Productive human immunodeficiency virus type 1 assembly takes place at the plasma membrane

Gag proteins are necessary and sufficient to direct human immunodeficiency virus type 1 (HIV-1) particle assembly and budding. Recent evidence suggests that Gag targeting to late endosomal/multivesicular body (LE/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE/MVB, where it accumulated, thus accounting for the majority of LE/MVB-associated Gag. Importantly, this accumulation of Gag in LE/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-beta-cyclodextrin. These results point towards the PM as being the primary site of productive HIV-1 assembly in cells that also support Gag accumulation in intracellular compartments.     

Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines

The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium-dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis.     

HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress.

Retroviral Gag proteins encode sequences, termed late domains, which facilitate the final stages of particle budding from the plasma membrane. We report here that interactions between Tsg101, a factor involved in endosomal protein sorting, and short peptide motifs in the HIV-1 Gag late domain and Ebola virus matrix (EbVp40) proteins are essential for efficient egress of HIV-1 virions and Ebola virus-like particles. EbVp40 recruits Tsg101 to sites of particle assembly and a short, EbVp40-derived Tsg101-binding peptide sequence can functionally substitute for the HIV-1 Gag late domain. Notably, recruitment of Tsg101 to assembling virions restores budding competence to a late-domain-defective HIV-1 in the complete absence of viral late domain. These studies define an essential virus-host interaction that is conserved in two unrelated viruses. Because the Tsg101 is recruited by small, conserved viral sequence motifs, agents that mimic these structures are potential inhibitors of the replication of these lethal human pathogens.     

Cell-type-dependent targeting of human immunodeficiency virus type 1 assembly to the plasma membrane and the multivesicular body

The human immunodeficiency virus type 1 (HIV-1) assembly-and-release pathway begins with the targeting of the Gag precursor to the site of virus assembly. The molecular mechanism by which Gag is targeted to the appropriate subcellular location remains poorly understood. Based on the analysis of mutant Gag proteins, we and others have previously demonstrated that a highly basic patch in the matrix (MA) domain of Gag is a major determinant of Gag transport to the plasma membrane. In this study, we determined that in HeLa and T cells, the MA mutant Gag proteins that are defective in plasma membrane targeting form virus particles in a CD63-positive compartment, defined as the late endosome or multivesicular body (MVB). Interestingly, we find that in primary human macrophages, both wild-type (WT) and MA mutant Gag proteins are targeted specifically to the MVB. Despite the fact that particle assembly in macrophages occurs at an intracellular site rather than at the plasma membrane, we observe that WT Gag expressed in this cell type is released as extracellular virions with high efficiency. These results demonstrate that Gag targeting to and assembly in the MVB are physiologically important steps in HIV-1 virus particle production in macrophages and that particle release in this cell type may follow an exosomal pathway. To determine whether Gag targeting to the MVB is the result of an interaction between the late domain in p6(Gag) and the MVB sorting machinery (e.g., TSG101), we examined the targeting and assembly of Gag mutants lacking p6. Significantly, the MVB localization of Gag was still observed in the absence of p6, suggesting that an interaction between Gag and TSG101 is not required for Gag targeting to the MVB. These data are consistent with a model for Gag targeting that postulates two different cellular binding partners for Gag, one on the plasma membrane and the other in the MVB.     

HIV-1 Vpu inhibits accumulation of the envelope glycoprotein within clathrin-coated, Gag-containing endosomes

Several viruses encode ion channels that both modulate the trafficking of envelope glycoprotein(s) and stimulate the release of virions from cells. HIV-1 Vpu enhances virion release and inhibits the endosomal accumulation of the viral structural protein Gag. We investigated whether Vpu affects the subcellular distribution of Env as well as Gag. Env and Vpu colocalized with each other, in part within the trans -Golgi network. In the absence of Vpu, Env accumulated more extensively within clathrin-coated endosomal structures. These structures had several features consistent with an endosomal viral assembly domain: they contained Gag, including proteolytically processed viral matrix protein; the tetraspanins CD63 and CD81; the adaptor protein complex AP-3; and AIP1/ALIX, a cellular cofactor for viral budding. These endosomes labelled incompletely with Env derived from the cell surface, suggesting that some Env reaches this compartment without transiting the plasma membrane. Consistent with this, endosomal accumulation of Env was not blocked by dominant-negative Eps15, an inhibitor of AP-2-mediated endocytosis. Although these data are potentially explained by greater endocytosis of mature virions in the absence of Vpu, they also raise the possibility that Vpu inhibits the transport of Env and Gag to late endosomes, leading to viral assembly at the plasma membrane.     

Annexin 2: a novel human immunodeficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages

Human immunodeficiency virus (HIV) replication in the major natural target cells, CD4+ T lymphocytes and macrophages, is parallel in many aspects of the virus life cycle. However, it differs as to viral assembly and budding, which take place on plasma membranes in T cells and on endosomal membranes in macrophages. It has been postulated that cell type-specific host factors may aid in directing viral assembly to distinct destinations. In this study we defined annexin 2 (Anx2) as a novel HIV Gag binding partner in macrophages. Anx2-Gag binding was confined to productively infected macrophages and was not detected in quiescently infected monocyte-derived macrophages (MDM) in which an HIV replication block was mapped to the late stages of the viral life cycle (A. V. Albright, R. M. Vos, and F. Gonzalez-Scarano, Virology 325:328-339, 2004). We demonstrate that the Anx2-Gag interaction likely occurs at the limiting membranes of late endosomes/multivesicular bodies and that Anx2 depletion is associated with a significant decline in the infectivity of released virions; this coincided with incomplete Gag processing and inefficient incorporation of CD63. Cumulatively, our data suggest that Anx2 is essential for the proper assembly of HIV in MDM.     

Structure of the myristylated human immunodeficiency virus type 2 matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in membrane targeting

During the late phase of retroviral replication, newly synthesized Gag proteins are targeted to the plasma membrane (PM), where they assemble and bud to form immature virus particles. Membrane targeting by human immunodeficiency virus type 1 (HIV-1) Gag is mediated by the PM marker molecule phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂], which is capable of binding to the matrix (MA) domain of Gag in an extended lipid conformation and of triggering myristate exposure. Here, we show that, as observed previously for HIV-1 MA, the myristyl group of HIV-2 MA is partially sequestered within a narrow hydrophobic tunnel formed by side chains of helices 1, 2, 3, and 5. However, the myristate of HIV-2 MA is more tightly sequestered than that of the HIV-1 protein and does not exhibit concentration-dependent exposure. Soluble PI(4,5)P₂ analogs containing truncated acyl chains bind HIV-2 MA and induce minor long-range structural changes but do not trigger myristate exposure. Despite these differences, the site of HIV-2 assembly in vivo can be manipulated by enzymes that regulate PI(4,5)P₂ localization. Our findings indicate that HIV-1 and HIV-2 are both targeted to the PM for assembly via a PI(4,5)P₂-dependent mechanism, despite differences in the sensitivity of the MA myristyl switch, and suggest a potential mechanism that may contribute to the poor replication kinetics of HIV-2.     

Chimeric Human Immunodeficiency Virus Type 1 Containing Murine Leukemia Virus Matrix Assembles in Murine Cells

Murine cells do not support efficient assembly and release of human immunodeficiency virus type 1 (HIV-1) virions. HIV-1-infected mouse cells that express transfected human cyclin T1 synthesize abundant Gag precursor polyprotein, but inefficiently assemble and release virions. This assembly defect may result from a failure of the Gag polyprotein precursor to target to the cell membrane. Plasma membrane targeting of the precursor is mediated by the amino-terminal region of polyprotein. To compensate for the assembly block, we substituted the murine leukemia virus matrix coding sequences into an infectious HIV-1 clone. Transfection of murine fibroblasts expressing cyclin T1 with the chimeric proviruses resulted in viruses that were efficiently assembled and released. Chimeric viruses, in which the cytoplasmic tail of the transmembrane subunit, gp41, was truncated to prevent potential interference between the envelope glycoprotein and the heterologous matrix, could infect human and murine cells. They failed to further replicate in the murine cells, but replicated with delayed kinetics in human MT-4 cells. These findings may be useful for establishing a murine model for HIV-1 replication.     

AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly

Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.     

The host protein Staufen1 participates in human immunodeficiency virus type 1 assembly in live cells by influencing pr55Gag multimerization

Human immunodeficiency virus type 1 (HIV-1) requires the sequential activities of virus-encoded proteins during replication. The activities of several host cell proteins and machineries are also critical to the completion of virus assembly and the release of infectious virus particles from cells. One of these proteins, the double-stranded RNA-binding protein Staufen1 (Stau1), selectively associates with the HIV-1 genomic RNA and the viral precursor Gag protein, pr55Gag. In this report, we tested whether Stau1 modulates pr55Gag assembly using a new and specific pr55Gag oligomerization assay based on bioluminescence resonance energy transfer (BRET) in both live cells and extracts after cell fractionation. Our results show that both the overexpression and knockdown of Stau1 increase the pr55Gag-pr55Gag BRET levels, suggesting a role for Stau1 in regulating pr55Gag oligomerization during assembly. This effect of Stau1 on pr55Gag oligomerization was observed only in membranes, a cellular compartment in which pr55Gag assembly primarily occurs. Consistently, expression of Stau1 harboring a vSrc myristylation signal led to a 6.5-fold enrichment of Stau1 in membranes and a corresponding enhancement in the Stau1-mediated effect on pr55Gag-pr55Gag BRET, demonstrating that Stau1 acts on assembly when targeted to membranes. A role for Stau1 in the formation of particles is further supported by the detection of membrane-associated detergent-resistant pr55Gag complexes and the increase of virus-like particle release when Stau1 expression levels are modulated. Our results indicate that Stau1 influences HIV-1 assembly by modulating pr55Gag-pr55Gag interactions, as shown in a live cell interaction assay. This likely occurs when Stau1 interacts with membrane-associated assembly intermediates.     

The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.     

Gag regulates association of human immunodeficiency virus type 1 envelope with detergent-resistant membranes

Assembly of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein on budding virus particles is important for efficient infection of target cells. In infected cells, lipid rafts have been proposed to form platforms for virus assembly and budding. Gag precursors partly associate with detergent-resistant membranes (DRMs) that are believed to represent lipid rafts. The cytoplasmic domain of the envelope gp41 usually carries palmitate groups that were also reported to confer DRM association. Gag precursors confer budding and carry envelope glycoproteins onto virions via specific Gag-envelope interactions. Thus, specific mutations in both the matrix domain of the Gag precursor and gp41 cytoplasmic domain abrogate envelope incorporation onto virions. Here, we show that HIV-1 envelope association with DRMs is directly influenced by its interaction with Gag. Thus, in the absence of Gag, envelope fails to associate with DRMs. A mutation in the p17 matrix (L30E) domain in Gag (Gag L30E) that abrogates envelope incorporation onto virions also eliminated envelope association with DRMs in 293T cells and in the T-cell line, MOLT 4. These observations are consistent with a requirement for an Env-Gag interaction for raft association and subsequent assembly onto virions. In addition to this observation, we found that mutations in the gp41 cytoplasmic domain that abrogated envelope incorporation onto virions and impaired infectivity of cell-free virus also eliminated envelope association with DRMs. On the basis of these observations, we propose that Gag-envelope interaction is essential for efficient envelope association with DRMs, which in turn is essential for envelope budding and assembly onto virus particles.     

Visualization of retroviral replication in living cells reveals budding into multivesicular bodies

Retroviral assembly and budding is driven by the Gag polyprotein and requires the host-derived vacuolar protein sorting (vps) machinery. With the exception of human immunodeficiency virus (HIV)-infected macrophages, current models predict that the vps machinery is recruited by Gag to viral budding sites at the cell surface. However, here we demonstrate that HIV Gag and murine leukemia virus (MLV) Gag also drive assembly intracellularly in cell types including 293 and HeLa cells, previously believed to exclusively support budding from the plasma membrane. Using live confocal microscopy in conjunction with electron microscopy of cells generating fluorescently labeled virions or virus-like particles, we observed that these retroviruses utilize late endosomal membranes/multivesicular bodies as assembly sites, implying an endosome-based pathway for viral egress. These data suggest that retroviruses can interact with the vps sorting machinery in a more traditional sense, directly linked to the mechanism by which cellular proteins are sorted into multivesicular endosomes.     

Citron kinase, a RhoA effector, enhances HIV-1 virion production by modulating exocytosis

RhoGTPases play important roles in the regulation of protein transport and membrane recycling. Little is known, however, about how RhoGTPases affect HIV-1 virion production, which is dependent on the endosomal sorting pathway. We report that ectopic expression of citron kinase (citron-K), a RhoA effector, preferentially enhances HIV-1 virion production. Depletion of endogenous citron-K inhibits HIV-1 virion production. Citron-N, which lacks the kinase domain, also enhances HIV-1 virion production. The leucine zipper, Rho-binding and zinc finger domains of citron-N are necessary for the enhancement activity. Citron-K also enhances murine leukemia virion production and the HIV-1 late domain is not required for the citron-K-mediated enhancement. Ectopic expression of citron-K leads to the formation of cytoplasmic structures containing citron-K and HIV-1 Gag proteins. HIV-1 and citron-K cooperatively enhance acidic endosome and lysosome compartments. Finally, citron-K promotes exocytosis of microvesicles or exosomes that co-purify with HIV-1 virions. We conclude that citron-K enhances HIV-1 virion production by stimulating the endosomal compartments and exocytosis.     

Matrix domain modulates HIV-1 Gag's nucleic acid chaperone activity via inositol phosphate binding

Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA(3)(Lys) serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA(3)(Lys) placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA(3)(Lys) annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing.     

Vif and the p55(Gag) polyprotein of human immunodeficiency virus type 1 are present in colocalizing membrane-free cytoplasmic complexes

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is a potent regulator of viral infectivity. Current data posit that Vif functions late in replication to modulate assembly, budding, and/or maturation. Consistent with this model, earlier indirect immunofluorescence analyses of HIV-1-infected cells demonstrated that Vif and Gag colocalize to a substantial degree (J. H. M. Simon, R. A. M. Fouchier, T. E. Southerling, C. B. Guerra, C. K. Grant, and M. H. Malim, J. Virol. 71:5259-5267, 1997). Here, we describe a series of subcellular fractionation studies which indicate that Vif and the p55(Gag) polyprotein are present in membrane-free cytoplasmic complexes that copurify in sucrose density gradients and are stable in nonionic detergents. Both Vif and Gag are targeted to these complexes independent of each other, and their association with them appears to be mediated by protein-protein interactions. We propose that these complexes may represent viral assembly intermediates and that Vif is appropriately localized to influence the final stages of the viral life cycle and, therefore, the infectivity of progeny virions.     

Human Immunodeficiency Virus Type 1 Gag Polyprotein Multimerization Requires the Nucleocapsid Domain and RNA and Is Promoted by the Capsid-Dimer Interface and the Basic Region of Matrix Protein

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein directs the formation of virions from productively infected cells. Many gag mutations disrupt virion assembly, but little is known about the biochemical effects of many of these mutations. Protein-protein interactions among Gag monomers are believed to be necessary for virion assembly, and data suggest that RNA may modify protein-protein interactions or even serve as a bridge linking Gag polyprotein monomers. To evaluate the primary sequence requirements for HIV-1 Gag homomeric interactions, a panel of HIV-1 Gag deletion mutants was expressed in bacteria and evaluated for the ability to associate with full-length Gag in vitro. The nucleocapsid protein, the major RNA-binding domain of Gag, exhibited activity comparable to that of the complete polyprotein. In the absence of the nucleocapsid protein, relatively weak activity was observed that was dependent upon both the capsid-dimer interface and basic residues within the matrix domain. The relevance of the in vitro findings was confirmed with an assay in which nonmyristylated mutant Gags were assessed for the ability to be incorporated into virions produced by wild-type Gag expressed in trans. Evidence of the importance of RNA for Gag-Gag interaction was provided by the demonstration that RNase impairs the Gag-Gag interaction and that HIV-1 Gag interacts efficiently with Gags encoded by distantly related retroviruses and with structurally unrelated RNA-binding proteins. These results are consistent with models in which Gag multimerization involves indirect contacts via an RNA bridge as well as direct protein-protein interactions.     

Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein.

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.     

The Vif and Gag proteins of human immunodeficiency virus type 1 colocalize in infected human T cells.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses is required for efficient replication in primary cells and certain immortalized cell lines in vitro and, in all likelihood, for the establishment of pathogenic infections in vivo. Current hypotheses concerning Vif's mechanism of action posit that it operates in virus-expressing cells during virion assembly, budding, or maturation such that released virions are modified in a manner that enables them to undergo productive infection in subsequent viral challenges. To gain further insight into the mechanism of action of lentivirus Vif proteins, we have performed a variety of in situ localization and biochemical fractionation studies using cells in which Vif is essential for efficient replication. Double-label immunofluorescence analyses of cells productively infected with HIV-1 or feline immunodeficiency virus revealed dramatic patterns of colocalization between Vif and the virally encoded Gag proteins. Subcellular fractionations of human T cells expressing HIV-1 Vif performed in the absence of any detergent demonstrated that greater than 90% of Vif is associated with cellular membranes. Additional purification using a continuous density gradient indicated that the majority of the membrane-bound Vif copurifies with the plasma membrane. Taken together, these observations suggest that lentivirus Vif and Gag proteins colocalize at the plasma membrane as virion assembly and budding take place. As a result, Vif is able to exert its modulatory effect(s) on these late steps of the virus life cycle.