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Transportin-SR2 (TRN-SR2, TNPO3, transportin 3) was previously identified as an interaction partner of human immunodeficiency virus type 1 (HIV-1) integrase and functions as a nuclear import factor of HIV-1. A possible role of capsid in transportin-SR2-mediated nuclear import was recently suggested by the findings that a chimeric HIV virus, carrying the murine leukemia virus (MLV) capsid and matrix proteins, displayed a transportin-SR2 independent phenotype, and that the HIV-1 N74D capsid mutant proved insensitive to transportin-SR2 knockdown.
Our present analysis of viral specificity reveals that TRN-SR2 is not used to the same extent by all lentiviruses. The DNA flap does not determine the TRN-SR2 requirement of HIV-1. We corroborate the TRN-SR2 independent phenotype of the chimeric HIV virus carrying the MLV capsid and matrix proteins. We reanalyzed the HIV-1 N74D capsid mutant in cells transiently or stably depleted of transportin-SR2 and confirm that the N74D capsid mutant is independent of TRN-SR2 when pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Remarkably, although somewhat less dependent on TRN-SR2 than wild type virus, the N74D capsid mutant carrying the wild type HIV-1 envelope required TRN-SR2 for efficient replication. By pseudotyping with envelopes that mediate pH-independent viral uptake including HIV-1, measles virus and amphotropic MLV envelopes, we demonstrate that HIV-1 N74D capsid mutant viruses retain partial dependency on TRN-SR2. However, this dependency on TRN-SR2 is lost when the HIV N74D capsid mutant is pseudotyped with envelopes mediating pH-dependent endocytosis, such as the VSV-G and Ebola virus envelopes.
Here we discover a link between the viral entry of HIV and its interaction with TRN-SR2. Our data confirm the importance of TRN-SR2 in HIV-1 replication and argue for careful interpretation of experiments performed with VSV-G pseudotyped viruses in studies on early steps of HIV replication including the role of capsid therein.
Retroviruses stably integrate the DNA copy of their RNA genome into the host cell chromatin. However, there are marked differences between the distinct families of retroviruses regarding their capacity to replicate in non-dividing cells. The lentivirinae such as the human immunodeficiency virus type 1 (HIV-1) can infect dividing and non-dividing cells such as macrophages, dendritic cells or CD4+ memory T-cells [1]. Rous sarcoma virus (RSV) can also infect non-dividing cells such as neurons or growth-arrested cells, but with less efficiency than HIV [2]. In contrast, the γ-retrovirus Moloney murine leukemia virus (MLV) infects only dividing cells efficiently [3]. To date, this difference cannot be explained. The prevailing hypothesis has been that lentiviruses adopt a specific mechanism for active nuclear import through the nucleopore, and that other retroviruses must depend on the breakdown of the nuclear membrane during mitosis for chromatin access in order to achieve integration [3-5]. More recently, a role for retroviral capsid was proposed in replication determination in non-dividing cells [6,7]. After HIV entry in the target cell, the viral core is released into the cytoplasm. On its way to the nucleus, viral capsid (CA) is shed from this nucleoprotein complex, containing both viral and cellular proteins, in an ill-defined process called uncoating (for a recent overview see [8]). Meanwhile the viral enzyme reverse transcriptase (RT) transcribes the RNA genome into a cDNA copy.
After reverse transcription, the preintegration complex (PIC) is transported through the nuclear pore complex (NPC). The NPC is a specialized channel ~40 nm in diameter [9] that supports passive diffusion of small molecules and ions and facilitates receptor-mediated translocation of proteins and ribonucleoprotein complexes above 40 kDa. Since the HIV-1 PIC is a nucleoprotein complex with an estimated diameter of 56 nm [10], it requires conformational changes and active transport through the NPC. Many attempts have been made to determine the viral and cellular factors mediating nuclear import of the HIV PIC (for a review see [11]). Viral protein R (Vpr), matrix protein (MA), integrase (IN) and the DNA flap have each been proposed as the main viral determinant for nuclear trafficking of the PIC, but these findings were not readily reproduced in subsequent studies. As cellular cofactors, importin-α/importin-β [12-15] and importin-7 [16-19] have been investigated as PIC transporters, but their role in HIV replication has not been thoroughly validated or confirmed. Also, importin-α3 has very recently been implicated in HIV nuclear import [20].
Recently, we identified the cellular protein transportin-SR2 (TRN-SR2, TNPO3, transportin 3), encoded by the TNPO3 gene, as the nuclear import factor of HIV [21]. Two genome-wide RNAi screens [22,23], but not others [24,25] also identified TRN-SR2 as a cofactor of HIV replication. Transportin-SR2 (TRN-SR2) was first identified as an important nuclear import factor for phosphorylated splicing factors of a family of serine/arginine-rich proteins (SR proteins) [26-28]. It has also been shown that TRN-SR2 imports other proteins not belonging to the SR protein family [29]. We identified TRN-SR2 as a binding partner of HIV-1 integrase in a yeast two-hybrid screen [21], and reverse yeast two-hybrid screening demonstrated that none of the other HIV proteins directly interacts with TRN-SR2. In cells transiently or stably depleted of TRN-SR2, HIV replication was severely hampered due to a defect in the nuclear import of the HIV PIC [21]. Using GFP-labeled HIV, a direct effect of TRN-SR2 on the nuclear import of PICs was also visualized. Finally, TRN-SR2 was required for HIV infection of both dividing and non-dividing cells, implying that a similar nuclear import pathway is used in different stages of the cell cycle.
A recent study confirmed the effect of TRN-SR2 knockdown on HIV-1 vector transduction [30]. In that study the specificity for different retroviral vectors and the direct interaction of TRN-SR2 with the integrase proteins from different retroviruses were examined, and the authors corroborated the direct interaction between recombinant TRN-SR2 and HIV-1 IN. Although TRN-SR2 was found to be a rather prolific IN binder, displaying affinity for multiple retroviral integrases, no clear correlation between the interactions of various integrases with TRN-SR2 and dependence on TRN-SR2 during viral vector transductions was observed. In addition, a chimeric reporter virus composed of both HIV and MLV proteins (MHIV) carrying the MLV MA, p12 and CA proteins instead of the HIV-1 MA and CA proteins [6,31], which was also pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) envelope, appeared to be insensitive to TRN-SR2 knockdown. Although no evidence was provided that TRN-SR2 and CA physically interact, it was proposed that the TRN-SR2 dependency of HIV-1 infection is mediated by CA and not by HIV-1 integrase [30]. In a follow up study, the role of CA in the TRN-SR2 requirement of HIV-1 replication was examined in more detail [32]. Ectopic expression of a C-terminally truncated version of the cleavage and polyadenylation specific factor 6 (CPSF6) resulted in a block of HIV replication. An HIV-1 strain with a mutation in CA (N74D) was capable of escaping this phenotype. Interestingly, the VSV-G pseudotyped HIV-1 N74D CA mutant virus appeared to be independent of TRN-SR2 for infection of both dividing and non-dividing cells [32]. Here we enter the debate by re-examining whether HIV CA is involved in the TRN-SR2 requirement of HIV. We compared wild type and VSV-G pseudotyped viral vectors and studied the N74D CA mutant which was reported to be independent of TRN-SR2. To our surprise, the phenotype of the N74D CA mutant virus appeared to be dependent on the viral entry route. Whereas the mutant virus was insensitive to TRN-SR2 depletion when pseudotyped with VSV-G, the same mutant proved to be still dependent on TRN-SR2, although to a somewhat lesser extent, when retaining the HIV envelope. Our results are suggestive of a role for capsid mutations having an indirect effect on the interaction between HIV and TRN-SR2, probably by affecting the processes of uncoating or docking to the nuclear pore that precede the previously demonstrated interaction between IN and TRN-SR2.
We previously identified TRN-SR2 as an important cellular cofactor mediating HIV-1 nuclear import [21]. HIV-2 was also dependent on TRN-SR2, although MLV did not appear to be dependent. Here we verified whether TRN-SR2 acts as a lentivirus-specific nuclear import factor. We transduced HeLaP4 cell lines transiently depleted of TRN-SR2 with different retroviral vectors (Figure (Figure1),1), and a mismatch siRNA was run in parallel to exclude off-target effects while mock transfected cells were used as controls for the transfection procedure. TRN-SR2 knockdown was verified by western blot (Figure (Figure1A).1A). Three days after siRNA transfection cells were transduced with concentrated VSV-G pseudotyped viral vectors derived from HIV-1, SIV (simian immunodeficiency virus), EIAV (equine infectious anemia virus), FIV (feline immunodeficiency virus) or MLV. Vector preparations were adjusted to yield 30-60% GFP positivity in mock transfected cells. Three days after transduction cells were fixed and analyzed for the overall GFP fluorescence by flow cytometry (Figure (Figure1B).1B). After TRN-SR2 knockdown, transduction by either HIV-1 or SIV vectors was inhibited up to 90% and 95%, respectively. The EIAV vector was also sensitive to TRN-SR2 depletion, although to a lesser extent (50% inhibition of transduction efficiency). Transductions by the FIV and MLV vectors were modestly affected (12% and 29% inhibition, respectively) when compared to mismatch siRNA-transfected cells. From this analysis, we conclude that TRN-SR2 is a cellular cofactor important for transduction by some, but not all VSV-G pseudotyped lentiviral vectors.
The central DNA flap is a structure in the reverse transcribed DNA genome of lentiviruses that is absent from retroviruses like MLV [33,34]. Since the DNA flap has been implicated in HIV nuclear import [35-40], we examined whether the central DNA flap might be important for the TRN-SR2 requirement of HIV-1. HeLaP4 cell lines transiently depleted of TRN-SR2 and control cells were challenged with 3 dilutions of HIV-1-derived VSV-G pseudotyped lentivectors carrying a cPPT/CTS sequence in sense (WT) or antisense orientation (Flap-). In antisense orientation, the cPPT/CTS sequence does not yield a functional flap. Vectors lacking a functional flap are known to display a 3- to 6-fold reduction in transduction efficiency [37]. Three days after transduction, overall GFP fluorescence was measured by flow cytometry (Additional file 1: Figures S1A and S1B). The vector dilutions yielded 90%, 60% or 20% GFP positive control cells, respectively. Transduction by the lentiviral vectors with (Additional file 1: Figure S1A) or without DNA flap (Additional file 1: Figure S1B) was inhibited up to 70% in TRN-SR2 depleted cells and at all dilutions used. Next, we tested the effect of DNA flap mutations on the multiple-round infectivity of HIV-1 virus strain NL4-3 in TRN-SR2 depleted HeLaP4 cells (Additional file 1: Figures S1C and S1D). We infected HeLaP4 cells transiently depleted of TRN-SR2 and control cells with 3 dilutions of infectious HIV-1NL4-3LAI cPPT wild type virus (WT) (Additional file 1: Figure S1C) and HIV-1NL4-3LAI cPPTD (Flap-) virus (Additional file 1: Figure S1D). The latter contains a mutated cPPT sequence which prevents formation of the DNA flap during reverse transcription and shows a 10- to 100-fold replication defect depending on the viral infection dose [35,38]. The HeLaP4 cells contain a β-galactosidase (β-gal) reporter gene under control of the HIV-1 LTR promoter. Three days after infection β-galactosidase activity was measured. The data demonstrate reduced infectivity of the flap-negative virus, which becomes more apparent at lower MOIs as was previously reported [35,38]. TRN-SR2 knockdown inhibited replication of wild type and flap-negative virus to the same extent (up to 80% inhibition) demonstrating that the DNA flap is not required for the TRN-SR2 dependency of HIV-1 replication.
Next we investigated which viral proteins, present in the PIC, are responsible for the TRN-SR2 independent phenotype displayed by the MLV vector. We used the HIV-MLV chimeric viruses (MHIV) previously constructed by the Emerman group [6,31]. In MHIV-mMA12CA the HIV MA and CA proteins are replaced by the MLV MA, CA and p12 proteins. In MHIV-mIN, the HIV IN protein is replaced by the MLV IN. MHIV-mMA12CA cannot infect non-dividing cells, but MHIV-mIN infects non-dividing cells as well as dividing cells [6,31]. Since these viruses are poorly infectious, VSV-G pseudotyping of the chimeric viruses during productions is absolutely required to obtain infectious virions. Construction of MLV-based chimeric proviruses did not generate infectious virions [6].
We evaluated the effect of siRNA-mediated TRN-SR2 knockdown in HeLaP4 cells on infection by both MHIV chimeric viruses (Figure (Figure2A).2A). We used VSV-G pseudotyped single-round MHIV-mMA12CA and MHIV-mIN viruses and their parental HIV-1 and MLV vector, all expressing the firefly luciferase reporter gene (Fluc). HeLaP4 cells transiently depleted of TRN-SR2 and control cells were infected with concentrated VSV-G pseudotyped viral stocks. Luciferase activities were measured 3 days post infection and were normalized to the levels in mock transfected control cells (Figure (Figure2A).2A). As previously reported [21-23,30], the MLV vector displayed only modest sensitivity to TRN-SR2 knockdown (33% inhibition of infectivity in TRN-SR2 knockdown cells compared to mismatch siRNA transfected cells). Surprisingly, the MHIV-mMA12CA chimeric virus was only partially sensitive to TRN-SR2 knockdown (34% inhibition in TRN-SR2 knockdown cells compared to mismatch siRNA transfected cells). In contrast, both the MHIV-mIN virus and the parental HIV-1 reporter virus were severely impaired by TRN-SR2 knockdown (77% and 87% inhibition, respectively). These results are comparable to those described by Krishnan and colleagues [30]. Swapping of HIV MA and CA proteins with those of MLV apparently interferes with the requirement for TRN-SR2 during infection but replacing the IN of HIV-1 by that of MLV does not alter the TRN-SR2 dependency of the chimeric virus.
Two alternative explanations for these results are possible. The viral CA may determine the interaction between TRN-SR2 and the HIV-1 PIC as was proposed by Krishnan et al. [30]; and by replacing the HIV-1 CA and MA proteins by their non-interacting MLV counterparts, this interaction could be inhibited, rendering infection of the MHIV-mMA12CA virus partially independent of TRN-SR2. Substituting the integrases in this case would have no effect. Alternatively, TRN-SR2 can interact with both HIV-1 and MLV IN and, as a result, the MHIV-mIN virus would remain dependent on TRN-SR2. However, recombinant His-tagged HIV-1 IN could pull down endogenous TRN-SR2 in cellular lysates, but recombinant His-tagged MLV IN could not [21]. Still, this interaction could have gone undetected due to low concentrations of endogenous TRN-SR2 in the cell lysate which are difficult to detect by western blot alone. Therefore, we reinvestigated the direct protein-protein interaction using AlphaScreen technology and recombinant GST-TRN-SR2 and IN-His6 (Figure (Figure2B).2B). As negative controls for binding to GST-TRN-SR2, we used two different His6-tagged proteins; His6-GαoA, the His6-tagged human heterotrimeric G protein α oA subunit [41], and His6-Roc-COR, a His6-tagged GTPase Ras of complex proteins (Roc) domain in tandem with its C-terminal domain of Roc (COR) of the leucine rich repeat kinase 2 protein (LRRK2) from Chlorobium tepidum [42]. The different His6-tagged proteins were titrated against a fixed concentration of GST-TRN-SR2 (10 nM). As expected, no interaction between His6-GαoA or His6-Roc-COR and GST-TRN-SR2 was detected under our assay conditions (Figure (Figure2B).2B). In this assay, we did observe binding of GST-TRN-SR2 to both His6-tagged HIV-1 IN and MLV IN with an apparent Kd of 36.3 ± 2.3 nM for HIV-1 IN and an even lower Kd of 17.5 ± 0.5 nM for MLV IN, an interaction also observed by Krishnan et al. [30]. Our data are consistent with the hypothesis that TRN-SR2 binds to both HIV-1 and MLV IN in the context of a viral PIC, explaining the TRN-SR2 dependency of MHIV-mIN, the chimeric HIV virus containing MLV IN. However, this does not explain the TRN-SR2 independent phenotype of the MLV vector and the MHIV-mMA12CA virus. Moreover, a CA mutant virus (HIV-1 N74D) has recently been described to be insensitive to TRN-SR2 knockdown [32]. These findings prompted us to investigate in more detail a possible role of HIV-1 CA in the TRN-SR2 requirement of HIV-1 replication.
A VSV-G pseudotyped HIV-1 N74D CA mutant virus was recently reported to be insensitive to TRN-SR2 knockdown [32]. Krishnan et al. [30] hypothesized that the TRN-SR2 dependency of HIV-1 is dictated by HIV-1 CA instead of IN. To test this hypothesis, we infected HeLaP4 cells transiently depleted of TRN-SR2 with VSV-G pseudotyped wild type and N74D CA mutant luciferase reporter viruses (Figures (Figures3A3A and and3B)3B) or with replication competent HIV-1 NL4-3 wild type and N74D mutant virus (Figures (Figures3C3C and and3D).3D). The infectivity of the VSV-G pseudotyped wild type and N74D CA mutant luciferase reporter viruses was measured by Fluc activity. Interestingly, after normalization of the virus stocks based on p24 measurements (PerkinElmer, HIV-1 p24 ELISA kit), the VSV-G pseudotyped N74D CA mutant virus appeared 5-fold more infectious (compare Figures Figures3A3A and and3B).3B). In repeated infection experiments, the VSV-G pseudotyped N74D mutant virus consistently displayed 5- to 10-fold higher luciferase counts than pseudotyped wild type virus (data not shown). We confirmed the TRN-SR2 independent phenotype of the VSV-G pseudotyped N74D CA mutant (Figure (Figure3B)3B) in comparison to the pseudotyped wild type virus (75% reduction of viral infectivity on average in the TRN-SR2 knockdown cells compared to the mismatch siRNA transfected cells) (Figure (Figure3A).3A). Similar results were obtained with a β-galactosidase readout (data not shown).
Subsequently, we infected HeLaP4 cells transiently depleted of TRN-SR2 with replication competent wild type and N74D mutant viruses. Both viruses carried the HIV-1 envelope proteins gp120 and gp41. To allow multiple round replication, three days after infection β-galactosidase activity was measured as readout for viral infectivity. Virus stocks were normalized for p24 content (PerkinElmer p24 ELISA kit). The N74D mutant again yielded higher β-galactosidase counts (compare Figures Figures3C3C and and3D),3D), although the difference in infectivity was less pronounced (1.5-fold higher infectivity than wild type virus) than with the VSV-G pseudotyped N74D CA mutant (5- to 10-fold higher infectivity than wild type virus, compare Figures Figures3A3A and and3B).3B). In repeated experiments using viruses carrying the HIV-1 envelope, we consistently observed a 1.5- to 3-fold higher infectivity of the N74D CA mutant measured via β-galactosidase activity (data not shown). We observed that both HIV-1 NL4-3 WT virus (Figure (Figure3C)3C) and the N74D CA mutant (Figure (Figure3D)3D) required TRN-SR2 for efficient infection of HeLaP4 cells (average reduction of infectivity of 80% and 50% in TRN-SR2 knockdown cells, respectively), although the N74D CA mutant virus was less sensitive to TRN-SR2 depletion. Nevertheless, a prominent alteration in the TRN-SR2 dependency of the N74D CA mutant was observed when using the VSV-G envelope (complete insensitivity to TRN-SR2 knockdown, Figure Figure3B)3B) or the HIV-1 envelope (intermediate sensitivity to TRN-SR2 knockdown, Figure Figure3D).3D). As these findings suggest, the difference in TRN-SR2 dependency displayed by the N74D CA mutant in the multiple round compared with the single round infections was dependent on the envelope proteins; we investigated prolonged multiple round replication of the HIV-1 N74D CA mutant carrying a wild type envelope using HeLaP4 cells stably depleted of TRN-SR2 knockdown.
We generated stable TRN-SR2 knockdown cell lines by transducing HeLaP4 cells with one of two different lentiviral vectors expressing shRNA targeting the TRN-SR2 mRNA (shTR3 and shTR4) and a control cell line using a control vector expressing a scrambled shRNA (shSCR). TRN-SR2 knockdown was verified with Western blotting (Figure (Figure4A)4A) and QPCR (Figure (Figure4B).4B). When visualized by Western blotting, TRN-SR2 knockdown in the shTR3 or shTR4 HeLaP4 cells was comparable to the level of knockdown obtained by transient transfection of siTRN-SR_2 (compare Figures Figures4A4A and and1A).1A). When measured by QPCR, expression of shTR3 or shTR4 decreased the amount of TRN-SR2 mRNA copies for 80% or 70% respectively in comparison with control cells (Figure (Figure4B).4B). Using immunostaining and FACS analysis of the CD4 surface receptor expressed by the TRN-SR2 depleted cells and control cells, comparable CD4 expression levels in the knockdown cells and control cells were observed (Figure (Figure4C).4C). As expected, no CD4 expression was observed in 293T cells which were used as a negative control to exclude non-specific binding of the anti-CD4 antibody. The stable TRN-SR2 knockdown and control cell lines were challenged with wild type HIV-1 NL4-3 and N74D CA mutant virus in a multiple-round infection (Figure (Figure4D),4D), with the inocula normalized for p24 content. Both viruses replicated with similar kinetics in the shSCR control cells. The replication of both the HIV-1 wild type virus and the HIV-1 N74D CA mutant was severely impaired up to 10 days post infection in both shTR3 and shTR4 HeLaP4 cell lines stably depleted of TRN-SR2, although the N74D CA mutant was somewhat less sensitive to TRN-SR2 knockdown (10-fold inhibition in the shTR3 HeLaP4 cells compared to shSCR cells) than the wild type virus (70-fold inhibition in the shTR3 expressing cells compared to shSCR cells). To exclude a non-specific effect of TRN-SR2 knockdown or the expression of the different shRNAs on the late steps of viral replication, we transfected the shSCR, shTR3 and shTR4 HeLaP4 cell lines with the viral molecular clone pNL4-3 and measured the p24 levels in the supernatant 24 hours after transfection (Figure (Figure4E).4E). Although a slight inhibition of the p24 production was observed in the TRN-SR2 knockdown cell lines, this difference could not account for the potent inhibition of HIV replication in the TRN-SR2 depleted cells. These results confirm our previous finding that TRN-SR2 depletion does not inhibit the late steps of HIV replication [21] and exclude non-specific effects on the late steps of HIV replication in the shTR3 and shTR4 expressing HeLaP4 cells compared to the shSCR control cell line. Together, these results show that the HIV-1 N74D CA mutant virus still requires TRN-SR2 for efficient infection in HeLaP4 cells.
Next, we wondered whether the inhibition of the N74D CA mutant by stable TRN-SR2 knockdown in the multiple round infection experiments could be mirrored in single round infection assays. We tested the infectivity of VSVG-pseudotyped wild type and N74D CA mutant virus, or replication competent wild type and N74D CA mutant viruses in single-round infection experiments in the HeLaP4 cells stably depleted of TRN-SR2 and in control cells. For these experiments, we normalized the virus stocks for p24 content and for RT activity (see the materials and methods section, paragraph infection and transduction). Various amounts of VSV-G pseudotyped HIV-1 NL4-3 and HIV-1 NL4-3 N74D CA mutant luciferase reporter viruses were used to infect the shSCR-, shTR3- and shTR4-expressing HeLaP4 cells (Figures (Figures5A5A and and5B).5B). Infectivity was measured by Fluc activity. In these experiments, the pseudotyped N74D CA mutant virus again proved to be more infectious than the wild type reporter virus (typically 10- to 15-fold). The results were comparable to the experiments using HeLaP4 cells transiently depleted of TRN-SR2 (Figure (Figure3),3), excluding possible non-specific effects on viral infectivity in the stable TRN-SR2 knockdown cells due to off-target effects or selection. The VSV-G pseudotyped HIV-1 N74D CA mutant (Figure (Figure5B),5B), in contrast to the VSV-G pseudotyped wild type virus (Figure (Figure5A),5A), did not require TRN-SR2 for infection as was described in [32]. Single round infectivity of the VSV-G pseudotyped wild type virus was less inhibited in the shTR4 TRN-SR2 knockdown cells (50% inhibition on average) compared to the shTR3 expressing TRN-SR2 knockdown cells (80% inhibition on average). This difference is likely due to the difference in the extent of TRN-SR2 knockdown in these different cell lines as measured by QPCR (compare Figures Figures5A5A and and4B).4B). Next, we infected the HeLaP4 cells stably depleted of TRN-SR2 and control cells with two different dilutions of replication competent HIV-1 NL4-3 and N74D CA mutant virus normalized for p24 values (and RT activity) (Figures (Figures5C5C and and5D).5D). Single round infections were performed in the presence of 5 μM of the protease inhibitor ritonavir. β-galactosidase activity was measured as readout for viral infectivity. The N74D CA mutant was 2.5-fold more infectious than wild type HIV-1 in these experiments, corresponding to the increase in infectivity we observed in the experiments using transient siRNA-mediated knockdown of TRN-SR2 (Figures (Figures3C3C and and3D).3D). In the shTR3 HeLaP4 cells, we observed an average reduction of 70% of wild type HIV-1 virus infection compared to shSCR cells, and in the shTR4 HeLaP4 cells an average reduction of 50% (Figure (Figure5C),5C), comparable to the experiments using VSV-G pseudotyped wild type virus (Figure (Figure5A).5A). Infection by the N74D CA mutant was inhibited 40% on average in the shTR3 cells, and 35% on average in the shTR4 cells (Figure (Figure5D).5D). These results point to a partial TRN-SR2 dependency of the N74D CA mutant virus when carrying the HIV-1 envelope.
The major endocytic pathways include pH-dependent clathrin-mediated endocytosis, pH-independent caveolae-mediated endocytosis, clathrin- and caveolae-independent endocytosis, macropinocytosis and phagocytosis (for a review see [43]). To investigate whether endocytosis in general renders the HIV-1 N74D CA mutant insensitive to TRN-SR2 knockdown, we produced wild type and N74D CA mutant NL4-3 luciferase reporter virus pseudotyped with the HIV-1 envelope glycoproteins, VSV-G, or viral envelopes derived from the measles virus, amphotropic MLV or Ebola virus. Both the HIV-1 envelope and the measles virus envelope mediate viral entry via pH-independent fusion of the viral and cellular membranes [44], although HIV-1 virions are also proposed to enter cells via pH-independent endocytosis leading to unproductive infection [45-47], or via endocytosis and subsequent dynamin-dependent fusion with endosomes [48]. The modes of entry of amphotropic MLV (MLVampho) and the highly pathogenic Ebola virus have been the subject of debate, but recent studies showed that MLVampho enters the cell via a pH-independent, caveola-dependent endocytic pathway [49] while the Ebola virus enters through pH-dependent clathrin-mediated endocytosis [50]. VSV-G pseudotyped viral particles enter target cells via pH-dependent endocytosis, although the role of clathrin in this process is not well understood [50-52]. Although we observed previously (Figure (Figure4D)4D) that the TRN-SR2 dependent phenotype of HIV-1 is more pronounced in prolonged multiple round infections, pseudotyping of the wild type and N74D CA mutant reporter viruses with different viral envelopes obliged single round infection experiments.
HeLaP4 cells transiently depleted of TRN-SR2 and control cells were challenged with the differently pseudotyped WT and N74D CA mutant reporter viruses and infectivity was measured using the Fluc reporter protein activity as readout. The infectivities of the pseudotyped viruses were in a similar range in control cells when envelopes mediating the same entry route were used (Figure (Figure6).6). When the HIV-1 N74D CA mutant reporter virus was pseudotyped with VSV-G (Figure (Figure6D)6D) or the Ebola envelope (Figure (Figure6E),6E), infections were not impaired by TRN-SR2 knockdown in contrast to wild type reporter virus. However, when the viral particles were pseudotyped with the HIV-1 envelope (Figure (Figure6A),6A), the MLVampho envelope (Figure (Figure6B)6B) or the measles virus envelope (Figure (Figure6C),6C), the N74D CA mutant was still dependent on TRN-SR2 (50% inhibition of infection in TRN-SR2 depleted cells), although not as dependent as the wild type virus (80-90% inhibition of infection). These findings show that the HIV-1 N74D CA mutant virus relies much less on TRN-SR2 when entering the target cells via pH-dependent endocytosis (VSV-G and Ebola envelope). After membrane fusion (HIV-1 and measles virus envelope) or pH-independent endocytosis (MLVampho envelope), the N74D CA mutant still requires TRN-SR2 for infection of HeLaP4 cells.
We initially identified TRN-SR2 in a yeast two-hybrid screen searching for cellular binding partners of HIV-1 IN, and in the reverse screen no interaction with capsid was detected. Subsequently we showed that TRN-SR2 mediates nuclear import of the HIV-1 PIC [21]. Since one of the key features of lentiviruses is their ability to infect non-dividing cells, we questioned whether TRN-SR2 is a lentiviral-specific cofactor of HIV-1 replication. We challenged HeLaP4 cells depleted of TRN-SR2 with VSV-G pseudotyped retroviral vectors derived from HIV-1, SIV, EIAV, FIV or MLV, and the results obtained were comparable with recently reported data [30]. We also observed a TRN-SR2 independent phenotype for the FIV vector, and the MLV vector was only slightly sensitive to TRN-SR2 depletion (Figure (Figure1).1). TRN-SR2 independent transduction by FIV, MLV and RSV derived vectors has been shown before [21-23,30]. Lee et al. reported that a pseudotyped FIV vector was insensitive to TRN-SR2 knockdown as well [32]. Together these data suggest that TRN-SR2 acts as a lentivirus-specific cofactor, although not all lentiviruses utilize it to the same extent. Because of our finding that VSV-G pseudotyping masks the TRN-SR2 requirement of the HIV-1 N74D CA mutant virus, it would be prudent to extend this study in a follow up project by using native viral envelopes or at least viral envelopes mimicking the natural entry pathway of each specific virus under study. HIV-1 and SIV are related lentiviruses and both enter target cells predominantly via membrane fusion. VSV-G pseudotyping did not alter the requirement of wild type HIV-1 for TRN-SR2 [21], although a single mutation in the CA protein (N74D) abolished the TRN-SR2 dependency of VSV-G pseudotyped HIV-1. Although VSV-G pseudotyped SIV was highly sensitive to TRN-SR2 knockdown, it would be interesting to test SIV with its viral envelope which mediates fusion-based entry. The EIAV virus is believed to enter the cell through pH-dependent, clathrin-mediated endocytosis [53], implying that VSV-G pseudotyping may actually resemble the natural entry pathway for this virus; the sensitivity for TRN-SR2 depletion of the pseudotyped EIAV vector may well reflect its natural dependency on TRN-SR2. The fusion-based mechanism of entry used by the FIV virus closely resembles that of HIV and SIV [54]. Both ecotropic and amphotropic MLV enter the cell through a pH-independent endocytic pathway [49,55]. When pseudotyping MLV and FIV one should take the specific entry pathways into account. At this stage, we cannot entirely exclude that FIV and MLV require TRN-SR2 for their replication since all experiments were performed with VSV-G pseudotyped vectors (our data and [30,32]). Krishnan and colleagues tried to correlate the binding affinities of recombinant TRN-SR2 for different retroviral integrases with the dependency on TRN-SR2 displayed by the corresponding viral vectors [30]. In the absence of any correlation, the authors concluded that IN must not play a dominant role in the TRN-SR2 requirement of HIV-1. However, our findings put into question the TRN-SR2-related phenotypes observed for the different viral vectors since all experiments described so far were performed using VSV-G pseudotyped viral particles and not the natural virus envelopes. Therefore, the conclusion that the requirement for TRN-SR2 during infection does not map to HIV-1 IN [30] may be premature, and requires further study on the retroviral specificity of TRN-SR2-mediated nuclear import using replicating viruses instead of vectors and the use of the respective host cells. Alternatively, one could pseudotype viral vectors while mimicking the natural entry mechanism to determine the role of TRN-SR2 in the replication of each particular virus. Although we don't provide direct evidence for an interaction between TRN-SR2 and HIV-1 IN in the context of a viral PIC during infection, we believe our results do not refute the hypothesis that IN plays a direct role in the TRN-SR2-mediated nuclear import of HIV.
Since the DNA flap has been implicated in nuclear import [35-40], we compared the infectivity of both HIV-1-derived VSV-G pseudotyped vectors and infectious viruses with or without a functional DNA flap in HeLaP4 cells depleted of TRN-SR2 (Additional file 1). The flap-negative vectors and viruses were as sensitive for TRN-SR2 depletion as the vectors and viruses harboring a functional DNA flap, ruling out an important role for the DNA flap in the TRN-SR2 requirement of HIV-1 replication.
To understand the TRN-SR2 independent phenotype of the pseudotyped MLV vector observed in our previous experiments, we used pseudotyped MHIV chimeric viruses [6,31] to analyze the role of different viral factors in the TRN-SR2 dependency of HIV-1 (Figure (Figure2).2). Both the pseudotyped MLV vector and the MHIV-mMA12CA chimera carrying the MLV CA and MA proteins in place of the corresponding HIV proteins were much less dependent on TRN-SR2 than the HIV-1 parental vector. Contrarily, the pseudotyped MHIV-mIN chimeric virus still required TRN-SR2 for infection of HeLaP4 cells. Similar results were recently reported [30].
There are two possible explanations for our observations: TRN-SR2 may bind to the HIV-1 CA and/or MA proteins but not to MLV CA and/or MA, or TRN-SR2 may bind to both HIV-1 IN and MLV IN. To investigate the latter hypothesis, we measured the direct protein-protein interaction between GST-TRN-SR2 and His-tagged HIV-1 IN or MLV IN. Both integrases strongly interact with TRN-SR2 and display similar dissociation constants (HIV-1 IN Kd: 36.3 nM, MLV IN Kd: 17.5 nM). While no physical interaction of TRN-SR2 and CA has yet been demonstrated, our data are consistent with TRN-SR2 interacting with the integrase protein during viral replication in the cell. We have also previously ruled out binding by TRN-SR2 to any other viral protein apart from HIV-1 IN by reverse yeast two-hybrid screening [21]. Although this result can explain the TRN-SR2 dependency of MHIV-mIN, it does not explain the TRN-SR2 independent phenotype of the MHIV-mMA12CA virus and the MLV vector. It is well known that the uncoating steps of HIV and MLV are quite different [56]. During the early steps of retroviral infection most of the CA proteins dissociate from the HIV nucleoprotein complexes of incoming virions, whereas a large amount of CA remains bound to the MLV nucleoprotein complexes [57,58]. The uncoating process may be the rate-limiting step determining further downstream events such as the interaction with TRN-SR2 and through that, the nuclear entry of the PIC. According to this hypothesis, the MLV capsid may mask or shield integrase from interaction with TRN-SR2. Alternatively, the presence of this capsid may alter docking of the PIC to the appropriate nucleoporin required for TRN-SR2 interaction, and, in so doing, the capsid may determine the vehicle selected to cross the nucleopore tunnel. Pseudotyping with VSV-G is required in studies with chimeric MHIV viruses to overcome their poor infectivity and to reach measurable titers [6,31]. As we have determined that pseudotyping of viruses may mask the role of TRN-SR2 during viral infection, we cannot exclude the possibility that MHIV-mMA12CA still requires TRN-SR2 for infection after viral entry mediated by fusion instead of endocytosis. Therefore, we cannot conclude, based on our experiments using the chimeric MHIV viruses and AlphaScreen protein-protein interaction assays, that the requirement for TRN-SR2 during infection does not map to HIV-1 IN, as was proposed by Krishnan et al., based on their comparable results [30].
In an elegant study, Lee and colleagues describe how an HIV-1 CA mutant, N74D, renders HIV-1 resistant to a block in replication imposed by the ectopic expression of a C-terminally truncated version of CPSF6 [32]. Surprisingly, VSV-G pseudotyped HIV-1 N74D CA mutant virus was shown to efficiently infect both dividing and non-dividing cells depleted of TRN-SR2. In the same study, the nucleoporins (Nups) selected by pseudotyped HIV-1 were shown to also depend on the single residue change in CA. While pseudotyped wild type HIV-1 virus was inhibited by knockdown of Nup153 and Nup160, the pseudotyped HIV-1 N74D CA mutant virus was impaired to a greater extent by knockdown of Nup85, Nup155 and Nup160.
We have now demonstrated that the TRN-SR2 requirement for replication of the HIV-1 N74D CA mutant strictly depends on the viral envelope chosen (Figures (Figures3,3, ,4,4, ,55 and and6).6). In a multiple round infection experiment using HeLaP4 cells stably depleted of TRN-SR2, which more closely resembles a natural occurring HIV infection than single round infection assays, the N74D CA mutant virus proved to be still dependent on TRN-SR2 (Figure (Figure4D),4D), although to a somewhat lesser extent than wild type HIV-1. When we pseudotyped viral particles with the VSV-G (Figures 3A, B, 5A, B and and6D)6D) or the Ebola virus envelope (Figure (Figure6E),6E), which both mediate entry through pH-dependent endocytosis, the mutant virus proved completely independent of TRN-SR2. However, after pseudotyping the N74D CA mutant with envelopes derived from MLVampho (Figure (Figure6B)6B) or measles virus (Figure (Figure6C)6C) or in the presence of the wild type HIV-1 envelope (Figures 3C, D, 5C, D and and6A),6A), the N74D CA mutant was intermediately impaired by TRN-SR2 knockdown. HIV-1 and measles virus enter cells by membrane fusion, while MLVampho uses a pH-independent, caveola-dependent endocytic route. Both the multiple round infections using p24 as readout (Figure (Figure4D)4D) and the infection experiments using bioluminescent reporter proteins to measure infectivity (Figures (Figures33 and and5)5) suggest that the TRN-SR2 independent phenotype displayed by the VSV-G pseudotyped HIV-1 N74D CA mutant depends on the viral envelope mediating entry into the target cell and the trafficking route, rather than on a reduced interaction between CA and TRN-SR2 as was hypothesized by Krishnan et al. [30]. Intriguingly, the partial dependence on TRN-SR2 suggests that the N74D CA mutant is able to use an alternative nuclear import pathway besides TRN-SR2-mediated nuclear trafficking, especially when entering the cell via pH-dependent endocytosis.
The reason why certain CA mutations abolish the requirement for TRN-SR2 of HIV-1, especially when pseudotyped, is currently unclear. It is known that the HIV-1 CA protein interacts with different cellular cofactors like cyclophilin A and restriction factors like TRIM5α [59]. It is plausible that the N74D CA mutation could inhibit or stimulate certain interactions of the incoming viral cores with cellular cofactors or restriction factors, thereby altering the structure of the PIC and shielding the interaction with TRN-SR2. This may also explain the higher infectivity of the HIV-1 N74D CA mutant virus compared to wild type virus (Figures (Figures3,3, ,4,4, ,55 and and6).6). Overall, the N74D CA mutant is more infectious than the wild type virus in our hands, especially when pseudotyped with the VSV-G envelope, and this higher infectivity seems more pronounced in single round infection assays using bioluminescence to measure infectivity.
It is also known that endocytosis-based uptake of virions in target cells, mediated by the VSV-G envelope, changes the trafficking route followed by the incoming viral particles during early steps of infection. After entering the cell, viruses need to penetrate cortical actin, the dense layer of actin microfilaments underneath the plasma membrane [60]. Endocytosis naturally by-passes the cortical actin barrier and transports the maturing viral cores deeper into the cytoplasm before they are released from the endocytic endosomes [60,61]. It is possible that this different way of entry guides the incoming viral cores to other trafficking routes along the microtubuli. These re-routed PICs may dock eventually to different nucleoporins and interact with other importins regulating nuclear import. Anderson and Hope also suggest that the HIV-1 CA may direct viral nucleoprotein complexes to a cytoplasmic pathway leading to efficient NPC trafficking before being shed from the incoming viral nucleoprotein complex [61]. Hypothetically, the N74D mutant CA could direct PICs to an alternative transport pathway and a different compartment of the nuclear membrane where an unknown alternative import pathway is used. This may also explain the reduced TRN-SR2 dependency of the N74D CA mutant when carrying the HIV-1 envelope.
The velocity of particle trafficking and the speed of uncoating might also affect the status of the PIC when it reaches the nuclear membrane. Accelerated VSV-G induced trafficking through the cytoplasm, combined with a slower kinetics of uncoating due to CA mutations, may result in the shielding of TRN-SR2 nuclear localization signals when reaching the nuclear pore, forcing alternative import pathways to be selected. This could explain why the N74D mutant loses its dependency on TRN-SR2 in particular when combined with a VSV-G envelope.
Finally, CA may be directly involved in docking of PICs to the nuclear pore. Altered requirements of VSV-G pseudotyped HIV-1 Q63A/Q67A, E45A and N74D CA mutants for certain Nups, compared to wild type virus, were indeed demonstrated [32]. If the N74D mutation alters the selection of the specific Nup, alternative importins may be chosen for nuclear import, which could also explain the reduced TRN-SR2 dependency of the N74D CA mutant when carrying the HIV-1 envelope. The nature of these import factors still has to be revealed.
Our data demonstrate that HIV-1 CA modulates nuclear entry of VSV-G pseudotyped viruses. The role of CA in nuclear import of viruses which retain their natural envelopes also warrants further study, since the N74D CA mutant is less dependent on TRN-SR2 for infection. Our results argue for careful interpretation of experiments performed with VSV-G pseudotyped viral particles when studying early steps in viral replication and the role of CA in uncoating, trafficking, docking at the NPCs and/or nuclear import. By playing a role in uncoating, trafficking and/or docking, steps that all precede the site-specific interaction between IN and TRN-SR2, CA may exert an indirect effect on the process of nuclear import. PIC nuclear import remains a bottleneck process in the viral life cycle and the interaction between HIV-1 IN and TRN-SR2 may provide an exciting new drug target [62]. In order to design drugs that can specifically interfere with the nuclear import of the PIC, a better understanding of the exact mechanisms of TRN-SR2-mediated nuclear import and possible other import pathways is required. The data presented here will aid in reaching this objective.
HeLaP4 cells and 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 50 μg/ml gentamicin (Gibco, BRL) and 10% fetal calf serum (International Medical, Belgium). Cells were incubated at 37°C and 5% CO2 in a humidified atmosphere. Two different HeLaP4 cell lines stably knocked down for TRN-SR2 were generated by transduction with two different lentiviral vectors expressing shRNAs targeting the TRN-SR2 mRNA, called shTR3 and shTR4 (Sigma, clone-id NM_012470.2-867s21c1 and NM_012470.2-2084s21c1). A control cell line was established using a control vector expressing a scrambled shRNA referred to as shSCR (Sigma, product number SHC002). These lentiviral transfer plasmids were a kind gift from Dr. R. Hoeben (Leiden University Medical Center, The Netherlands). The shRNA expressing transfer plasmids are based on the pLKO.1 plasmid (Sigma) containing a puromycin resistance cassette. After transduction cells were selected and grown in DMEM as described above with the addition of 1 μg/ml puromycin.
For siRNA transfections, 6 × 105 HeLaP4 cells were plated per well in 6-well plates the day before transfection. The next day, 60 pmol of siRNA were transfected using 15 μl siLentFect (Bio-Rad, Belgium) in a total volume of 1 ml DMEM (10% serum, no antibiotics) per well according to the manufacturer's instructions. The siRNA siTRN-SR_2 targeting the TRN-SR2 mRNA (5' UCGGCGCACAGAAAUUAUAdTdT 3') (Qiagen, The Netherlands) was previously characterized [21]. As a control for non-specific effects, we used siTRN-SR_2MM targeting the same sequence but harboring 4 mutations (bold) (5' UCGGCGCAGUCUAAUUAUAdTdT 3') as described [21]. Mock transfected cells were always analyzed in parallel.
The viral molecular clones pNL4-3 and pNL4-3.Luc.R-E- were obtained through the NIH AIDS Research and Reagent Reference program, division of AIDS, NIAID, NIH, and were used to produce wild type HIV-1 virus and HIV-1 single-round luciferase reporter virus, respectively. The construct pNL4-3.Luc.R-E- N74D CA encoding a N74D CA mutant reporter virus was a kind gift of Dr. V. KewalRamani (National Cancer Institute, Maryland). Plasmid pNL4-3 N74D CA was created by digesting pNL4-3.Luc.R-E- N74D CA with BssHII and SpeI and cloning this fragment into pNL4-3 digested with BssHII and SpeI. Plasmids pNL4.3LAI cPPT and pNL4.3LAI cPPTD were used to produce wild type control viruses or viruses harboring an inactive DNA flap respectively as previously studied [38]. The following luciferase-encoding single-round reporter viruses were generous gifts from Dr. M. Emerman (Fred Hutchinson Cancer Research Center, Washington) [6,31]. Plasmid pLNCLuc is a MLV-derived retroviral transfer plasmid for production of MLV-based vector particles. Plasmid pLai3ΔenvLuc2 is an Env-deleted HIV-1 provirus and was the parental construct for the following HIV-based chimeric viruses (MHIV). The chimeric clone pMHIV-mMA12CA harbors the MLV MA, p12 and CA coding sequences instead of the HIV-1 MA and CA coding sequences [6]. In the chimeric clone pMHIV-mIN the HIV-1 IN coding sequence is replaced by the MLV IN coding sequence [31].
Viral vector production was performed as described earlier [63,64]. Briefly, VSV-G pseudotyped viral vectors were produced by triple polyethylenimine (PEI)-mediated transfection of 293T cells using per 10 cm dish 5 μg of the envelope expression plasmid pMD.G [65] for expression of VSV-G, 10 μg of a packaging plasmid and 20 μg of a transfer plasmid carrying a reporter gene flanked by 2 long terminal repeats. Two and three days after transfection, the supernatant was harvested, filtered with 0.45 μm pore-size syringe filters (Sartorius) and concentrated by centrifugal filtration using vivaspin concentrators (Sartorius). To produce viral vectors derived from HIV-1 we used pCHMWS_eGFP-T2A-Fluc [64] and pCMVΔR8.91 [66] as transfer and packaging plasmid respectively; for simian immunodeficiency virus (SIV) vectors we used pGAE-CAG-eGFP-Wpre [67] and pAd-SIV3+ [68] (a kind gift from Didier Nègre, Ecole Normale Supérieure, Lyon); for feline immunodeficiency virus (FIV) pSM32 and pSP26 [69] (a kind gift from Yea-Lih Lin, Institut de Génétique Humaine, Montpellier) were used; equine infectious anemia virus (EIAV) vectors were produced with p6.1G3CeGFPw (unpublished) and pEV53B [70] (kind gifts from M. Patel and J.C. Olsen, University of North Carolina, Chapel Hill) and for MLV-derived vectors we used pLNC_eGFP-T2A-Fluc and pCMVgagpol (Cell Biolabs, San Diego, CA). pLNC_eGFP-T2A-Fluc was constructed by cloning the eGFP-T2A-Fluc expression cassette from pCHMWS_eGFP-T2A-Fluc into the multiple cloning site in pLNCX (Clontech, Saint-Germain-en-Laye, France). Transfer plasmids pCSGFPW and pCASGFPW were used to produce lentiviral vectors harboring a DNA flap in sense or antisense orientation, respectively [37].
For production of Env-deleted pseudotyped single-round reporter viruses 20 μg of a viral molecular clone plasmid and 5 μg of an envelope expression plasmid were cotransfected per 10 cm Petri dish. Plasmids pMD.G [65] and pDOLHIVEnv [71] were used for pseudotyping viral particles with VSV-G or the HIV-1 envelope, respectively. Plasmid pAmpho [72] was a kind gift from Dr. M. Sena-Esteves (Massachusetts General Hospital, Boston) and was used for pseudotyping viral particles with the amphotropic MLV envelope. Plasmid pEboZΔ6GP (NTDL6) [73] was a kind gift from Dr. J. Wilson (Penn University, Philadelphia) and was used to pseudotype viruses with the Ebola Zaire (EboZ) envelope glycoprotein. Plasmids pCG-HcΔ18 and pCG-FcΔ30 [74] were a kind gift from Dr. C. Buchholz (Paul-Ehrlich-Institut, Langen), and were cotransfected (0.5 μg and 3.5 μg per Petri dish, respectively) during virus productions for pseudotyping with the measles virus envelope hemagglutinin and fusion protein, respectively. For productions of single-round viral particles pseudotyped by other envelopes than VSV-G, and for productions of the MHIV chimeric viruses, concentrated virus preps were used freshly and undiluted for infection because of the poor infectivity of these viral preparations. For production of wild type HIV-1 NL4-3 and NL4-3 N74D CA, the cells were transfected with 20 μg plasmid encoding the viral molecular clone. These viruses carry the wild type HIV-1 envelope; they were not concentrated but stored at -80°C after harvest and filtration through 0.45 μm pore-size syringe filters (Sartorius).
1 × 104 HeLaP4 cells transiently or stably depleted of TRN-SR2 and control cells were seeded into 96-well plates. Two days later cells were infected in triplicate with 3 dilutions (typically 5 × 104, 1 × 104 and 2 × 103 pg p24 unless described otherwise) of HIV-1 virus or VSV-G pseudotyped single-round HIV-1 virus in a total volume of 100 μl per well. p24 measurements were done with the HIV-1 p24 ELISA kit of PerkinElmer, or the INNOTEST HIV Antigen mAb kit of Innogenetics for the experiments presented in Figure Figure5.5. Virus stocks used in Figure Figure55 were also normalized for RT activity using the commercially available HS-Lenti RT Activity kit (Cavidi). We measured equal ratios of wild type and N74D mutant virus in both the pseudotyped and replication competent virus stocks using both the Innogenetics p24 ELISA and the RT activity kit. The inocula were replaced by fresh medium 24 h after infection. Each well was lysed 72 h after infection using 50 μl of lysis buffer (50 mM Tris/HCl, pH 7.3, 200 mM NaCl, 0.2% NP40, 5% glycerol) and was analyzed for β-galactosidase activity (chemiluminescent β-Gal Reporter Gene Assay, Roche Applied Science, Belgium) or firefly luciferase activity (ONE-Glo™, Promega, Belgium) according to the manufacturer's protocols. Chemiluminescence was measured with a Glomax luminometer (Promega, Belgium). The protein concentration of each sample was determined (BCA Protein Assay Kit, Thermo Scientific Pierce) and readouts were normalized to 1 μg total protein. The MHIV chimeric viruses and single-round viral particles pseudotyped with other envelopes than VSV-G were used freshly and undiluted for infection after concentration.
When retroviral vectors expressing GFP were used for transductions the inocula were adjusted to yield 30-60% GFP positive control cells. Transduction was performed 3 days post transfection of siRNAs and vectors were replaced by medium 24 h later. Three days post transduction, cells were trypsinized and fixed in 2% paraformaldehyde prior to analysis with a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ). Results are expressed as overall GFP fluorescence, i.e.% transduced cells × mean fluoresence intensity. For multiple-round infections of the stable TRN-SR2 knockdown HeLaP4 cells and control cells, 2 × 104 cells per well were plated the day before infection in a 6-well plate. The next day cells were infected with HIV-1 in a total volume of 1 ml corresponding to 6 × 104 pg p24 as determined by p24 measurements (HIV-1 p24 ELISA kit, PerkinElmer) of the viral stocks. Inocula were replaced by 4 ml of fresh medium containing only 5% fetal calf serum 24 h after infection. Four days after infection sampling of the supernatants for p24 analysis was started on a daily basis until cells reached complete confluency.
For the analysis of the late steps of viral replication, 5 × 105 HeLaP4 cells stably depleted of TRN-SR2 and control cells were seeded in a 6-well plate and grown until 50% confluency. Next 1 μg of pNL4-3 was transfected using 5 μl Fugene-6 according to the manufacturer's protocol. 6 hours after transfection cells were replenished with 1 ml fresh growth medium, and 24 hours after transfection supernatants were collected to measure p24 production (HIV-1 p24 ELISA kit, PerkinElmer). As a negative control, 5 μM of the protease inhibitor ritonavir was added to the shSCR expressing control cells.
TRN-SR2 cDNA was amplified from pGEX-6P-1-TRN-SR2 (kind gift from Dr. W.-Y. Tarn, Institute of Biomedical Sciences, Taiwan) using a forward primer (5' AAAAGGATCCATGGAAGGAGCAAAGC 3') containing a BamHI site and a reverse primer (5' AAAACTCGAGCTACTATCGAAACAACCTGG 3') containing a XhoI site. The PCR fragment was digested with BamHI and XhoI and inserted into the pGEX-6P-2 plasmid digested with BamHI and XhoI. GST-TRN-SR2 was purified using Glutathione Sepharose Resin (GE Healthcare, Belgium) as described previously [21] with minor modifications. Plasmids pKBIN6H [75] and pETINH1 [76] were used for expression of recombinant C-terminally His6-tagged integrases derived from HIV-1 and MLV respectively. C-terminally His6-tagged integrases were purified by affinity and ion exchange chromatography as described [75]. The expression construct for His6-tagged human heterotrimeric G protein α oA subunit (His6-GαoA) was kindly provided by Dr. D. Siderovski (University of North Carolina) and purified as described in [41]. His6-Roc-COR was purified via affinity and ion exchange chromatography as described in [42].
The AlphaScreen binding assay (PerkinElmer) was performed in a total volume of 25 μl per well in 384-well Optiwell™ microtiter plates (PerkinElmer). Appropriate dilutions of recombinant GST-TRN-SR2 and IN-His6 were added to 5 μl of the assay buffer (25 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.1% (v/v) Tween-20, 0.1% (w/v) BSA) to a total volume of 15 μl and the microtiter plates were incubated for 1 h at 4°C. Next, 10 μl of a mix of glutathione donor and nickel-chelate acceptor beads (PerkinElmer) (final concentration of 20 μg beads/ml) were added. Plates were then incubated for 1.5 h at room temperature and analyzed using an EnVision Multilabel Reader (PerkinElmer) according to the manufacturer's instructions. The assay was initially optimized for the interaction between GST-TRN-SR2 and HIV-1 IN-His6 by cross-titrations. A final concentration of 10 nM GST-TRN-SR2 was determined as optimal to avoid binding curve perturbation while still yielding a high signal-to-noise ratio (> 35). Subsequently, concentrations ranging from 1 to 150 nM of recombinant His6-tagged GαoA, Roc-COR, HIV-1 IN or MLV IN proteins were used to titrate GST-TRN-SR2. Each titration was performed in duplicate and assays were independently repeated. The equilibrium dissociation constants (Kd) were calculated with GraphPad Prism 5.
For western blotting, protein concentrations of 1% SDS whole cell extracts were determined using the BCA protein assay (Thermo Scientific Pierce) and after separation by SDS-PAGE 30 μg of each extract was electroblotted onto polyvinylidene difluoride membranes. Membranes were probed with monoclonal antibodies against TRN-SR2 (1:100 dilution) (Genway biotech, USA), β-tubulin (1:10000 dilution) (Sigma) antibodies were used to confirm equal loading. Detection was performed using horseradish peroxidase-conjugated goat anti-mouse antibody (Dako) and chemiluminescence (ECL+, Amersham Biosciences). For FACS analysis of CD4 expression levels, 4 × 105 HeLaP4 or 293T cells were incubated with 5 μl R-phycoerythrin-conjugated anti-human CD4 antibody (Miltenyi Biotec) in a total volume of 150 μl PBS for 0.5 hour at 4°C. After incubation cells were washed with PBS, resuspended in 2% paraformaldehyde and analyzed by FACS.
To determine TRN-SR2 mRNA levels, RNA was extracted from 2 × 106 HeLaP4 cells using the Aurum total RNA Mini Kit (BioRad). 5 μg of total RNA was reverse transcribed to cDNA in 100 μl total volume using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA corresponding to 25 ng of RNA was used to amplify TRN-SR2 mRNA or β-actin mRNA using the iQ Supermix (BioRad) and in-house designed primers and probes. Reactions and analysis were performed with the iQ5 Multicolor RT PCR Detection system and iQ5 Optical System software (BioRad). Each reaction contained 12.5 μl iQ Supermix, 5 μl cDNA, 100 nM or 200 nM forward primer, 100 nM or 200 nM reverse primer, 100 nM or 200 nM probe and water in a total volume of 25 μl for the detection of β-actin or TRN-SR2 mRNA, respectively. Primers and probe used for TRN-SR2 mRNA amplification and detection were: forward primer, 5'-CTACCAGATGTGGCTGAAGT-3'; reverse primer, 5'-ACAAAAAGTCGGTCTGTCAA-3'; probe, 5'-FAM-GCTCTGGGAGATCATGCAGG-TAMRA-3'. For β-actin mRNA detection we used: forward primer, 5'-CACTGAGCGAGGCTACAGCTT-3'; reverse primer, 5'-TTGATGTCGCGCACGATTT-3'; probe, 5'-HEX-ACCACCACGGCCGAGCGG-TAMRA-3'. Each sample was run in triplicate for 3 minutes at 95°C followed by 50 cycles of 10 seconds at 95°C and 30 seconds at 55°C.
The authors declare that they have no competing interests.
WT carried out the infection experiments, characterized the stable knockdown cells and wrote the manuscript. SDH generated the stable knockdown cell lines and carried out viral and vector experiments. JD performed the alphascreen protein-protein binding assays. OT, RV and MG purified the proteins used in the AlphaScreen assays. JDR helped with FACS analysis and drafting the manuscript. RG characterized and produced the different viral vectors used in this study. FC and ZD participated in the design and coordination of the study and wrote and edited the manuscript.
Supplementary Figure 1. The DNA flap does not affect the TRN-SR2 dependency of HIV-1 replication. (A) HeLaP4 cells depleted of TRN-SR2 (siTRN-SR_2) and control cells (mock and siTRN-SR_2MM) were challenged using 3 dilutions of an HIV-1-derived VSV-G pseudotyped viral vector harboring a functional wild type DNA flap (WT) or (B) with a flap-negative (Flap-) vector. Both vectors express GFP. Three days post transduction the overall GFP fluorescence in the cells was analyzed by FACS. Graphs show the mean values of GFP fluorescence ± SD of one representative experiment out of two performed in triplicate. (C) Same experiment as in (A), but using multiple-round HIV-1 NL4-3 (WT) or (D) HIV-1 NL4-3 flap-negative (Flap-) infectious viruses. Three days post infection β-gal activity was measured and normalized to the total amount of protein in the cell lysates. Graphs show the mean values of β-gal light units per μg protein (β-gal LU/μg protein) ± SD of one representative experiment out of two performed in triplicate.
The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL4-3 from Dr. Malcolm Martin and pNL4-3.Luc.R-E- from Dr. Nathaniel Landau. We thank R. Hoeben for kindly providing the NM_012470.2-867s21c1, NM_012470.2-2084s21c1 and SHC002 clones from the Sigma Mission human shRNA library, V. KewalRamani for providing construct pNL4-3.Luc.R-E- N74D CA, M. Emerman for providing plasmids pLai3ΔenvLuc2, pMHIV-mMA12CA and pMHIV-mIN, D. Nègre for providing constructs pGAE-CAG-eGFP-Wpre and pAd-SIV3+, Y.-L. Lin for providing plasmids pSM32 and pSP26, M. Patel for providing constructs p6.1G3CeGFPw and pEV53B, M. Sena-Esteves for providing plasmid pAmpho, J. Wilson for providing plasmid pEboZΔ6GP (NTDL6), C. Buchholz for providing constructs pCG-HcΔ18 and pCG-FcΔ30, W.-Y. Tarn for providing the expression construct pGEX-6p-1-TRN-SR2 and D. Siderovski for providing the His6-GαoA expression construct. We thank Melissa McNeely for critically reading the manuscript. Research was funded by grants from the IWT (SBO), the FWO and the EU (FP7 THINC). JDR was supported by a Mathilde Krim Fellowship from the Foundation for AIDS Research (amfAR).
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Sophie's at Saks Fifth Avenue has quickly climbed to the top of my favorite places to eat list! The amazing menu caters perfectly to all of my pregnancy cravings and I have yet to order something that I don't like. With under 30 days to go, I am feeling VERY ready for Baby Z to arrive! See how I'm preparing for her arrival on The Chicago Life Blog, plus get a sneak peek inside of her nursery.
Throughout my pregnancy, one of my favorite places to shop has been Seraphine. I fell in love with them just a little bit more when I heard about their #JustforLove campaign benefiting Baby2Baby. This incredible organization provides low-income children with diapers, clothing, and all of their basic necessities. Click here to see how you can help support this awesome cause!

subset: commoncrawl


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Announced Dec. 6, 2016 by Armin
From previous mentions here and on Twitter/Instagram you might have been aware that the 2017 Brand New Conference was planned to take place in New York but due to previously unscheduled construction at the New York venue we were forced to look for another one. Unfortunately, New York is so difficult to find a venue that matches our needs, budget, and preferred dates that, with all the pros and cons taken into account, it was better to change cities altogether. Apologies to those that were counting on BNConf coming back to New York — we were looking forward to it as well.
So… The 2017 Brand New Conference will be in Chicago, NOT in New York. The dates remain the same, September 14 - 15, 2017. We will be back at the crisp Harris Theater in Millennium Park.
Pre-sale tickets are available now through late April when prices go up by $100. If you have some budget left from 2016, this is a good time to avoid giving that money to the IRS.
The first round of speaker invitations have gone out and if all goes as planned, 2017's BNConf will be excitingly eccentric-ish.


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Ah, we made it! This post means it is the weekend! But we are thinking ahead to …Read more
Product Review: Goodbyn Lunchbox from UnCommon Goods is easy to pack and Made in the USA
02.29.12 by Comments 0
USA Love List loves shopping with UnCommon Goods. Although they are not specifically a Made in USA catalog, they …Read more


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Aino Vonge Corry
Retrospective Facilitation, København
5.000,00 kr.
Target Audience: The target audience are retrospectives facilitators, either novices or experienced who need new input.
Prerequisites: We recommend that you have read Diana Larsen and Esther Derby's book 'Agile Retrospectives' before the workshop.
Aino Vonge Corry is a teacher, a technical conference editor and retrospectives facilitator. She holds a masters degree and a ph.d. in computer science. She has 12 years of experience with Patterns in Software Development, and teaches OO design, software architecture and development in academia and industry. She also teaches how to teach Computer Science to teachers, and thus lives up to the name of her company; Metadeveloper. In her spare time, she runs and sings (but not at the same time).
Twitter: @apaipi
Video presentation: Functional Design Patterns
GOTO Recommends Aino because...
Aino is a distinguished GOTO speaker and teacher. She has a unique ability to observe and extract patterns and antipatterns in software organisations and she presents these in an enlightening way.
Varighed: 1 dag, Marts 15, fra 8.30 til 16.30
Lokation: Nordea, Strandgade 3, 1401 København K.
Sprog: Engelsk


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£79 for an overnight 4* city break for two plus a bottle of Prosecco, breakfast and late check out, £149 for two nights at The Richmond Hotel, Liverpool - save up to 68%
Found 413 days ago on Wowcher
Spend an unforgettable night for two at the Richmond Hotel, Liverpool Winner of a coveted TripAdvisor Certificate of Excellence. Upgrade to enjoy a two-night stay! Includes a bottle of crisp Prosecco, late check out and buffet breakfast. Located in the heart of Liverpool. Valid for stays until 28th Feb 2017.
4* Liverpool Break with Breakfast & Prosecco for 2 @ The Richmond


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To all those in the US, Australia, New Zealand and any other parts of the world who are celebrating Mother's Day today . . . . . .
Happy Mother's Day!
Should you want to bake your Mum a little something to brighten her day, see my posts here and here.
Our thoughts are with you if you are away from or missing your Mum on Mother's Day for whatever reason. It is not a happy day for everyone so hugs and thoughts from my family to yours if that is the case.


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Training on The Teaching of French For Specific Purposes – University and Tertiary Colleges in Kenya
- Details
- Friday, 10 June 2016
The above named training organized by the French Embassy in conjunction with the Alliance Française took place on 31st May to 4th June 2016 at the Alliance Française, Nairobi. It targeted participants from Kenyan Public Universities and colleges offering French language in their various academic programmes. Professional French seeks to equip learners with the linguistic and intercultural competency to interact in a professional context. The theme of the training was "CURRICULUM DEVELOPMENT AND IMPLEMENTATION OF FRENCH FOR PROFESSIONAL INTERACTION IN BUSINESS, TOURISM, HOTEL AND RESTAURANT, INTERNATIONAL RELATIONS AND DIPLOMACY "
It was facilitated by Mr. Alexandre Holle from the Center of French language Institute in Paris- France. The training recognized the need to tailor a curriculum in line with the professional demands and industry expectations necessary for the accomplishment of professional tasks
La Journée de la Francophonie (The Universal French Speakers' Day)
- Details
- Wednesday, 23 March 2016
Held on the 20th March 2016 at The Kenya School of Monetary Studies
The Kenyan chapter of this annual event organised by : French Embassy, Kenya Association of Teachers'of French (KATF) and Francophonie Organisation in Kenya, attracts participation from kenyan universities, tertiary institutions, NGOs, secondary and primary schools offering French as teaching and learning subject. In addition, country representatives of Francophone countries like Burundi, Congo Brazaville, Comoro Islands, Mayote island, Madagascar were present this year to showcase their traditions and culture. The event brings together French speakers in Kenya with the aim of promoting the use of French language. The students have an opportunity to nework with native French speakers, recite poems, sing songs, act and dance to French music. The Technical University of Kenya seized the opportunity to market its academic programmes to the participants. More than a thousand clients visited the university stand.
« A TU-K le français, c'est pratique», Vincent Mukoshi a 2nd Year student in the School of Hospitality.


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Former World's Fastest Supercomputer Of 2009 Now To Be Decommissioned
Roshan Ashraf Shaikh / 5 years ago
IBM's Roadrunner which at one time held #1 spot in Top 500 Supercomputer list in June 2008, November 2008 and June 2009 will now be decommissioned. IBM built this super computer 5 years ago to model the decay of United States' arsenal of Nuclear Weapons and had the power to do 1 quadrillion floating point operations per second and currently in Department of Energy's Los Alamos National Library, New Mexico.
Roadrunner uses 296 server racks, using 122,400 processing cores with the combination of IBM's PowerXCell 8i and AMD Opteron dual-core processors. and occupying 6,000 Square feet, the total cost was more than $120 Million. AMD's Opteron were used for "basic" tasks, but IBM's PoweXCell were used as a "computational accelerator" as it was handling most intense part of the calculations.
Los Alamos lab said in an announcement "During its five operational years, Roadrunner, part of the National Nuclear Security Administration's Advanced Simulation and Computing (ASC) program to provide key computer simulations for the Stockpile Stewardship Program, was a workhorse system providing computing power for stewardship of the US nuclear deterrent, and in its early shakedown phase, a wide variety of unclassified science."
It should be noted that although it doesn't hold the #1 spot, it still is one of 22 fastest Super computers in the world, however the power consumption of Roadrunner is not very energy efficient, thus the reason of being taken offline.The former world's #1 supercomputer "Roadrunner" will first be studied for sometime before its being dismantled.
Los Alamos added,""Future supercomputers will need to improve on Roadrunner's energy efficiency to make the power bill affordable. Future supercomputers will also need new solutions for handling and storing the vast amounts of data involved in such massive calculations."
Source: ArsTechnica


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Christopher Rushton
PhD ResearcherRoom: 2.10 / LG.12
Email: firstname.lastname@example.com_
Research Group: Spatial Modelling and Dynamics
PhD Research Topic: Analysing Remote Sensing Data to Redesign Emission factor Models.
My key research interest is to measure the emissions of vehicles in real driving environments. I am particularly interested in:
- Modelling of vehicle emissions
- Impact of Taxis and Light Commercial Vehicles
- Impact of Euro 6 Technology
- Deriving strategies for Low Emission Zones
2012 - Present: PhD Student at ITS Leeds
MPhys and MSc from University of Leeds
This page will be updated from our publications database.


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It's Megabucks Friday!
4 Mar
It's Megabucks Friday today on Swagbucks!
I love Megabucks Friday because that means I have more chances to win swagbucks [they're "extra generous" on Fridays]. Normally you'll be awarded 20 – 50 pts….but on Fridays you have chances of earning 100 points or more per search!
Go search away on Swagbucks.com.
Haven't heard of Swagbucks? Click here to read about it.


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One man is dead after a single-vehicle rollover southwest of Melville, Sask. on Saturday night.
It happened on Highway 22 one kilometre west of Neudorf, Sask. at about 7:40 p.m. CST.
Police said a man driving a pickup truck was heading eastbound when he lost control of the vehicle, causing it to roll several times down the centre of the highway.
The man was the only person in the truck.
He was thrown out of the pickup and was pronounced dead at the scene.
Road conditions were good at the time of the accident and are not a factor in the crash, police said.
The RCMP are investigating. The driver's name will not be released.
Neudorf is located 135 kilometres northeast of Regina.


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Discussion in 'Geoff's Search Engine.' started by geoff501, Aug 11, 2009.
Morning Geoff,
Cheers, many thanks for updating.
I have just tried to search for a post WW2 Armed Forces death on the Armed Forces Memorial roll of honour and it says:
This part of GOV.UK is being rebuilt – find out what this means
This is on the following website page:
I believe the serviceman I was looking for died of 'natural causes' rather than due to involvement in a war or conflict. I had an idea service post-war personnel who died due to accidents or natural causes were commemorated on this website, but did not have their names engraved on the memorial at the National Arboretum?
It is rather difficult to check when the official means to do so is unavailable. Does anyone have any information on this please?
Links broke again. Think I'll throw the towel in on this one.....
Now on line again... but a bit out of date perhaps:
Geoff's AFM Search Engine
The official MOD search is here, but you can only search on name unit number:
AFM Start Page
Separate names with a comma.


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Want to follow along with this writing? Sign up Today, it's free and easy
over 5 years ago Janae Elizabeth said:
*For the Swap* Oh my word I love you're writing style! The introduction was brilliant. It was confusing and dark and yet made so much sense and...I probably sound kind of crazy right now but the gist of it is I loved it. Great job!
over 5 years ago Gaby S. said:
This was just was just so exciting! It was fun and flowed well. Great job with this, and keep it up.
over 5 years ago J. Hammer said:
This is really good and has a lot of potential. I really hope you continue it. It kind of reminds me of Inception but I get the feeling that it's different. It kept me interested and kept me reading, which is very good. I also could feel something. Some of this was very well written, while other bits may need some work. I would consider rewriting the first part (not the prologue) of the story, especially the first sentence, because it was confusing. Try to break it up more, and make your individual sentences flow more. I think that even if you don't do this, the story will only get better. I hope you continue.
over 5 years ago Nicole said:
This is fantastic writing! I found myself being soaked into every word you have placed on the screen of my laptop. Its descriptive, full of emotion, and wonderfully crafted. I really enjoyed this piece and hope you don't give up on it!
about 5 years ago RAY. said:
Well for starters, you're a really talented writer. This is honestly one of the better pieces I've read on this website.
Anyway, there are a few minor speed bumps. You're storyline is extremely complicated. You're trying to keep an ample amount of mystery within the plot, which is what ultimately is going to keep the reader's attention. There is nothing wrong with this, in fact that's what impresses me most about you're writing. You're really good at keeping me intrigued, and I actually sort of love how confusing everything is. However, at some points there is almost too much confusion. Some information we dont need this early in the story, like Avery's feelings on her parents' death. Yeah she's upset, but we already know that and I feel as though you should save her exact feelings and emotions for later as the story progresses. On the contrary,there's a lack of information in other points. You're giving us too much info on Avery, but not enough info on the main character. I would either take away from Avery, or add in info about the main character fairly soon once you continue.
And as far as continuing goes, I'm not totally sure what advice I can give you. I have no idea where you're going with the plot, and if you're anything like me, you don't know either. Stay focused though. It helps me to overcome writer's block when I step back and reevaluate each of the character's backgrounds. Start with their parents and work your way through their lives, and into the main character, and Avery's lives. Completely design the character from their history to their characteristics. Then decide where that information is going to lead them. I never know what happens next when I write, I sort of just sit back and let the scenario play out in my head as a movie would.
If you have any other questions please feel free to ask. Normally it's a pain to write reviews (I mean let's be honest), but I've really taken a genuine interest in your story. Definitely keep it up. Good luck!
over 5 years ago Summer Hill said:
I really like this story and think it could be really amazing as you review it more and more.
First off, I like how the introduction is kind of free flowing, but it gets almost to off track and distant. I would just try to pull it in to convey exactly what you want.
Also, in attempts to make it better, it seems like your using sort of obscure sentence structures at times. Sometimes it works, but I think you should read over for strange wordings. On the contrary, some sentences are to plain, simple, and almost unnecessary (just restating something in a more simple way). Let the reader put the facts together a bit!
This is a no brainer teachers always say... SHOW DON"T TELL! But really, at some parts you could use a bit more showing.
I find the end part of "The Beginning, The Middle, and The End" to be misplaced. You could add the info about the little girl feeling her parents death is her fault in some other place. And the Aric stuff is unnecsarry and obvious. Of course she doesn't want him to die, we all see that.
I still can't get a picture of either girl. I can't even picture the main girls age.
How did the two girls find one another? How do they know it's the same killer?
Where is this?
Aric's speech in the beginning is so awkward. The "My fair Amanda" and "Stay safe" are so not things people would typically say. Especially in the given situation. Plus, Amanda would have totally freaked out more judging by her character, or so it seems to me.
So is it a dream or reality? I truthfully can not tell. Maybe I just didnt read close enough but I can not tell.
As for going forward, I would clear up if its a dream ro reality and WHY things are going on. I'm pretty confused at parts to be honest.
All the being said, I like this alot. Don;'t take the criticm negatively. Its just because saying "Yay! Wow! Amzing :)" wont make the piece any better. I want it to be the best possible! :) Great job on it.


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GraphicRiver Nail Polish with Silver Cap 9902568
Stock Vector \ Conceptual \ Commercial \ Shopping \ Services
Polish \ Vector \ Simple \ Spatter \ Pretty \ Symbol \ Traditional \ Silver \ WetNail
Stock Vector - Nail Polish with Silver Cap 9902568 by Lyusjen @ GraphicRiver
Stock Vector \ Conceptual \ Commercial \ Shopping \ Services
Nail polish with silver cap. Vector set
Layered: No; Graphics Files Included: Photoshop PSD, Transparent PNG, JPG Image, Vector EPS, InDesign INDD, Quark QXP, TIFF Image; Minimum Adobe CS Version: CS.
Floral Frame; Bows; Little Princess in a Purple Dress; Little Princess in a Yellow Dress; Green Christmas Wreath; Heart Rose; Mouse and Cat Holding a Heart.
Keywords: art, beautician, beauty, bottle, brush, cosmetics, element, fashion, female, gel, glamour, glass, icon, illustration, isolated, makeup, manicure, nail, paint, polish, pretty, salon, shop, sign, simple, spatter, symbol, traditional, vector, wet.


text:
Hi there.
I just wanted to say that the deck is great and the service was fantastic. That's all I can say.
Thanks again.
Read More ->
Hi MJM. thanks for all your help. Your web site is easy navigate through and the shipping time is great. It doesnt take long te recieve things from...
Read More ->


text:
So my writers block is still in full swing so i thought that today i would tell you the story of my life. I know this is normally done through a video but i thought i would write it all instead, so i guess let's get to it. This is going to be one long story.
I was born on the 18th August 1997. I was going to be called Rebecca Sophie but the women in the bed next to my mom called her daughter Rebecca so my mom decided to swap the names around and that is how i got my name Sophie Rebecca.
Thinking about it now i could never imagine myself being called Rebecca it wouldn't suit me at all.
When i first started primary school, in reception i absolutely hated it. I was so upset my teacher had to sit me down with a jigsaw just to make me be quiet. I soon settled into school and i loved it. As i went through school i got more and more close to my friends and they soon became my best friends. I grew up with them and we knew everything about each other. Throughout primary school I was bullied a lot by one girl and even after my mom went to see the principle nothing got done because they didn't believe her ( obviously i would make up being bullied right?!). I remember the one time in the school holidays when i was out with one of my friends and the girl who bullied me put the outside spikey part of an conker down my back and pushed me against the wall so the spikes stabbed me.
I stayed friends with the two girls i was friends with until we left for secondary school where we split up and went to different schools. I loved secondary school, well for the most part anyway. When i joined in year 7 i got settled into the wrong crowd but i soon got out of it. I became what most people will call a geek or nerd. I was part of a science club and i loved it. After separating out of the wrong crown in year 7 i became friends with a group of girls in my classes. We became really close and i was finally happy and on the right track. I stayed in that group of friends until year 8 when my worst nightmare happened (well my worst nightmare at the time). The girl who had bullied me for my whole primary school years joined my secondary school AND became buddies with my best friends. We all know how this ended right? I was pushed out and i became part of another group. Honestly although i hated it at the time this was probably the best thing that happened to me.
I became friends with a whole different group of people and i was so much happier. Over the course of the year i became closer with one girl in the group and as time went on we formed a friendship group with a group of girls from different classes. This was when i met my old best friend. I got really close with one of the girls from a different class and we did everything together, we did the cheesy 'facebook best friend marriage' and we were inseparable. At the end of year 8 and the start of year 9 i became close with yet another group of people and our friendship groups blended. It was this year that i met my best friend. My cousin was dating Greg and we became really good friends, even after they broke up we still remained really good friends. We didn't have any classes together but spoke constantly through text and saw each other at break and lunch. As time went on and we went into year 10, we were pretty much inseparable, we text 24/7 and his girlfriend hated it ( i found it funny but hey i get why she was mad).
At the end of year 10, me, Greg and my other friend Lucy were chosen to be prefects. We were so happy but the other people in out group turned bitter about it. It was at this time that me and my 'best friend' stopped talking, she hated how close i was with Greg and honestly just turned nasty. This was also the time the longest argument of my life happened. I don't even know what started the argument but it lasted for over a year. Facebook comments, mediation's and full blown arguments, it was then when i realised who my true friends were. Looking back i don't regret the argument because it did make me stronger as a person.
At the start end of year 10 was when me and Greg fell out, we went through the whole 'more than friends' moment and argued after and well that turned into not talking for weeks. But even after the whole argument we still ended up friends. It was then a few weeks into year 11 when Greg introduced me to my other best friend, Brit.
I will never be able to thank him enough for introducing me to a girl who has changed my whole life.
This was also one of the worst times in my life. I felt like i should have been so happy, i had great friends, i was doing well in school and nothing seemed wrong. But i constantly fell down, i felt like i was constantly drowning without a reason why, i couldn't seem to feel happy and i soon believed i just didn't deserve to be. To this day i still don't know what happened that year but i constantly felt down and this was when i self harmed for the first time, something i still haven't spoken to my parents about. Looking back now i wish i never had, i guess all people do but getting over it made me stronger. At the end of year 11 i went to prom, i wasn't going to go to prom. I'm not much of a dress kind of girl, but i was so glad i went i had so many laughs and jokes and it's definitely a night i won't forget. It was also just before this that i chose to attend sixth form at the same school i was at. I started sixth form with the same friends but soon me and Greg fell out again, some of our other friends thought we we're 'too' close and it caused a huge argument. I look back now and realise the argument was ridiculous, but at the time i was so dramatic.
However over the year we became friends again due to studying the same A-Level. I studied three A-Levels and a BTEC during my first year, I still self harmed and i couldn't help but feel down all the time. This was when my best friend Lucy found out. I still remember the day that she sat me down and spoke to me about what was happening and there was a lot of tough love involved but it got through to me. It was after that that i decided that i needed to find every way i could to stop. And i did, it didn't happen overnight but i got through it.
I then entered my second year and then dropped down to two A-Levels and a BTEC. During my second year i started applying for University, i was so excited but looking back now i think i was only excited because it was something i was doing with my friends. As the year continued i stayed close with my four best friends, Lucy, Greg, Brit and Emma and ended up accepting a university offer from Nottingham Trent, as did Greg. I booked my accommodation and started buying things for my room. I was so excited about the future.
When i finished Sixth form i was so excited. I left with a B in Media Studies, a C in sociology and a Distinction * (A*) in ICT. But as i got closer to the university start date i realised it wasn't at all what i wanted. I was on holiday in Cornwall and i made myself ill because i was so worried about going. It was then that i decided to transfer to Coventry University. However as the week went on i decided i didn't want to go at all and decided to drop out altogether.
Looking back now, i do miss school. Growing up my parents would always say school will be the best days of your life, i always laughed it off but they were right.
While all of this was going on, i was on holiday. Probably the best holiday of my life. I spent two weeks in Cornwall with a group of people I've known my whole life. I have never laughed so much in my whole life. I had so much fun and i truly learnt the meaning of family. You might not get to pick your biological family, but your heart does get to pick some family and my heart picked an amazing bunch of people for me to call family.
When i came back from holiday, i knew i didn't just want to sit around and do nothing so i applied for a job at my local grocery store and got an 8hr a week contract. By the end of the first week my contracted was upped to 12hrs and by the second week it was upped to 16hrs. And this is where i am now. On the 7th February 2016 i posted my first blog post and i have been writing ever since i love blogging and hope to keep blogging for as long as possible.
Blogging is a way for me to get my thoughts out, a way for me to talk about things I'm passionate about and i love it.
I hope you enjoyed this lengthy, write my life, if you have any questions feel free to comment.
Thank You for reading,
Soph xoxo
Disclaimer- All names have been changed for privacy reasons.


text: I tried the old technique of unplugging and replugging the battery pack in until it stayed red for longer, but this didn't work for me. This is how to completely drain the battery pack and reset it so that it will start to charge again! What you need: Rechargeable battery packs (the ones we are trying to fix) a few paper-clips A charging station or whatever you use to recharge your packs Preparation First bend your paper clip into a horse shoe shape, it is easier if you do a few Draining the Battery pack You'll see 6 pins on the battery of the battery pack. [] [] [] [] [] [] You need to use the horseshow paper-clip to touch pins 2 and 5 together (in red) The paper clip will get hot, when it does either change paper clips or let it cool Repeat this until there is no charge left in the battery (the paper clips will no longer get hot) *When you first do this, you may find the paper clip doesn't get hot, keep pressing 2 and 5 together and you will begin to feel the heat* To Reset the battery indicator circuit [][] [] [] [][] First touch pin 1 and 5 together (blue) [] [][] [] [] [] Then touch pin 2 and 6 together (red) Now you should be finished! plug it into the dock and good luck! (if you have problems with the light not coming on at all, repeat the last step, or drain a bit more before repeating the last step)


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Falling into the severe category of parental alienation are those parents who become obsessed with destroying the child's relationship with the other parent and that parent's family and friends. Dr. Frank Williams describes this goal of cutting a parent out of a child's life as a "parentectomy." In these cases, a child will succumb to the alienator's programming or brainwashing and experience fear, anger, and hatred toward the target parent. When parental alienation is severe enough, children have no choice but to align with the disturbed parent against the target parent, thus destroying their relationship with the target parent. These children no longer have free will or the ability to continue loving the target parent. PAS describes the child's behavior in response to the brainwashing that has occurred; it does not describe actions on the part of a parent. The focus of this article in on children who are being severely alienated or who are already experiencing PAS...It is unfortunate that too many people will believe a dramatic story more than they will listen to evidence. Drama is the hallmark of people who are psychologically disturbed. Individuals with these severe mental health issues are under-diagnosed. We need more research and clarity on the effects of PA/PAS. The costs are staggering to children, the target parent, and that person's family. The damage is severe and has long-reaching effects. The whole fabric of our society is undermined by the behaviors of these severely disturbed individuals. Both men and women with obsessed thinking create PA/PAS situations with children and their target parent. Their irresponsible behaviors siphon off a staggering amount of social resources to stabilize the chaos they create. Any protocol that we use for the regular population is woefully inadequate in making them normal. Every year, hundreds of thousands of children and parents are experiencing the phenomenon of PA/PAS and the resulting devastation it causes. Millions of people are ending up damaged because, up to now, we have not even recognized the phenomenon or truly considered its impact. We all need to take action to educate and help people who have this terrible problem that does such severe damage to children.
By Jayne A. Major, Ph.D
Sunday, September 14, 2008
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Just replace my clutch master cylinder because I was losing some pressure in my clutch pedal and could not get it into gear. I had a clutch kit put into my truck about a year ago.I bench-bled the entire clutch assembly. I put everything back together and I have pressure once again and can shift. The problem is now when I press on the clutch pedal to shift I get a whining noise. Is it the throwout bearing?
Very likely. What year is this Ranger and how many miles do you have on this clutch?
It is pretty likely that you need to replace the clutch plate and the throwout bearing along with it. You might as well, since most of the cost involved is labor. The parts are relatively inexpensive.
If the noise disappears when the clutch is fully released and occurs as soon as you depress the pedal, it's the throw-out bearing..
It is a 2001 with about 110,000 miles.I had a clutch kit put in at 100,000. Who knows what type of work the mechanic did. His shop is closed now. I know he didn't put a new slave in because I asked him why he didn't when I picked i up and he said he didn't need to. What an answer right.
Since the clutch plate was just replaced last year do you think I should replace it again or just put in a new slave w/ throwout bearing? Is this job easy enough to do for a weekend warrior? I done a lot of reading and watched some videos on how to replace the throw out and clutch assembly.
Will driving around with a throw out bearing that is going out cause any further damage.I don't think I'll be able to work on it for a few weeks.
Why do you think you need a slave cylinder? You possibly could replace that without pulling the transmission, but it doesn't sound like your problem.
You need to pull the transmission to do the bearing, that's why it's not much more work to do the clutch plate at the same time, but since it only has 10k on it I would probably not relace it.
I've seen people drive around with a failing throwout bearing for far longer than a few weeks. Just go easy on the clutch, don't make any long trips, and you should be fine for a few weeks.
The release bearing is a "thrust bearing". Its load is along its rotating axis. Basically, the only time the outer "race" and the inner "race" are spinning relative to one another is when the flywheel (to which the pressure plate assembly is bolted) and the tranny input shaft are spinning at different speeds, and the only time it"s under axial load is when it's pushing the pressure plate levers in. Both of these conditions only occur during shifting or holding the clutch in. As long as you're cruising along with the car in gear, or sitting at the light with the tranny in neutral and the pedal released, the bearing is not doing anything nor is it under load. You ahould be fne for the next few weeks as long as you go easy. And don't hold the pedal in at stoplights.
The reason I was thinking of replacing the slave too, was that my slave is internal(inside the bell housing). So since when I had the clutch kit put in and the knuckle head didn't replace the slave I thought it might be a good time to do it.


text:
LAHORE, Dec 14: The Lahore High Court issued on Friday notices to Attorney-General Malik Muhammad Qayyum for the alleged involvement of his son, Muhammad Ahmad Qayyum, and 13 others in a fraudulent execution of the sale deed of a plot of land.
The division bench, comprising Justice Syed Shabbar Raza Rizvi and Justice Muhammad Muzammal Khan, issued notices in a contempt of court petition by Zahid Mehmood. The case will be taken up on Feb 4.
The petitioner said influential parties were involved in litigation of expensive land on Main Boulevard Gulberg-III namely 'Qasr-i-Zauq'. He said Mr Qayyum's law firm conducted cases pending in various courts pertaining to the land on behalf of various plaintiffs and defendants.
He said Mr Qayyum committed fraud with courts and also instituted a suit through his maternal uncle, Malik Zulfiqar, regarding the property on the basis of a fake compromise.
"But the suit was dismissed on technical grounds and Mr Qayyum's son filed an appeal with the LHC," the petitioner said.
He said an LHC division bench on Nov 30, 2006, issued an injunction against the sale and transfer of the property.
"Only after 160 days of court's orders, Malik Qayyum sold the property to the son of Senator Gulzar for a mere Rs80 million," he added.
He prayed that the respondents along with Mr Qayyum be ordered to be detained for six months. He prayed that the impugned sale deeds dated Dec 16, 2006, registered by the Gulberg sub-registrar be declared illegal.
He also prayed that respondent M/s Asian Gas Pvt Ltd be directed to present the unlawful sale deeds in original and be restrained permanently from using the impugned deeds.
He said the sale deeds were prepared and executed at Malik Qayyum's office. He said the law firm also got public notices published in national dailies confirming the sale of the property.


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