated from breast milk were IgA isotype, in stark contrast to the overwhelming predominance of IgG isotype Env-specific B cells in breast milk of chronically HIV-infected women. A clonal relationship was identified between Env-specific blood and breast milk B cells, suggesting trafficking of that cell population between the two compartments. Furthermore, IgA and IgG monoclonal antibodies isolated from Env-specific breast milk B cells demonstrated diverse Env epitope specificities and multiple effector functions, including tier 1 neutralization, antibody-dependent cellular cytotoxicity (ADCC), infected cell binding, and inhibition of viral attachment to epithelial cells. Thus, maternal i.n./i.m. combined immunization is a novel strategy to enhance protective Env-specific IgA in milk, which is subsequently transferred to the infant via breastfeeding. IMPORTANCE Efforts to increase the availability of antiretroviral therapy to pregnant and breastfeeding women in resource-limited areas
Inability of Plasmacytoid Dendritic Cells To Directly Lyse HIV-Infected Autologous CD4+ T Cells despite Induction of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand ▿
Chehimi, Jihed; Papasavvas, Emmanouil; Tomescu, Costin; Gekonge, Bethsebah; Abdulhaqq, Shaheed; Raymond, Andrea; Hancock, Aidan; Vinekar, Kavita; Carty, Craig; Reynolds, Griffin; Pistilli, Maxwell; Mounzer, Karam; Kostman, Jay; Montaner, Luis J.
The function of plasmacytoid dendritic cells (PDC) in chronic human immunodeficiency virus type 1 (HIV-1) infection remains controversial with regard to its potential for sustained alpha interferon (IFN-α) production and induction of PDC-dependent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity of HIV-infected cells. We address these areas by a study of chronically HIV-1-infected subjects followed through antiretroviral therapy (ART) interruption and by testing PDC cytolytic function against autologous HIV-infected CD4+ T cells. Rebound in viremia induced by therapy interruption showed a positive association between TRAIL and viral load or T-cell activation, but comparable levels of plasma IFN-α/β were found in viremic ART-treated and control subjects. While PDC from HIV-infected subjects expressed less interferon regulator factor 7 (IRF-7) and produced significantly less IFN-α upon Toll-like receptor 7/9 (TLR7/9) engagement than controls, membrane TRAIL expression in PDC from HIV+ subjects was increased. Moreover, no significant increase in death receptor 5 (DR5) expression was seen in CD4+ T cells from viremic HIV+ subjects compared to controls or following in vitro infection/exposure to infectious and noninfectious virus or exogenous IFN-α, respectively. Although activated PDC killed the DR5-expressing HIV-infected Sup-T1 cell line, PDC did not lyse primary autologous HIV+ CD4+ T cells yet could provide accessory help for NK cells in killing HIV-infected autologous CD4+ T cells. Taken together, our data show a lack of sustained high levels of soluble IFN-α in chronic HIV-1 infection in vivo and document a lack of direct PDC cytolytic activity against autologous infected or uninfected CD4+ T cells. PMID:20042498
Virion Structure of Iflavirus Slow Bee Paralysis Virus at 2.6-Angstrom Resolution
Kalynych, Sergei; Přidal, Antonín; Pálková, Lenka; Levdansky, Yevgen; de Miranda, Joachim R.
ABSTRACT The western honeybee (Apis mellifera) is the most important commercial insect pollinator. However, bees are under pressure from habitat loss, environmental stress, and pathogens, including viruses that can cause lethal epidemics. Slow bee paralysis virus (SBPV) belongs to the Iflaviridae family of nonenveloped single-stranded RNA viruses. Here we present the structure of the SBPV virion determined from two crystal forms to resolutions of 3.4 Å and 2.6 Å. The overall structure of the virion resembles that of picornaviruses, with the three major capsid proteins VP1 to 3 organized into a pseudo-T3 icosahedral capsid. However, the SBPV capsid protein VP3 contains a C-terminal globular domain that has not been observed in other viruses from the order Picornavirales. The protruding (P) domains form "crowns" on the virion surface around each 5-fold axis in one of the crystal forms. However, the P domains are shifted 36 Å toward the 3-fold axis in the other crystal form. Furthermore, the P domain contains the Ser-His-Asp triad within a surface patch of eight conserved residues that constitutes a putative catalytic or receptor-binding site. The movements of the domain might be required for efficient substrate cleavage or receptor binding during virus cell entry. In addition, capsid protein VP2 contains an RGD sequence that is exposed on the virion surface, indicating that integrins might be cellular receptors of SBPV. IMPORTANCE Pollination by honeybees is needed to sustain agricultural productivity as well as the biodiversity of wild flora. However, honeybee populations in Europe and North America have been declining since the 1950s. Honeybee viruses from the Iflaviridae family are among the major causes of honeybee colony mortality. We determined the virion structure of an Iflavirus, slow bee paralysis virus (SBPV). SBPV exhibits unique structural features not observed in other picorna-like viruses. The SBPV capsid protein VP3 has a large C
HIV-1 vaccine-specific responses induced by Listeria vector vaccines are maintained in mice subsequently infected with a model helminth parasite, Schistosoma mansoni.
Shollenberger, Lisa M; Bui, Cac T; Paterson, Yvonne; Nyhoff, Lindsay; Harn, Donald A
In areas co-endemic for helminth parasites and HIV/AIDS, infants are often administered vaccines prior to infection with immune modulatory helminth parasites. Systemic Th2 biasing and immune suppression caused by helminth infection reduces cell-mediated responses to vaccines such as tetanus toxoid and BCG. Therefore, we asked if infection with helminthes post-vaccination, alters already established vaccine induced immune responses. In our model, mice are vaccinated against HIV-1 Gag using a Listeria vaccine vector (Lm-Gag) in a prime-boost manner, then infected with the human helminth parasite Schistosoma mansoni. This allows us to determine if established vaccine responses are maintained or altered after helminth infection. Our second objective asked if helminth infection post-vaccination alters the recipient's ability to respond to a second boost. Here we compared responses between uninfected mice, schistosome infected mice, and infected mice that were given an anthelminthic, which occurred coincident with the boost or four weeks prior, as well as comparing to un-boosted mice. We report that HIV-1 vaccine-specific responses generated by Listeria vector HIV-1 vaccines are maintained following subsequent chronic schistosome infection, providing further evidence that Listeria vector vaccines induce potent vaccine-specific responses that can withstand helminth infection. We also were able to demonstrate that administration of a second Listeria boost, which markedly enhanced the immune response, was minimally impacted by schistosome infection, or anthelminthic therapy. Surprisingly, we also observed enhanced antibody responses to HIV Gag in vaccinated mice subsequently infected with schistosomes.
HIV-1 Accessory Proteins: Vpu and Vif
Andrew, Amy; Strebel, Klaus
HIV-1 Vif and Vpu are accessory factors involved in late stages of viral replication. Vif regulates viral infectivity by preventing virion incorporation of APOBEC3G and other members of the family of cytidine deaminases, while Vpu causes degradation of CD4 and promotes virus release by functionally inactivating the host factor BST-2. This chapter described techniques used for the characterization of Vif and Vpu and their functional interaction with host factors. Many of the techniques are, however, applicable to the functional analysis of other viral proteins. PMID:24158820
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Flow virometric sorting and analysis of HIV quasispecies from plasma
Jones, Jennifer C.; Keele, Brandon F.; Jenkins, Lisa M. Miller; Demberg, Thorsten
Flow cytometry is utilized extensively for cellular analysis, but technical limitations have prevented its routine application for characterizing virus. The recent introduction of nanoscale fluorescence-activated cytometric cell sorting now allows analysis of individual virions. Here, we demonstrate staining and sorting of infectious HIV. Fluorescent antibodies specific for cellular molecules found on budding virions were used to label CCR5-tropic Bal HIV and CXCR4-tropic NL4.3 HIV Env-expressing pseudovirions made in THP-1 cells (monocyte/macrophage) and H9 cells (T cells), respectively. Using a flow cytometer, we resolved the stained virus beyond isotype staining and demonstrated purity and infectivity of sorted virus populations on cells with the appropriate coreceptors. We subsequently sorted infectious simian/human immunodeficiency virus from archived plasma. Recovery was approximately 0.5%, but virus present in plasma was already bound to viral-specific IgG generated in vivo, likely contributing to the low yield. Importantly, using two broadly neutralizing HIV antibodies, PG9 and VRC01, we also sorted virus from archived human plasma and analyzed the sorted populations genetically and by proteomics, identifying the quasispecies present. The ability to sort infectious HIV from clinically relevant samples provides material for detailed molecular, genetic, and proteomic analyses applicable to future design of vaccine antigens and potential development of personalized treatment regimens. PMID:28239654
Different HIV pox viral vector-based vaccines and adjuvants can induce unique antigen presenting cells that modulate CD8 T cell avidity.
Trivedi, Shubhanshi; Jackson, Ronald J; Ranasinghe, Charani
The lung-derived dendritic cell (LDC) recruitment following intranasal (i.n.) vaccination of different poxviral vector-based vaccines/adjuvants were evaluated to decipher how these factors influenced CD8(+) T cell avidity. Compared to the standard i.n. recombinant fowlpox virus (FPV)-HIV vaccination, the FPV-HIV IL-13Rα2 or IL-4Rα antagonist adjuvanted vaccines that induced higher avidity CD8(+) T cells, also recruited significantly elevated MHCII(+) CD11c(+) CD11b(+) CD103(-) CD64(-) MAR-1(-) conventional DC (cDCs) to the lung mucosae (hierarchy: IL-4R antagonist>IL-13Rα2>unadjuvanted). In contrast, elevated CD11b(-) CD103(+) LDCs were detected in animals that received recombinant HIV vaccinia virus (rVV) or Modified Vaccinia Ankara virus (MVA) vector-based vaccines. Adoptive transfer studies indicated that CD11b(-) CD103(+) LDCs significantly dampened HIV-specific CD8(+) T cell avidity compared to CD11b(+) CD103(-) LDCs. Collectively; our observations revealed that rFPV vector prime and transient inhibition of IL-4/IL-13 at the vaccination site favoured the recruitment of unique LDCs, associated with the induction of high quality immunity.
Transcriptome analysis of rainbow trout in response to non-virion (NV) protein of viral haemorrhagic septicaemia virus (VHSV).
Chinchilla, Blanca; Encinas, Paloma; Estepa, Amparo; Coll, Julio M; Gomez-Casado, Eduardo
The non-virion (NV) protein of viral haemorrhagic septicaemia virus (VHSV), an economically important fish novirhabdovirus, has been implicated in the interference of some host innate mechanisms (i.e. apoptosis) in vitro. This work aimed to characterise the immune-related transcriptome changes in rainbow trout induced by NV protein that have not yet been established in vivo. For that purpose, immune-targeted microarrays were used to analyse the transcriptomes from head kidney and spleen of rainbow trout (Oncorhynchus mykiss) after injection of recombinant NV (rNV). Results showed the extensive downregulation (and in some cases upregulation) of many innate and adaptive immune response genes not related previously to VHSV infection. The newly identified genes belonged to VHSV-induced genes (vigs), tumour necrosis factors, Toll-like receptors, antigen processing and presentation, immune co-stimulatory molecules, interleukins, macrophage chemotaxis, transcription factors, etc. Classification of differentially downregulated genes into rainbow trout immune pathways identified stat1 and jun/atf1 transcription factor genes as the most representative of the multipath gene targets of rNV. Altogether, these results contribute to define the role and effects of NV in trout by orchestrating an immunosuppression of the innate immune responses for favouring viral replication upon VHSV infection. Finally, these transcriptome results open up the possibility to find out new strategies against VHSV and better understand the interrelationships between some immune pathways in trout.
Virology: The Next Generation from Digital PCR to Single Virion Genomics
White, Richard A.; Brazelton De Cardenas, Jessica N.; Hayden, Randall T.
In the past 25 years, virology has had major technology breakthroughs stemming first from the introduction of nucleic acid amplification testing, but more recently from the use of next-generation sequencing, digital PCR, and the possibility of single virion genomics. These technologies have and will improve diagnosis and disease state monitoring in clinical settings, aid in environmental monitoring, and reveal the vast genetic potential of viruses. Using the principle of limiting dilution, digital PCR amplifies single molecules of DNA in highly partitioned endpoint reactions and reads each of those reactions as either positive or negative based on the presence or absence of target fluorophore. In this review, digital PCR will be highlighted along with current studies, advantages/disadvantages, and future perspectives with regard to digital PCR, viral load testing, and the possibility of single virion genomics.
Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions.
Holland, J J; de la Torre, J C; Steinhauer, D A; Clarke, D; Duarte, E; Domingo, E
Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach. Images PMID:2479770
Arakelyan, Anush; Fitzgerald, Wendy; Zicari, Sonia; Grivel, Murad Vagida Jean-Charles; Margolis, Leonid
To characterize antigenic spectra of individual viruses or extracellular vesicles we immunocapture them with 15nm magnetic nanoparticles (MNPs) coupled to antibodies that recognize a surface antigen of interest. The captured virions or vesicles are labeled with fluorescent antibodies against other surface antigens and the resultant complexes are separated from unbound antibodies in a high magnetic field followed by analysis on conventional flow cytometer triggered on fluorescence. PMID:28190041
Productive replication and evolution of HIV-1 in ferret cells.
Fadel, Hind J; Saenz, Dyana T; Guevara, Rebekah; von Messling, Veronika; Peretz, Mary; Poeschla, Eric M
A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 dependency factors. All Feliformia species and at least one Caniformia species also lack a major lentiviral restriction mechanism (TRIM5α/TRIMCyp proteins). Here we investigated cells from two species in another carnivore family, the Mustelidae, for permissiveness to the HIV-1 life cycle. Mustela putorius furo (domesticated ferret) primary cells and cell lines did not restrict HIV-1, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or N-tropic murine leukemia virus (MLV) postentry and supported late HIV-1 life cycle steps comparably to human cells. The ferret TRIM5α gene exon 8, which encodes the B30.2 domain, was found to be pseudogenized. Strikingly, ferret (but not mink) cells engineered to express human HIV-1 entry receptors supported productive spreading replication, amplification, and serial passage of wild-type HIV-1. Nevertheless, produced virions had relatively reduced infectivity and the virus accrued G→A hypermutations, consistent with APOBEC3 protein pressure. Ferret cell-passaged HIV-1 also evolved amino acid changes in the capsid cyclophilin A binding loop. We conclude that the genome of this carnivore can provide essential nonreceptor HIV-1 dependency factors and that ferret APOBEC3 proteins with activity against HIV-1 are likely. Even so, unlike in cat cells, HIV-1 can replicate in ferret cells without vif substitution. The virus evolves in this novel nonprimate cell adaptive landscape. We suggest that further characterization of HIV-1 adaptation in ferret cells and delineation of Mustelidae restriction factor repertoires are warranted, with a view to the potential for an HIV-1
Drug-induced haemolysis, renal failure, thrombocytopenia and lactic acidosis in patients with HIV and cryptococcal meningitis: a diagnostic challenge.
Camara-Lemarroy, Carlos R; Flores-Cantu, Hazael; Calderon-Hernandez, Hector J; Diaz-Torres, Marco A; Villareal-Velazquez, Hector J
Patients with HIV are at risk of both primary and secondary haematological disorders. We report two cases of patients with HIV and cryptococcal meningitis who developed severe haemolytic anaemia, thrombocytopenia, renal failure and lactic acidosis while on treatment with amphotericin B and co-trimoxazole.
Comparative clonal analysis of human immunodeficiency virus type 1 (HIV- 1)-specific CD4+ and CD8+ cytolytic T lymphocytes isolated from seronegative humans immunized with candidate HIV-1 vaccines
The lysis of infected host cells by virus-specific cytolytic T lymphocytes (CTL) is an important factor in host resistance to viral infection. An optimal vaccine against human immunodeficiency virus type 1 (HIV-1) would elicit virus-specific CTL as well as neutralizing antibodies. The induction by a vaccine of HIV-1-specific CD8+ CTL in humans has not been previously reported. In this study, CTL responses were evaluated in HIV-1-seronegative human volunteers participating in a phase I acquired immune deficiency syndrome (AIDS) vaccine trial involving a novel vaccine regimen. Volunteers received an initial immunization with a live recombinant vaccinia virus vector carrying the HIV-1 env gene and a subsequent boost with purified env protein. An exceptionally strong env-specific CTL response was detected in one of two vaccine recipients, while modest but significant env-specific CTL activity was present in the second vaccinee. Cloning of the responding CTL gave both CD4+ and CD8+ env-specific CTL clones, permitting a detailed comparison of critical functional properties of these two types of CTL. In particular, the potential antiviral effects of these CTL were evaluated in an in vitro system involving HIV-1 infection of cultures of normal autologous CD4+ lymphoblasts. At extremely low effector-to-target ratios, vaccine-induced CD8+ CTL clones lysed productively infected cells present within these cultures. When tested for lytic activity against target cells expressing the HIV-1 env gene, CD8+ CTL were 3-10-fold more active on a per cell basis than CD4+ CTL. However, when tested against autologous CD4+ lymphoblasts acutely infected with HIV-1, CD4+ clones lysed a much higher fraction of the target cell population than did CD8+ CTL. CD4+ CTL were shown to recognize not only the infected cells within these acutely infected cultures but also noninfected CD4+ T cells that had passively taken up gp120 shed from infected cells and/or free virions. These results were
BLyS-mediated modulation of naïve B cell subsets impacts HIV Env-induced antibody responses1
Dosenovic, Pia; Soldemo, Martina; Scholz, Jean L.; O'Dell, Sijy; Grasset, Emilie K.; Pelletier, Nadège; Karlsson, Mikael C. I.; Mascola, John R.; Wyatt, Richard T.; Cancro, Michael P.; Karlsson Hedestam, Gunilla B.
Neutralizing Abs provide the protective effect of the majority of existing human vaccines. For a prophylactic vaccine against HIV-1, broadly neutralizing Abs (bNAbs) targeting conserved epitopes of the viral envelope glycoproteins (Env) are likely required, as the pool of circulating HIV-1 variants is extremely diverse. The failure to efficiently induce bNAbs by vaccination may be due to the use of sub-optimal immunogens or immunization regimens, or it may indicate that B cells specific for broadly neutralizing Env determinants are selected against during peripheral checkpoints, either before or after antigen encounter. To investigate if perturbation of B cell subsets prior to immunization with recombinant Env protein affects the vaccine-induced Ab response in mice, we used B Lymphocyte Stimulator (BLyS), a cytokine that regulates survival and selection of peripheral B cells. We show that the transient BLyS treatment used here substantially affected naïve B cell populations; in particular, it resulted in an increased number of B cells surviving counter-selection at the transitional stages. We also observed an increased number of mature naïve B cells, especially marginal zone B cells, in BLyS-treated mice. Intriguingly, provision of excess BLyS prior to immunization led to a consistent improvement in the frequency and potency of HIV-1 Env vaccine-induced neutralizing Ab responses, without increasing the number of Env-specific Ab-secreting cells or the Ab binding titers measured after boosting. The results presented here suggest that an increased understanding of BLyS-regulated processes may help the design of vaccine regimens aimed at eliciting improved neutralizing Ab responses against HIV-1. PMID:22561155
Cryo-electron microscopy of hepatitis B virions reveals variability in envelope capsid interactions
Seitz, Stefan; Urban, Stephan; Antoni, Christoph; Böttcher, Bettina
Hepatitis B virus (HBV) is a major human pathogen causing about 750 000 deaths per year. The virion consists of a nucleocapsid and an envelope formed by lipids, and three integral membrane proteins. Although we have detailed structural insights into the organization of the HBV core, the arrangement of the envelope in virions and its interaction with the nucleocapsid is elusive. Here we show the ultrastructure of hepatitis B virions purified from patient serum. We identified two morphological phenotypes, which appear as compact and gapped particles with nucleocapsids in distinguishable conformations. The overall structures of these nucleocapsids resemble recombinant cores with two α-helical spikes per asymmetric unit. At the charged tips the spikes are contacted by defined protrusions of the envelope proteins, probably via electrostatic interactions. The HBV envelope in the two morphotypes is to some extent variable, but the surface proteins follow a general packing scheme with up to three surface protein dimers per asymmetric unit. The variability in the structure of the envelope indicates that the nucleocapsid does not firmly constrain the arrangement of the surface proteins, but provides a general template for the packing. PMID:17762862
Proteomic Analysis of Mamestra Brassicae Nucleopolyhedrovirus Progeny Virions from Two Different Hosts
Hou, Dianhai; Chen, Xi
Mamestra brassicae nucleopolyhedrovirus (MabrNPV) has a wide host range replication in more than one insect species. In this study, a sequenced MabrNPV strain, MabrNPV-CTa, was used to perform proteomic analysis of both BVs and ODVs derived from two infected hosts: Helicoverpa armigera and Spodoptera exigua. A total of 82 and 39 viral proteins were identified in ODVs and BVs, respectively. And totally, 23 and 76 host proteins were identified as virion-associated with ODVs and BVs, respectively. The host proteins incorporated into the virus particles were mainly involved in cytoskeleton, signaling, vesicle trafficking, chaperone and metabolic systems. Some host proteins, such as actin, cyclophilin A and heat shock protein 70 would be important for viral replication. Several host proteins involved in immune response were also identified in BV, and a C-type lectin protein was firstly found to be associated with BV and its family members have been demonstrated to be involved in entry process of other viruses. This study facilitated the annotation of baculovirus genome, and would help us to understand baculovirus virion structure. Furthermore, the identification of host proteins associated with virions produced in vivo would facilitate investigations on the involvement of intriguing host proteins in virus replication. PMID:27058368
Exocytosis of Alphaherpesvirus Virions, Light Particles, and Glycoproteins Uses Constitutive Secretory Mechanisms
Hogue, Ian B.; Scherer, Julian
ABSTRACT Many molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV) exocytosis, based on total internal reflection fluorescence (TIRF) microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i) trafficking of viral glycoproteins to the plasma membrane, (ii) exocytosis of viral light particles, and (iii) exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway. PMID:27273828
The evolution of structured illumination microscopy in studies of HIV.
Marno, Kelly; Al'Zoubi, Lara; Pearson, Matthew; Posch, Markus; McKnight, Áine; Wheeler, Ann P
The resolution limit of conventional light microscopy has proven to be limiting for many biological structures such as viruses including Human immunodeficiency virus (HIV). Individual HIV virions are impossible to study using confocal microscopy as they are well below the 200 nm resolution limit of conventional light microscopes. Structured illumination microscopy (SIM) allows a twofold enhancement in image resolution compared to standard widefield illumination and so provides an excellent tool for study of HIV. Viral capsids (CAs) vary between 110 and 146 nm so this study challenges the performance of SIM microscopes. SIM microscopy was first developed in 2000, commercialised in 2007 and rapidly developed. Here we present the changes in capabilities of the SIM microscopes for study of HIV localisation as the instrumentation for structured illumination microscopy has evolved over the past 8 years.
Emerging studies of human HIV-specific antibody repertoires
Hicar, Mark D.; Kalams, Spyros A.; Spearman, Paul W.; Crowe, James E.
There has been an explosion of interest in the human B cell response to HIV infection of late. Recent advances in techniques for isolation of human antibodies and antibody secreting cell lines have facilitated a rapid expansion in the number of antibodies available for study. Early analysis of these repertoires reveals interesting features of the HIV-specific antibody response. HIV-specific repertoires exhibit a high level of clonality in circulating cells, and high levels of somatic mutations within the antibody variable gene segments. It appears that many if not most antibodies in circulation bind to virus envelope conformations that are found only in complex oligomeric structures on virion particles or virus-like particles. The rapid isolation of large panels of novel human neutralizing antibodies promises to reveal new insights into the fundamental principles underlying antibody-mediated neutralization of HIV. PMID:20510738
The Molecular Characterization of Intestinal Explant HIV Infection Using Polymerase Chain Reaction-Based Techniques
Janocko, Laura; Althouse, Andrew D.; Brand, Rhonda M.; Cranston, Ross D.
Abstract The ex vivo mucosal explant model is frequently used to test the efficacy of microbicides that have the potential for preventing HIV-1 transmission. The conventional assessment of product efficacy has been the extent of HIV-1 p24 suppression in supernatant fluids sampled up to day 14 after HIV-1 challenge ex vivo. The purpose of this study was to determine if measurement of HIV-1 nucleic acids by real-time PCR and HIV-1 integration by Alu-gag PCR provides advantages with regard to monitoring HIV-1 infection in explants. Rectal biopsies from HIV-1-negative individuals were challenged with 1 × 105 virions/ml of HIV-1BaL or HIV-1CH077 ex vivo. HIV-1 RNA and HIV-1 p24 in supernatant fluids and HIV-1 nucleic acids and integrated provirus in individual biopsies were measured at days 1–14 after infection. HIV-1 RNA and proviral DNA were measured by quantitative real-time PCR (qRT-PCR) while integrated virus was detected by Alu-gag PCR. Real-time PCR assays detecting HIV-1 DNA and RNA performed similarly provided that the infecting virus sequences were a good match with the sequences of the assay primers and probes. Increased HIV-1 nucleic acid levels and DNA integration were measurable on days 11 and 14 after infection. The magnitude of explant infection was similar after challenge with HIV-1BaL and HIV-1CH077, although the trajectory of infection was delayed in the HIV-1CH077-infected biopsies. In the majority of experiments, qRT-PCR did not appreciably shorten the time necessary to detect evidence of HIV-1 infection. PMID:26214703
Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus.
Roby, C; Gibson, W
Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of particles phosphorylated in vivo showed the following. Virions contain three predominant phosphoproteins (i.e., basic phosphoprotein and upper and lower matrix proteins) and at least nine minor phosphorylated species. NIEPs contain all of these and one additional major species, the assembly protein. Dense bodies contain only one (i.e., lower matrix) of the predominant and four of the minor virion phosphoproteins. Two-dimensional (charge-size) separations in denaturing polyacrylamide gels showed that the relative net charges of the predominant phosphorylated species ranged from the basic phosphoprotein to the more neutral upper matrix protein. In vitro assays showed that purified virions of human CMV have an associated protein kinase activity. The activity was detected only after disrupting the envelope; it had a pH optimum of approximately 9 to 9.5 and required a divalent cation, preferring magnesium to manganese. In vitro, this activity catalyzed phosphorylation of the virion proteins observed to be phosphorylated in vivo. Peptide comparisons indicated that the sites phosphorylated in vitro are a subset of those phosphorylated in vivo, underscoring the probable biological relevance of the kinase activity. Casein, phosvitin, and to a minor extent lysine-rich histones served as exogenous phosphate acceptors. Arginine-rich and lysine-rich histones and protamine sulfate, as well as the polyamines spermine and spermidine, stimulated incorporation of phosphate into the endogenous viral proteins. Virions of all human and simian CMV strains tested showed this activity. Analyses of other virus particles, including three intracellular capsid forms (i.e., A, B, and C capsids), NIEPs, and dense bodies, indicated that the active enzyme was not present in the capsid. Rate-velocity sedimentation of disrupted virions
T cells induced by recombinant chimpanzee adenovirus alone and in prime-boost regimens decrease chimeric EcoHIV/NDK challenge virus load.
Roshorm, Yaowaluck; Cottingham, Mathew G; Potash, Mary-Jane; Volsky, David J; Hanke, Tomáš
The popularity of nonreplicating adenoviruses of chimpanzee origin (ChAdVs) as vectors for subunit vaccines is on the rise. This is mainly for their excellent safety and impressive immunogenicity observed in human studies to date. Here, we recloned the chimpanzee adenovirus sero type 68 (ChAdV-68), also designated SAdV-25 and AdC68, genome and demonstrated its straightforward genetic manipulation facilitated by the use of bacterial artificial chromosome recombineering. To generate the ChAdV68.GagB vaccine, the HIV-1 consensus clade B Gag-derived Tg was inserted into the E1 region. In part confirming previous observations, the ChAdV68.GagB vaccine alone and in heterologous prime-boost regimens with plasmid DNA- and modified vaccinia virus Ankara (MVA)-vectored vaccines induced robust polyfunctional HIV-1-specific CD8(+) and CD4(+) T-cell responses with a gut-homing phenotype. Importantly, we showed that when a single epitope is expressed as an immunodominant CD8(+) T-cell determinant, responses elicited by ChAdV68.GagB alone and in combination lowered surrogate challenge EcoHIV/NDK (where EcoHIV is chimeric ecotropic HIV) virus load in mice both at the peak T-cell frequencies 2 weeks after vaccination and 16 weeks later indicating development of protective effector memory. These results parallel the immunogenicity of similar vaccine regimens in macaques and an ongoing phase I/IIa trial in humans, and support further development of vaccines vectored by ChAdVs.
HIV-1 protein Tat potentiation of methamphetamine-induced decreases in evoked overflow of dopamine in the striatum of the rat.
Cass, Wayne A; Harned, Michael E; Peters, Laura E; Nath, Avindra; Maragos, William F
HIV-1 infection of the brain can lead to the development of clinical syndromes reminiscent of Parkinson's disease, suggesting that HIV infection may damage nigrostriatal dopamine (DA) neurons. Although the responsible mechanisms have not been well defined, neurotoxic viral proteins, such as Tat, released from infected cells may be involved. Drug abuse is a major risk factor for contracting HIV infection. Methamphetamine (METH), a psychostimulant with high abuse potential, may also be toxic to brain DA neurons. Thus, the combination of METH abuse and HIV infection may lead to substantial alterations in DA neuron functioning. The present experiments examined how Tat, alone and with METH, affects DA release in the striatum. Male rats were given an intrastriatal injection of Tat (25 micro g) or vehicle 24 h before treatment with saline or neurotoxic doses of METH. Seven days later microdialysis studies were carried out to measure potassium- and amphetamine-evoked overflow of DA from the striatum. The Tat treatment alone led to no change in potassium-evoked overflow of DA, a 20% decrease in amphetamine-evoked overflow of DA, and a 16% decrease in striatal DA content. The METH alone led to a 37-42% decrease in striatal DA overflow and content. The combined treatment with Tat and METH led to significantly greater 70-78% decreases in striatal DA overflow and content. These results indicate that Tat enhances METH-induced reductions in striatal DA release and content, possibly in a synergistic manner, and suggest that METH abusers infected with HIV may be at increased risk for basal ganglia dysfunction.
An immunogen containing four tandem 10E8 epitope repeats with exposed key residues induces antibodies that neutralize HIV-1 and activates an ADCC reporter gene.
Sun, Zhiwu; Zhu, Yun; Wang, Qian; Ye, Ling; Dai, Yanyan; Su, Shan; Yu, Fei; Ying, Tianlei; Yang, Chinglai; Jiang, Shibo; Lu, Lu
After three decades of intensive research efforts, an effective vaccine against HIV-1 remains to be developed. Several broadly neutralizing antibodies to HIV-1, such as 10E8, recognize the membrane proximal external region (MPER) of the HIV-1 gp41 protein. Thus, the MPER is considered to be a very important target for vaccine design. However, the MPER segment has very weak immunogenicity and tends to insert its epitope residues into the cell membrane, thereby avoiding antibody binding. To address this complication in vaccine development, we herein designed a peptide, designated 10E8-4P, containing four copies of the 10E8 epitope as an immunogen. As predicted by structural simulation, 10E8-4P exhibits a well-arranged tandem helical conformation, with the key residues in the 10E8 epitope oriented at different angles, thus suggesting that some of these key residues may be exposed outside of the lipid membrane. Compared with a peptide containing a single 10E8 epitope (10E8-1P), 10E8-4P not only exhibited better antigenicity but also elicited neutralizing antibody response against HIV-1 pseudoviruses, whereas 10E8-1P could not induce detectable neutralizing antibody response. Importantly, antibodies elicited by 10E8-4P also possessed a strong ability to activate an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter gene, thus suggesting that they may have ADCC activity. Therefore, this strategy shows promise for further optimization and application in future HIV-1 vaccine design.
Estimating the stoichiometry of HIV neutralization.
Magnus, Carsten; Regoes, Roland R
HIV-1 virions infect target cells by first establishing contact between envelope glycoprotein trimers on the virion's surface and CD4 receptors on a target cell, recruiting co-receptors, fusing with the cell membrane and finally releasing the genetic material into the target cell. Specific experimental setups allow the study of the number of trimer-receptor-interactions needed for infection, i.e., the stoichiometry of entry and also the number of antibodies needed to prevent one trimer from engaging successfully in the entry process, i.e., the stoichiometry of (trimer) neutralization. Mathematical models are required to infer the stoichiometric parameters from these experimental data. Recently, we developed mathematical models for the estimations of the stoichiometry of entry [1]. In this article, we show how our models can be extended to investigate the stoichiometry of trimer neutralization. We study how various biological parameters affect the estimate of the stoichiometry of neutralization. We find that the distribution of trimer numbers-which is also an important determinant of the stoichiometry of entry-influences the estimated value of the stoichiometry of neutralization. In contrast, other parameters, which characterize the experimental system, diminish the information we can extract from the data about the stoichiometry of neutralization, and thus reduce our confidence in the estimate. We illustrate the use of our models by re-analyzing previously published data on the neutralization sensitivity [2], which contains measurements of neutralization sensitivity of viruses with different envelope proteins to antibodies with various specificities. Our mathematical framework represents the formal basis for the estimation of the stoichiometry of neutralization. Together with the stoichiometry of entry, the stoichiometry of trimer neutralization will allow one to calculate how many antibodies are required to neutralize a virion or even an entire population of
Intervention induced changes on parenting practices, youth self-pride and sexual norms to reduce HIV-related behaviors among rural African American youths.
Murry, Velma McBride; Berkel, Cady; Chen, Yi-Fu; Brody, Gene H; Gibbons, Frederick X; Gerrard, Meg
AIDS is the leading killer of African Americans between the ages of 25 and 44, many of whom became infected when they were teenagers or young adults. The disparity in HIV infection rate among African Americans youth residing in rural Southern regions of the United States suggests that there is an urgent need to identify ways to promote early preventive intervention to reduce HIV-related risk behavior. The Strong African American Families (SAAF) program, a preventive intervention for rural African American parents and their 11-year-olds, was specially designed to deter early sexual onset and the initiation and escalation of alcohol and drug use among rural African American preadolescents. A clustered-randomized prevention trial was conducted, contrasting families who took part in SAAF with control families. The trial, which included 332 families, indicated that intervention-induced changes occurred in intervention-targeted parenting, which in turn facilitated changes in youths' internal protective processes and positive sexual norms. Long-term follow up assessments when youth were 17 years old revealed that intervention-induced changes in parenting practices mediated the effect of intervention-group influences on changes in the onset and escalation of risky sexual behaviors over 65 months through its positive influence on adolescents' self-pride and their sexual norms. The findings underscore the powerful effects of parenting practices among rural African American families that over time serve a protective role in reducing youth's risk behavior, including HIV vulnerable behaviors.
Viral protein R of HIV type-1 induces retrotransposition and upregulates glutamate synthesis by the signal transducer and activator of transcription 1 signaling pathway.
Doi, Akihiro; Iijima, Kenta; Kano, Shigeyuki; Ishizaka, Yukihito
Viral protein R (Vpr) of HIV-1 plays an important role in viral replication in macrophages. Various lines of evidence suggest that expression of Vpr in macrophages causes immunopathogenesis; however, the underlying mechanism is not yet fully understood. In this study, it was shown that recombinant Vpr (rVpr) induces retrotransposition of long interspersed element-1 in RAW264.7, a macrophage-like cell line, and activates reverse transcriptase-dependent immunotoxic cascades including production of IFN-β and phosphorylation of signal transducer and activator of transcription 1 (STAT1). Knockout experiments based on the CRISPR/Cas9 nickase system further demonstrated that cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and stimulator of interferon gene (STING) are responsible for IFN-β production and STAT1 phosphorylation, respectively. Moreover, rVpr was found to increase production of glutaminase C, a regulator of glutamate synthesis, which is also dependent on the cGAS-STING pathway. Taken together with reports that glutaminase C is involved in the pathogenesis of HIV-associated neurocognitive disorder (HAND) and that Vpr is detectable in the cerebrospinal fluid of HIV-1-positive patients, a possible role of Vpr-induced L1-RTP and immunotoxic cascades in the development of HAND is discussed.
The virion of Cafeteria roenbergensis virus (CroV) contains a complex suite of proteins for transcription and DNA repair.
Fischer, Matthias G; Kelly, Isabelle; Foster, Leonard J; Suttle, Curtis A
Cafeteria roenbergensis virus (CroV) is a giant virus of the Mimiviridae family that infects the marine phagotrophic flagellate C. roenbergensis. CroV possesses a DNA genome of ~730 kilobase pairs that is predicted to encode 544 proteins. We analyzed the protein composition of purified CroV particles by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified 141 virion-associated CroV proteins and 60 host proteins. Data are available via ProteomeXchange with identifier PXD000993. Predicted functions could be assigned to 36% of the virion proteins, which include structural proteins as well as enzymes for transcription, DNA repair, redox reactions and protein modification. Homologs of 36 CroV virion proteins have previously been found in the virion of Acanthamoeba polyphaga mimivirus. The overlapping virion proteome of CroV and Mimivirus reveals a set of conserved virion protein functions that were presumably present in the last common ancestor of the Mimiviridae.
HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR
Vivarini, Áislan de Carvalho; Santos Pereira, Renata de Meirelles; Barreto-de-Souza, Victor; Temerozo, Jairo Ramos; Soares, Deivid C.; Saraiva, Elvira M.; Saliba, Alessandra Mattos; Bou-Habib, Dumith Chequer; Lopes, Ulisses Gazos
HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49–57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling. PMID:26608746
Antigenicity and immunogenicity of HIV-1 consensus subtype B envelope glycoproteins
Kothe, Denise L.; Decker, Julie M.; Li Yingying; Weng Zhiping; Bibollet-Ruche, Frederic; Zammit, Kenneth P.; Salazar, Maria G.; Chen, Yalu; Salazar-Gonzalez, Jesus F.; Moldoveanu, Zina; Mestecky, Jiri; Gao Feng; Haynes, Barton F.; Shaw, George M. ||; Muldoon, Mark; Korber, Bette T.M. |; Hahn, Beatrice H. |. E-mail: firstname.lastname@example.com
'Centralized' (ancestral and consensus) HIV-1 envelope immunogens induce broadly cross-reactive T cell responses in laboratory animals; however, their potential to elicit cross-reactive neutralizing antibodies has not been fully explored. Here, we report the construction of a panel of consensus subtype B (ConB) envelopes and compare their biologic, antigenic, and immunogenic properties to those of two wild-type Env controls from individuals with early and acute HIV-1 infection. Glycoprotein expressed from full-length (gp160), uncleaved (gp160-UNC), truncated (gp145), and N-linked glycosylation site deleted (gp160-201N/S) versions of the ConB env gene were packaged into virions and, except for the fusion defective gp160-UNC, mediated infection via the CCR5 co-receptor. Pseudovirions containing ConB Envs were sensitive to neutralization by patient plasma and monoclonal antibodies, indicating the preservation of neutralizing epitopes found in contemporary subtype B viruses. When used as DNA vaccines in guinea pigs, ConB and wild-type env immunogens induced appreciable binding, but overall only low level neutralizing antibodies. However, all four ConB immunogens were significantly more potent than one wild-type vaccine at eliciting neutralizing antibodies against a panel of tier 1 and tier 2 viruses, and ConB gp145 and gp160 were significantly more potent than both wild-type vaccines at inducing neutralizing antibodies against tier 1 viruses. Thus, consensus subtype B env immunogens appear to be at least as good as, and in some instances better than, wild-type B env immunogens at inducing a neutralizing antibody response, and are amenable to further improvement by specific gene modifications.
Ashwagandha (Withania somnifera) Reverses β-Amyloid1-42 Induced Toxicity in Human Neuronal Cells: Implications in HIV-Associated Neurocognitive Disorders (HAND)
Kurapati, Kesava Rao Venkata; Atluri, Venkata Subba Rao; Samikkannu, Thangavel; Nair, Madhavan P. N.
Alzheimer's disease (AD) is characterized by progressive dysfunction of memory and higher cognitive functions with abnormal accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles throughout cortical and limbic brain regions. At present no curative treatment is available, and research focuses on drugs for slowing disease progression or providing prophylaxis. Withania somnifera (WS) also known as 'ashwagandha' is used widely in Ayurvedic medicine as a nerve tonic and memory enhancer. However, there is a paucity of data on the potential neuroprotective effects of W.somnifera against β-Amyloid (1–42)-induced neuropathogenesis. In the present study, we have tested the neuroprotective effects of methanol:Chloroform (3:1) extract of ashwagandha against β-amyloid induced toxicity and HIV-1Ba-L (clade B) infection using a human neuronal SK-N-MC cell line. Our results showed that β-amyloid induced cytotoxic effects in SK-N-MC cells as shown by decreased cell growth when tested individually. Also, confocal microscopic analysis showed decreased spine density, loss of spines and decreased dendrite diameter, total dendrite and spine area in clade B infected SK-N-MC cells compared to uninfected cells. However, when ashwagandha was added to β-amyloid treated and HIV-1 infected samples, the toxic effects were neutralized. Further, the MTT cell viability assays and the peroxisome proliferator-activated receptor-γ (PPARγ) levels supported these observations indicating the neuroprotective effect of WS root extract against β-amyloid and HIV-1Ba-L (clade B) induced neuro-pathogenesis. PMID:24147038
Candidate multi-epitope vaccines in aluminium adjuvant induce high levels of antibodies with predefined multi-epitope specificity against HIV-1.
Ding, J; Lu, Y; Chen, Y
Some neutralizing epitopes on HIV-1 envelope proteins were identified to induce antibodies which could effectively inhibit the infection of different strains in vitro. But only very low levels of these antibodies were determined in the HIV-1 infected individuals. To increase the levels of protective antibodies in vivo, we suggested multi-epitope vaccine as a new strategy to induce high level of neutralization antibodies with predefined multi-epitope specificity. A synthesized epitope peptide MP (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) containing three neutralizing epitopes (GPGRAFY, ELDKWA, RILAVERYLKD) was conjugated to carrier protein KLH, and then used for immunization in mouse together with aluminium adjuvant or Freund's adjuvant (FA). The candidate MP-KLH multi-epitope vaccine in aluminium adjuvant could induce antibody response very strongly to the epitope peptide C-(RILAVERYLKD-G)2 and the immunosuppressive peptide (P1) (LQARILAVERYLKDQQL) (antibody titer: 1:51200), strongly to the epitope peptide C-(ELDKWA-G)4 and the C-domain peptide (P2) (1:12800), and moderately to the epitope peptide C-(GPGRAFY)4 and the V3 loop peptide (1:1600). The immunoblotting analysis demonstrated that the antibodies in sera could recognize P1, P2, V3 loop peptides and rsgp41 (aa 539-684). These results are similar with that in the case of PI-BSA in FA, and suggest that the multi-epitope vaccine in aluminium could induce high levels of antibodies of predefined multi-epitope specificity, which provides experimental evidence for the new strategy to develop an effective neutralizing antibody-based multi-epitope vaccine against HIV-1.
Soluble CD4 blocks the infectivity of diverse strains of HIV and SIV for T cells and monocytes but not for brain and muscle cells.
Clapham, P R; Weber, J N; Whitby, D; McIntosh, K; Dalgleish, A G; Maddon, P J; Deen, K C; Sweet, R W; Weiss, R A
The CD4 antigen has been subverted as a receptor by the human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV). Several groups have reported that recombinant, soluble forms of the CD4 molecule (sCD4) block the infection of T lymphocytes by HIV-1, as CD4 binds the HIV envelope glycoprotein, gp120, with high affinity. We now report that sCD4 blocks diverse strains of HIV-1, HIV-2 and SIV, but is less effective for HIV-2. The blocking effect is apparent even after adsorption of virions to CD4 cells. Soluble CD4 prevents HIV infection of T-lymphocytic and myelomonocytic cell lines, but neither sCD4 nor anti-CD4 antibodies inhibit infection of glioma and rhabdomyosarcoma cell lines.
HIV-1 Vif Versus the APOBEC3 Cytidine Deaminases: An Intracellular Duel Between Pathogen and Host Restriction Factors
Wissing, Silke; Galloway, Nicole L. K.; Greene, Warner C.
The Vif protein of HIV is essential for the effective propagation of this pathogenic retrovirus in vivo. Vif acts by preventing virion encapsidation of two potent antiviral factors, the APOBEC3G and APOBEC3F cytidine deaminases. Decreased encapsidation in part involves Vif-mediated recruitment of a ubiquitin E3 ligase complex that promotes polyubiquitylation and proteasome-mediated degradation of APOBEC3G/F. The resultant decline in intracellular levels of these enzymes leads to decreased encapsidation of APOBECG/F into budding virions. This review discusses recent advances in our understanding of the dynamic interplay of Vif with the antiviral APOBEC3 enzymes. PMID:20538015
Alteration of Methamphetamine-induced stereotypic behaviour in transgenic mice expressing HIV-1 envelope protein gp120.
Roberts, Amanda J; Maung, Ricky; Sejbuk, Natalia E; Ake, Christopher; Kaul, Marcus
The use of drugs for recreational purposes, in particular Methamphetamine, is associated with an increased risk of infection with human immunodeficiency virus (HIV)-1. HIV-1 infection in turn can lead to HIV-associated neurological disorders (HAND) that range from mild cognitive and motor impairment to HIV-associated dementia (HAD). Interestingly, post mortem brain specimens from HAD patients and transgenic (tg) mice expressing the viral envelope protein gp120 in the central nervous system display similar neuropathological signs. In HIV patients, the use of Methamphetamine appears to aggravate neurocognitive alterations. In the present study, we injected HIV/gp120tg mice and non-transgenic littermate control animals with Methamphetamine dissolved in Saline or Saline vehicle and assessed locomotion and stereotyped behaviour. We found that HIVgp120-transgenic mice differ significantly from non-transgenic controls in certain domains of their behavioural response to Methamphetamine. Thus this experimental model system may be useful to further study the mechanistic interaction of both the viral envelope protein and the psychostimulant drug in behavioural alterations and neurodegenerative disease.
Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges.
Bomsel, Morgane; Tudor, Daniela; Drillet, Anne-Sophie; Alfsen, Annette; Ganor, Yonatan; Roger, Marie-Gaëlle; Mouz, Nicolas; Amacker, Mario; Chalifour, Anick; Diomede, Lorenzo; Devillier, Gilles; Cong, Zhe; Wei, Qiang; Gao, Hong; Qin, Chuan; Yang, Gui-Bo; Zurbriggen, Rinaldo; Lopalco, Lucia; Fleury, Sylvain
Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS.
Strategies for Preventing Mucosal Cell-Associated HIV Transmission
Whaley, Kevin J.; Mayer, Kenneth H.
Human immunodeficiency virus (HIV) may be transmitted through either cell-free virions or leukocytes harboring intracellular HIV in bodily fluids. In recent years, the early initiation of combination antiretroviral therapy leading to virological suppression has resulted in decreased HIV transmission to uninfected partners. Additionally, the efficacy of primary chemoprophylaxis with oral or topical antiretroviral regimens containing tenofovir (with or without emtricitabine) has been demonstrated. However, the efficacy of these approaches may be compromised by suboptimal adherence, decreased drug concentrations in mucosal compartments in women, and genital inflammation. Furthermore, in vitro studies on the effects of tenofovir on cell-associated HIV transmission have produced conflicting results. Preclinical studies suggest that combination preventive approaches may be most effective in stopping the transmission of HIV after mucosal exposure. Since the development of antibodies were found to correlate with protection in the only effective HIV vaccine trial, the administration of preformed mucosal and systemic antibodies may inform the development of safe and effective antibody-based oral, topical, and/or systemic preexposure prophylaxis agents and provide guidance in the development of HIV vaccines that effectively block cell-associated HIV transmission. PMID:25414423
Membrane-Proximal External HIV-1 gp41 Motif Adapted for Destabilizing the Highly Rigid Viral Envelope
Apellániz, Beatriz; Ivankin, Andrey; Nir, Shlomo; Gidalevitz, David; Nieva, José L.
Electron microscopy structural determinations suggest that the membrane-proximal external region (MPER) of glycoprotein 41 (gp41) may associate with the HIV-1 membrane interface. It is further proposed that MPER-induced disruption and/or deformation of the lipid bilayer ensue during viral fusion. However, it is predicted that the cholesterol content of this membrane (∼45 mol %) will act against MPER binding and restructuring activity, in agreement with alternative structural models proposing that the MPER constitutes a gp41 ectodomain component that does not insert into the viral membrane. Here, using MPER-based peptides, we test the hypothesis that cholesterol impedes the membrane association and destabilizing activities of this gp41 domain. To that end, partitioning and leakage assays carried out in lipid vesicles were combined with x-ray reflectivity and grazing-incidence diffraction studies of monolayers. CpreTM, a peptide combining the carboxyterminal MPER sequence with aminoterminal residues of the transmembrane domain, bound and destabilized effectively cholesterol-enriched membranes. Accordingly, virion incubation with this peptide inhibited cell infection potently but nonspecifically. Thus, CpreTM seems to mimic the envelope-perturbing function of the MPER domain and displays antiviral activity. As such, we infer that CpreTM bound to cholesterol-enriched membranes would represent a relevant target for anti-HIV-1 immunogen and inhibitor development. PMID:22098741
Longitudinal imaging of HIV-1 spread in humanized mice with parallel 3D immunofluorescence and electron tomography
Kieffer, Collin; Ladinsky, Mark S; Ninh, Allen; Galimidi, Rachel P; Bjorkman, Pamela J
Dissemination of HIV-1 throughout lymphoid tissues leads to systemic virus spread following infection. We combined tissue clearing, 3D-immunofluorescence, and electron tomography (ET) to longitudinally assess early HIV-1 spread in lymphoid tissues in humanized mice. Immunofluorescence revealed peak infection density in gut at 10–12 days post-infection when blood viral loads were low. Human CD4+ T-cells and HIV-1–infected cells localized predominantly to crypts and the lower third of intestinal villi. Free virions and infected cells were not readily detectable by ET at 5-days post-infection, whereas HIV-1–infected cells surrounded by pools of free virions were present in ~10% of intestinal crypts by 10–12 days. ET of spleen revealed thousands of virions released by individual cells and discreet cytoplasmic densities near sites of prolific virus production. These studies highlight the importance of multiscale imaging of HIV-1–infected tissues and are adaptable to other animal models and human patient samples. DOI: http://dx.doi.org/10.7554/eLife.23282.001 PMID:28198699
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Selective elimination of HIV-1-infected cells by Env-directed, HIV-1-based virus-like particles
Peretti, Silvia; Schiavoni, Ilaria; Pugliese, Katherina; Federico, Maurizio . E-mail: email@example.com
We recently showed that both replicating and resting cells cultivated with ganciclovir (GCV) were killed when challenged with vesicular stomatitis virus G glycoprotein pseudotyped HIV-1-based virus-like particles (VLPs) carrying the Nef7 (i.e., an HIV-1 Nef mutant incorporating in virions at high levels)/herpes simplex virus-1 thymidine kinase (HSV-TK) fusion product. On this basis, a novel anti-HIV therapeutic approach based on Nef7/TK VLPs expressing X4 or R5 HIV cell receptor complexes has been attempted. We here report that (CD4-CXCR4) and (CD4-CCR5) Nef7-based VLPs efficiently enter cells infected by X4- or R5-tropic HIV-1 strains, respectively. Importantly, the delivery of the VLP-associated Nef7/TK led to cell death upon GCV treatment. Of interest, VLPs were effective also against non-replicating, HIV-1-infected primary human monocyte-derived macrophages. HIV-targeted VLPs represent a promising candidate for the treatment of persistently HIV-1-infected cells that are part of virus reservoirs resistant to HAART therapies.
Envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus.
Wissink, E H J; Kroese, M V; van Wijk, H A R; Rijsewijk, F A M; Meulenberg, J J M; Rottier, P J M
Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2a, E, GP3, and GP4. Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indicating that these proteins interact with each other and are assembled into virions as a multimeric complex. Independent evidence for such complexes was obtained by coexpression of the minor envelope proteins in BHK-21 cells using a Semliki Forest virus expression system. By analyzing the maturation of their N-linked oligosaccharides, we found that the glycoproteins were each retained in the endoplasmic reticulum unless expressed together, in which case they were collectively transported through the Golgi complex to the plasma membrane and were even detected in the extracellular medium. As the PRRSV particles lacking the minor envelope proteins are not infectious, we hypothesize that the virion surface structures formed by these proteins function in viral entry by mediating receptor binding and/or virus-cell fusion.
Vaccinia Virus Extracellular Enveloped Virion Neutralization In Vitro and Protection In Vivo Depend on Complement▿
Benhnia, Mohammed Rafii-El-Idrissi; McCausland, Megan M.; Moyron, Juan; Laudenslager, John; Granger, Steven; Rickert, Sandra; Koriazova, Lilia; Kubo, Ralph; Kato, Shinichiro; Crotty, Shane
Antibody neutralization is an important component of protective immunity against vaccinia virus (VACV). Two distinct virion forms, mature virion and enveloped virion (MV and EV, respectively), possess separate functions and nonoverlapping immunological properties. In this study we examined the mechanics of EV neutralization, focusing on EV protein B5 (also called B5R). We show that neutralization of EV is predominantly complement dependent. From a panel of high-affinity anti-B5 monoclonal antibodies (MAbs), the only potent neutralizer in vitro (90% at 535 ng/ml) was an immunoglobulin G2a (IgG2a), and neutralization was complement mediated. This MAb was the most protective in vivo against lethal intranasal VACV challenge. Further studies demonstrated that in vivo depletion of complement caused a >50% loss of anti-B5 IgG2a protection, directly establishing the importance of complement for protection against the EV form. However, the mechanism of protection is not sterilizing immunity via elimination of the inoculum as the viral inoculum consisted of a purified MV form. The prevention of illness in vivo indicated rapid control of infection. We further demonstrate that antibody-mediated killing of VACV-infected cells expressing surface B5 is a second protective mechanism provided by complement-fixing anti-B5 IgG. Cell killing was very efficient, and this effector function was highly isotype specific. These results indicate that anti-B5 antibody-directed cell lysis via complement is a powerful mechanism for clearance of infected cells, keeping poxvirus-infected cells from being invisible to humoral immune responses. These findings highlight the importance of multiple mechanisms of antibody-mediated protection against VACV and point to key immunobiological differences between MVs and EVs that impact the outcome of infection. PMID:19019965
Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection.
Ye, Lin; Wang, Jiaming; Beyer, Ashley I; Teque, Fernando; Cradick, Thomas J; Qi, Zhongxia; Chang, Judy C; Bao, Gang; Muench, Marcus O; Yu, Jingwei; Levy, Jay A; Kan, Yuet Wai
Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9-mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.
HSV-2- and HIV-1- permissive cell lines co-infected by HSV-2 and HIV-1 co-replicate HSV-2 and HIV-1 without production of HSV-2/HIV-1 pseudotype particles
LeGoff, Jérôme; Bouhlal, Hicham; Lecerf, Maxime; Klein, Christophe; Hocini, Hakim; Si-Mohamed, Ali; Muggeridge, Martin; Bélec, Laurent
Background Herpes simplex virus type 2 (HSV-2) is a major cofactor of human immunodeficiency virus type 1 (HIV-1) sexual acquisition and transmission. In the present study, we investigated whether HIV-1 and HSV-2 may interact at the cellular level by forming HIV-1 hybrid virions pseudotyped with HSV-2 envelope glycoproteins, as was previously reported for HSV type 1. Methods We evaluated in vitro the production of HSV-2/HIV-1 pseudotypes in mononuclear CEM cells and epithelial HT29 and P4P cells. We analyzed the incorporation into the HIV-1 membrane of HSV-2 gB and gD, two major HSV-2 glycoproteins required for HSV-2 fusion with the cell membrane, in co-infected cells and in HIV-1-infected P4P cells transfected by plasmids coding for gB or gD. Results We show that HSV-2 and HIV-1 co-replicated in dually infected cells, and gB and gD were co-localized with gp160. However, HIV-1 particles, produced in HIV-1-infected cells expressing gB or gD after transfection or HSV-2 superinfection, did not incorporate either gB or gD in the viral membrane, and did not have the capacity to infect cells normally non-permissive for HIV-1, such as epithelial cells. Conclusion Our results do not support the hypothesis of HSV-2/HIV-1 pseudotype formation and involvement in the synergistic genital interactions between HIV-1 and HSV-2. PMID:17207276
Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways
Buckingham, Erin M.; Jarosinski, Keith W.; Jackson, Wallen; Carpenter, John E.
ABSTRACT Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome. IMPORTANCE VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an
Antiviral Innate Immune Activation in HIV-Infected Adults Negatively Affects H1/IC31-Induced Vaccine-Specific Memory CD4+ T Cells
Schindler, Tobias; Kagina, Benjamin M.; Zhang, Jitao David; Lukindo, Tedson; Mpina, Maxmillian; Bang, Peter; Kromann, Ingrid; Hoff, Søren T.; Andersen, Peter; Reither, Klaus; Churchyard, Gavin J.; Certa, Ulrich
Tuberculosis (TB) remains a global health problem, with vaccination being a necessary strategy for disease containment and elimination. A TB vaccine should be safe and immunogenic as well as efficacious in all affected populations, including HIV-infected individuals. We investigated the induction and maintenance of vaccine-induced memory CD4+ T cells following vaccination with the subunit vaccine H1/IC31. H1/IC31 was inoculated twice on study days 0 and 56 among HIV-infected adults with CD4+ lymphocyte counts of >350 cells/mm3. Whole venous blood stimulation was conducted with the H1 protein, and memory CD4+ T cells were analyzed using intracellular cytokine staining and polychromatic flow cytometry. We identified high responders, intermediate responders, and nonresponders based on detection of interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) expressing central (TCM) and effector memory CD4+ T cells (TEM) 182 days after the first immunization. Amplicon-based transcript quantification using next-generation sequencing was performed to identify differentially expressed genes that correlated with vaccine-induced immune responses. Genes implicated in resolution of inflammation discriminated the responders from the nonresponders 3 days after the first inoculation. The volunteers with higher expression levels of genes involved in antiviral innate immunity at baseline showed impaired H1-specific TCM and TEM maintenance 6 months after vaccination. Our study showed that in HIV-infected volunteers, expression levels of genes involved in the antiviral innate immune response affected long-term maintenance of H1/IC31 vaccine-induced cellular immunity. (The clinical trial was registered in the Pan African Clinical Trials Registry [PACTR] with the identifier PACTR201105000289276.) PMID:25924764
How conformational changes can affect catalysis, inhibition and drug resistance of enzymes with induced-fit binding mechanism such as the HIV-1 protease.
Weikl, Thomas R; Hemmateenejad, Bahram
A central question is how the conformational changes of proteins affect their function and the inhibition of this function by drug molecules. Many enzymes change from an open to a closed conformation upon binding of substrate or inhibitor molecules. These conformational changes have been suggested to follow an induced-fit mechanism in which the molecules first bind in the open conformation in those cases where binding in the closed conformation appears to be sterically obstructed such as for the HIV-1 protease. In this article, we present a general model for the catalysis and inhibition of enzymes with induced-fit binding mechanism. We derive general expressions that specify how the overall catalytic rate of the enzymes depends on the rates for binding, for the conformational changes, and for the chemical reaction. Based on these expressions, we analyze the effect of mutations that mainly shift the conformational equilibrium on catalysis and inhibition. If the overall catalytic rate is limited by product unbinding, we find that mutations that destabilize the closed conformation relative to the open conformation increase the catalytic rate in the presence of inhibitors by a factor exp(ΔΔGC/RT) where ΔΔGC is the mutation-induced shift of the free-energy difference between the conformations. This increase in the catalytic rate due to changes in the conformational equilibrium is independent of the inhibitor molecule and, thus, may help to understand how non-active-site mutations can contribute to the multi-drug-resistance that has been observed for the HIV-1 protease. A comparison to experimental data for the non-active-site mutation L90M of the HIV-1 protease indicates that the mutation slightly destabilizes the closed conformation of the enzyme. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.
CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus.
Kang, HyunJun; Minder, Petra; Park, Mi Ae; Mesquitta, Walatta-Tseyon; Torbett, Bruce E; Slukvin, Igor I
The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/C