regulated degradation of cell surface molecules , particularly receptors involved in myriad signaling pathways , is essential for the control of many biological processes , including cell growth , tissue morphogenesis , and host defense ( seto et al . , 2002 ) . endocytic membrane cargo can be partitioned either into recycling vesicles , for delivery back to the cell surface , or into late endosomes , for delivery to degradative compartments ( gruenberg , 2001 ) . recognition of cargo at the limiting membrane of the late endosome results in invagination of the bilayer , budding of cargo into intraluminal vesicles , and formation of mvbs . the vesicles and their contents are then degraded by proteases and hydrolases after fusion of the mvbs to lysosomes ( katzmann et al . , 2002 genetic and biochemical studies during the last several years have begun to elucidate the mechanism by which degradation of membrane cargo is regulated ( katzmann et al . , screens in saccharomyces cerevisiae have identified multiple genes involved in trafficking of proteins from early endosomes to the degradative vacuole , the lysosome equivalent in yeast . studies of mutants with defects in sorting of transmembrane proteins , such as the g protein  coupled ste2p pheromone receptor and carboxypeptidase s , from the plasma membrane and golgi , respectively , to the vacuolar lumen have led to the identification of class e vps proteins required at different stages of this process ( katzmann et al . , 2002 ) . these include components of the three biochemically characterized high molecular weight escrt complexes and additional proteins , notably vps27p and vps4p , an atpase required for the release of the escrt machinery at late stages of budding ( garrus et al . , 2001 ; conibear , 2002 ; katzmann et al . , 2002 ) . despite the identification of many of the components required for cargo delivery to the mvbs , there remains a gap in understanding how cargo is recognized and targeted for invagination in late endosomes . monoubiquitination of cargo has been proposed to be at least one of the signals involved in recognition by the vacuolar sorting machinery ( hicke , 2001 ; clague , 2002 ) , thus , mutant carboxypeptidase s that fails to be ubiquitinated is delivered to the limiting membrane of endosomes but does not invaginate into mvbs ( katzmann et al . , 2001 ) . in mammalian cells , appending ubiquitin to the cytoplasmic tail of transferrin receptor , which normally recycles to the cell surface , results in its diversion to late endosomes ( raiborg et al . , 2002 ) . several class e vps proteins have ubiquitin interaction motifs ( uims ) or uev domains that interact with ubiquitin in vitro ( katzmann et al . , 2002 ) . these include vps23p / tsg101 and vps27p / hrs ( pornillos et al . , 2002a ; shih et al . , 2002 ) the interactions of these proteins , resulting in recruitment of escrt - i to the limiting membrane of the late endosome , are described in the current articles . the recent characterization of the interaction of viral l domains with tsg101 has spurred efforts to identify cellular components that normally recruit the escrt - i complex in the process of mvb formation . the current works demonstrate that the yeast adaptor protein vps27p and its mammalian ortholog , hrs , serve as key links to deliver early endosomal cargo to the mvb and vacuole ( bache et al . , 2003 ; katzmann et al . , 2003 ; lu et al . , 2003 ; pornillos et al . , 2003 ) hrs had been shown to associate with several proteins involved in endocytosis , including clathrin and eps15 ( raiborg and stenmark , 2002 ) . it was also found to colocalize primarily with early endosomes and to contain a fyve domain , which interacts with phosphatidylinositol 3-phosphate ( pip ) , a marker of early endosomes ( fig . 1 ) ( komada et al . , 1997 ; raiborg et al . , 2001 ; stahelin et al . , hrs also has a uim , which is involved both in binding to monoubiquitinated proteins and in directing ubiquitination of hrs itself ( raiborg and stenmark , 2002 ; shih et al . , 2002 ) . in yeast , the uim is required for delivery of cargo to the vacuole ( bilodeau et al . , 2002 ) . mouse embryos homozygous for a targeted inactivation of hrs display abnormally large early endosomes , and hrs - deficient drosophila have defective endosomal invagination and mvb formation ( komada and soriano , 1999 ; lloyd et al . , 2002 ) . in addition , hrs and vps27p have several proline - rich motifs that resemble those in viral l domains . the new studies have approached the problem of how escrt - i is recruited to the site of mvb formation from different angles . emr and colleagues have used fluorescently tagged proteins to examine their relationship in yeast ( katzmann et al . , 2003 ) . they show that gfp - tagged vps27p colocalizes with ds - red  tagged fyve domain of eea1 , an early endosomal marker , and that this requires the fyve domain of vps27p ( fig . deficient vps34 mutant cells , consistent with a requirement for pi(3)p in the recruitment of vps27p to endosomes ( katzmann et al . , 2003 ) . vps23p was coprecipitated with vps27p from membrane fractions , but not soluble fractions , of cells transfected with epitope - tagged versions of both proteins , suggesting a direct interaction between vps27p and escrt - i only on endosomal membranes . this interaction did not require either the uim or the conserved vhs domain of unknown function in the nh2-terminal part of vps27p . gfp - vps27p was localized to endosomal structures in a vps23p - deficient strain , but vps23p - gfp was diffusely distributed in the cytoplasm in the absence of either vps27p or vps34p ( katzmann et al . , 2003 ) . these results suggest that vps27 acts upstream of vps23 , recruiting it to the endosomal limiting membrane once it is activated due to interaction with cargo protein ( fig . 2 ) . targeting of vps23p - gfp to endosomal membranes required expression of vps27p with an intact p / q - rich cooh - terminal domain , including psdp and ptvp sequences resembling those in retroviral l domains . . the normal function of hrs , shown in the left panel , is to recruit tsg101 and the escrt - i complex for sorting ubiquitinated ( ub ) cargo into the mvb . during hiv infection , hiv gag mimics hrs and redirects the escrt machinery to the site of viral budding . in t cells ( left panel ) , viral assembly and release occur at the plasma membrane , whereas in macrophages ( center ) hiv particles accumulate in late endosomes and mvbs . in dcs ( right panel ) , hiv undergoes receptor - mediated endocytosis after binding to dc - sign and is recycled to the cell surface . although the vesicular compartments involved have not been identified , macrophages and dcs may share a mechanism for releasing hiv particles upon contact with t lymphocytes . consistent results were obtained by stenmark 's group , which used rna interference to reduce levels of hrs in hela cells and showed that tsg101 association with membranes is dependent on hrs expression ( bache et al . , conversely , when hrs was overexpressed tsg101 was relocalized from lamp1 late endosomes to the eea1 early endosomal compartment . these results are consistent with the ability of hrs to interact with early endosomal cargo and subsequently recruit tsg101/escrt - i to the late endosome . when hrs levels are abnormally high , more of the protein may be expected to associate with pi(3)p in early endosomes , resulting in aberrant binding of tsg101 at an early stage of endosomal maturation ( bache et al . , 2003 ) . sundquist and colleagues approached the general problem of targeting of cargo to mvbs from the perspective of the tsg101-recruiting sequence in hiv gag ( pornillos et al . , 2003 ) . they surveyed vps proteins for the ptap or psap sequences present in p6 and found such a motif in hrs . using biacore and yeast two - hybrid analyses , they found that hrs interacts with tsg101 , although the psap - containing peptide from hrs bound with lower affinity than that from hiv p6 to the uev domain of tsg101 ( pornillos et al . , 2003 ) . yeast two - hybrid analyses of stenmark , sundquist , and cohen together show that the nh2-terminal vhs and fyve domains and the uim of hrs are dispensable for binding to tsg101 but that the psap motif and a second region encompassing the coiled - coil domain and part of the p / q - rich region are required for full binding activity ( fig . 1 ) ( bache et al . , 2003 ; lu et al . , 2003 ; pornillos et al . , when this segment of hrs was fused to the cooh terminus of a budding - defective hiv gag mutated in the p6 ptap motif , there was restoration of gag particle release . release of this fusion protein was abrogated by rnai depletion of tsg101 and was dependent on the ptap - binding sequence of the tsg101 uev ( pornillos et al . these studies are consistent with the view that hrs / vps27p has key roles in cargo recognition early in the process of endocytosis and in subsequent invagination of the endosomal limiting membrane . this protein is recruited to the early endosome by virtue of its fyve domain interaction with pi(3)p . the interaction of the hrs uim domain with ubiquitinated cargo and with other monoubiquitinated proteins involved in endocytosis , such as eps15 and epsins , may also contribute to its localization at the membrane of early endosomes . the uim of hrs directs its own monoubiquitination , and this may contribute to its regulated interaction with tsg101 , which results in recruitment of the escrt machinery and subsequent budding of the limiting membrane into the lumen to form mvbs . the principal cellular targets for infection with hiv are cd4 t helper cells and macrophages . although hiv budding is observed to occur at the plasma membrane in t cells , it has long been known that hiv particles accumulate in vacuolar compartments of infected macrophages ( orenstein et al . , 1988 ) . marsh and colleagues now demonstrate that in primary macrophages hiv accumulates in vacuoles containing late endosome / mvb markers ( pelchen - matthews et al . , 2003 ) . ( 2002 ) indicating that macrophages accumulate hiv particles in the miic compartment , the site where mhc class ii complexes assemble with peptides derived from late endosomes . using immunogold em , pelchen - matthews et al . ( 2003 ) found that p24/p55 immature viral particles are often near the limiting membrane of vesicles . by labeling with gold particles of different sizes , they found p24 in the same compartment as cd63 , lamp-1 , and other endosomal markers ( pelchen - matthews et al . , 2003 ) . infectious virus released from the macrophages was absorbed very efficiently with antibody specific for cd63 , a marker for vesicles within mvbs , suggesting that the viral envelope had incorporated this protein in the course of budding into this compartment . what advantage would hiv have by being sequestered in such vesicles ? ( 2002 ) propose that hiv particles are assembled within secretory vesicles in macrophages and are then released by exocytosis when these cells receive specific signals . mvbs of antigen - presenting cells are also sites for accumulation of exosomes , vesicles that have been proposed to efficiently prime t lymphocytes ( thery et al . , 2002 ) . hiv , like exosomes , may undergo regulated release in an environment enriched for t helper cells , thus facilitating viral spread . unlike macrophages , dendritic cells ( dcs ) are not readily infected with hiv . however , interaction of hiv with dcs results in substantial enhancement of infectivity of adjacent t cells , and this requires the endocytosis of hiv into a yet undefined vesicular compartment within the dcs ( kwon et al . , it is possible that regulated exocytosis in macrophages , which are productively infected with hiv , and in dcs , which may simply transcytose virions , has a similar outcome in both cell types , resulting in more efficient infection of t helper cells when these cells come into contact with the antigen - presenting cells ( kwon et al . , 2002 ; mcdonald et al . , it remains to be determined whether release of virus from macrophages is constitutive or regulated . it is also unclear if tsg101 and the escrt machinery have similar roles in hiv budding in t cells and macrophages . the role of these host cell molecules has been established in t cell lines , but similar studies have not yet been reported in macrophages or monocytes ( garrus et al . , 2001 ) . it is possible that interaction of hiv gag with a macrophage - specific protein overrides its targeting to the plasma membrane and allows it to assemble at the mvb , the normal site of action of the escrt complexes . although ubiquitination of p6 increases its affinity for the uev of tsg101 , abrogation of ubiquitination by mutating the relevant lysines in p6 had no effect on viral release from transfected hela cells ( freed , 2002 ) . the role of gag ubiquitination in release of hiv in primary t cells versus macrophages has not been reported and is a plausible explanation for how the virus employs different pathways to bud in different cell types . it will also be informative to learn whether depletion of hrs in t cells versus macrophages influences budding of hiv in these cells . the progress in understanding how hrs functions in endosomal sorting will very likely provide us with a deeper appreciation of the mechanisms employed for budding by diverse enveloped viruses . hiv and ebola virus , a filovirus , have ptap motifs that interact with tsg101 , but other viruses , such as rous sarcoma and moloney murine leukemia viruses , have l domains with pppy motifs that do not interact with tsg101 , but that , nonetheless , utilize the class e vps pathway ( freed , 2002 ) . further studies on how these viruses and hiv entrain the vps machinery during their assembly in different cell types will result in a better understanding of protein sorting and viral pathogenesis .
to efficiently bud off from infected cells , hiv and other enveloped viruses hijack the host cellular machinery that is normally involved in vacuolar protein sorting and multivesicular body ( mvb ) biogenesis . the hiv gag protein 
 mimics hepatocyte growth factor  regulated tyrosine kinase substrate ( hrs ) , a modular adaptor protein that links membrane cargo recognition to its degradation after delivery to mvbs . 
 in contrast to t cells , where hiv budding occurs at the plasma membrane , virus buds into vacuoles of macrophages , a process that may facilitate its spread within the infected host .