Highly Purified Human Immunodeficiency Virus Type 1 Reveals a Virtual Absence of Vif in Virions

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary blood-derived lymphocytes, macrophages, and certain human T-cell lines. It has been shown that Vif is associated with HIV-1 virions purified by sucrose density-equilibrium gradient analysis. However, the specificity of Vif incorporation into virions has not been determined. Moreover, recent studies have demonstrated that standard HIV-1 particle preparations created with sucrose density-equilibrium gradients are contaminated with cell-derived microvesicles. Here we demonstrate, as previously reported, that Vif cosediments with HIV-1 particles in sucrose density-equilibrium gradient analysis. However, we also found that, when Vif was expressed in the absence of all other HIV-1-encoded gene products and then isolated by sucrose density-equilibrium gradient centrifugation from extracellular supernatants, its sedimentation pattern was largely unaltered, suggesting that Vif can be secreted from cells. Using a newly developed OptiPrep velocity gradient method, we were able to physically separate most of the extracellular Vif from the HIV-1 virions without disrupting the infectivity of the virus. By titrating serial dilutions of purified Vif and Gag against the viral peak fraction in the OptiPrep gradient, we demonstrate that <1.0 Vif molecule per virion was present. This study shows that Vif is not significantly present in HIV-1 virions, a finding which is consistent with the idea that Vif functions predominantly in the virus-producing cells during virus assembly. The OptiPrep velocity gradient technique described here could be an easy and rapid way to purify HIV and other enveloped viruses from microvesicles and/or cell debris.     

Nef from Human Immunodeficiency Virus Type 1F12 Inhibits Viral Production and Infectivity

Human immunodeficiency virus type 1(F12) (HIV-1(F12)) interferes with the replication of other strains of HIV. Its accessory protein, Nef, is sufficient for this phenotype, where the production and infectivity of HIV are impaired significantly. The analysis of three rare mutations in this Nef protein revealed that these effects could be separated genetically. Moreover, the defect in virus production correlated with the lack of processing of the p55(Gag) precursor in the presence of Nef from HIV-1(F12). Importantly, the introduction of one of these mutations (E177G) into Nef from HIV-1(NL4-3) also created a dominant-negative Nef protein. Effects of Nef from HIV-1(F12) on virus production and Gag processing correlated with its altered subcellular distribution. Moreover, the association with two new cellular proteins with molecular masses of 74 and 75 kDa, which do not interact with other Nef proteins, correlated with the decreased virion infectivity. The identification of a dominant-negative protein for the production and infectivity of HIV suggests that Nef plays an active role at this stage of the viral replicative cycle.     

A Conserved Tryptophan-Rich Motif in the Membrane-Proximal Region of the Human Immunodeficiency Virus Type 1 gp41 Ectodomain Is Important for Env-Mediated Fusion and Virus Infectivity

Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an α-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.     

Annexin 2 Is Not Required for Human Immunodeficiency Virus Type 1 Particle Production but Plays a Cell Type-Dependent Role in Regulating Infectivity

During assembly and budding of retroviruses, host cell proteins are incorporated into viral particles. Identification of virion-associated proteins may help pinpoint key cellular components required for virus production and function. The cellular protein annexin 2 (Anx2) is incorporated into HIV-1 particles, and knockdown of Anx2 has been reported to cause defects in Gag processing and infectivity of HIV-1 particles in macrophages. Here, we tested whether Anx2 was required for HIV-1 production in other cell types capable of producing HIV-1 virions. Endogenous Anx2 levels were knocked down by ∼98% using lentivirus encoding short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting Anx2. Under these conditions, there was no reduction in HIV-1 virus-like particle (VLP) production in either COS-1, 293T, or Jurkat T cells or primary human monocyte-derived macrophages (MDMs). Murine embryonic fibroblasts derived from Anx2^−/− mice produced the same levels of VLPs as matched cells from wild-type mice. The calcium-mediated spike in VLP production still occurred in Anx2-depleted COS-1 cells, and there was no apparent alteration in the intracellular Gag localization. Overexpression of Anx2 in trans had no effect on Gag processing or VLP production. Neither Anx2 depletion nor Anx2 overexpression altered the infectivity of HIV-1 particles produced by COS-1 or 293T cells. However, supernatants containing virus from Anx2 siRNA-treated primary human MDMs exhibited decreased infectivity. These data indicate that Anx2 is not required for HIV-1 assembly or Gag processing but rather plays a cell type-dependent role in regulating production of infectious HIV-1 by macrophages.The Gag polyprotein generates the key structural proteins for all retroviruses. Gag is necessary and sufficient for the formation of virus-like particles (VLPs), which are morphologically similar to immature virions. Following its synthesis in the cytoplasm, HIV-1 Gag is trafficked to sites of particle production on membranes. Viral particle production depends on Gag-membrane interactions mediated by the myristoylated MA domain of Gag (18, 22, 31) and Gag-Gag interactions mediated by the CA and NC domains. Budding and release of the new virion are mediated by the Gag p6 domain. For successful particle production to occur, HIV-1 Gag must also interact with numerous host cell proteins and protein complexes. Identification of these interactions provides a crucial window into determining Gag trafficking intermediates as well as clues to the mechanism of virion production.The host cell protein annexin 2 (Anx2) has recently attracted attention for its potential to regulate key processes in both cells and viruses (9, 14, 17, 24). Anx2 belongs to a family of conserved calcium-regulated proteins and interacts with actin, membranes, and negatively charged phospholipids. The major protein binding partner for Anx2 is p11, also known as S100A10. Two populations of Anx2 have been identified: a heterotetrameric complex with two molecules of Anx2 and two molecules of p11 (found predominantly at the plasma membrane) and a monomeric form found mainly in the cytoplasm. Anx2 performs multiple functions in the cell, including regulation of actin-based dynamics, fibrinolysis, calcium-mediated exocytosis, and transport of intermediates from early to late endosomes (10, 14-16) Anx2 also enhances binding and fusion of cytomegalovirus with phospholipid membranes (21). In addition, Anx2 can be detected within influenza virus particles (28), where it has been shown to aid in virus replication (9).Several lines of evidence suggest that Anx2 may play a role in HIV-1 biogenesis. Both Anx2 and its binding partner p11 are incorporated in HIV-1 particles produced by macrophages (2). Anx2 interacts with Gag in macrophages, and annexin 2 knockdown has been reported to cause defective Gag processing and reduced infectivity of the released particles (24). Blockade of Anx2 function, with either anti-Anx2 antibody or small interfering RNA (siRNA)-mediated knockdown, results in suppression of HIV-1 infection in macrophages (11). Anx2 also binds to Gag in 293T cells, and expression of Anx2 in trans in these cells has been reported to lead to increased Gag processing and HIV-1 production (7). Taken together, these findings suggest that Anx2 might play a universal role in Gag trafficking and particle production. To test this hypothesis, we exploited methods to efficiently knock down Anx2 expression and determined the effect of Anx2 knockdown in a variety of cell lines capable of producing HIV-1 virions. Here we show that, in the absence of Anx2 expression, HIV-1 Gag is expressed, trafficked, and capable of mediating viral particle formation in a manner similar to that of control cells expressing Anx2. However, a cell type-dependent effect of Anx2 depletion on HIV-1 infectivity was detected in primary human monocyte-derived macrophages (MDMs). These findings suggest that Anx2 might be a macrophage-specific host cell factor that regulates HIV-1 infectivity.     

Cryoelectron Microscopic Examination of Human Immunodeficiency Virus Type 1 Virions with Mutations in the Cyclophilin A Binding Loop

The human immunodeficiency virus type 1 capsid protein contains a conserved P₂₁₇X₄PX₂PX₅P₂₃₁ motif. Mutation at Pro-222 decreases virion incorporation of cyclophilin A, while mutation at Pro-231 abolishes infectivity. Although viral RNA incorporation and protease cleavage of the Gag precursor were not affected by these mutations, cryoelectron microscopy revealed a loss of virion maturation in P231A particles.     

Lymphotropic Virions Affect Chemokine Receptor-Mediated Neural Signaling and Apoptosis: Implications for Human Immunodeficiency Virus Type 1-Associated Dementia

Chemokine receptors pivotal for human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes and macrophages (CCR3, CCR5, and CXCR4) are expressed on neural cells (microglia, astrocytes, and/or neurons). It is these cells which are damaged during progressive HIV-1 infection of the central nervous system. We theorize that viral coreceptors could effect neural cell damage during HIV-1-associated dementia (HAD) without simultaneously affecting viral replication. To these ends, we studied the ability of diverse viral strains to affect intracellular signaling and apoptosis of neurons, astrocytes, and monocyte-derived macrophages. Inhibition of cyclic AMP, activation of inositol 1,4,5-trisphosphate, and apoptosis were induced by diverse HIV-1 strains, principally in neurons. Virions from T-cell-tropic (T-tropic) strains (MN, IIIB, and Lai) produced the most significant alterations in signaling of neurons and astrocytes. The HIV-1 envelope glycoprotein, gp120, induced markedly less neural damage than purified virions. Macrophage-tropic (M-tropic) strains (ADA, JR-FL, Bal, MS-CSF, and DJV) produced the least neural damage, while 89.6, a dual-tropic HIV-1 strain, elicited intermediate neural cell damage. All T-tropic strain-mediated neuronal impairments were blocked by the CXCR4 antibody, 12G5. In contrast, the M-tropic strains were only partially blocked by 12G5. CXCR4-mediated neuronal apoptosis was confirmed in pure populations of rat cerebellar granule neurons and was blocked by HA1004, an inhibitor of calcium/calmodulin-dependent protein kinase II, protein kinase A, and protein kinase C. Taken together, these results suggest that progeny HIV-1 virions can influence neuronal signal transduction and apoptosis. This process occurs, in part, through CXCR4 and is independent of CD4 binding. T-tropic viruses that traffic in and out of the brain during progressive HIV-1 disease may play an important role in HAD neuropathogenesis.     

CD4+ T-Lymphocyte Depletion in Human Lymphoid Tissue Ex Vivo Is Not Induced by Noninfectious Human Immunodeficiency Virus Type 1 Virions

We tested infectious human immunodeficiency virus type 1 (HIV-1), noninfectious but conformationally authentic inactivated whole HIV-1 virions, and purified gp120 for the ability to induce depletion of CD4⁺ T cells in human lymphoid tissues ex vivo. Infectious CXCR4-tropic HIV-1, but not matched inactivated virions or gp120, mediated CD4⁺ T-cell depletion, consistent with mechanisms requiring productive infection.     

In Vitro Modification of Human Immunodeficiency Virus Type 1 (HIV-1) Infectivity by the U937 Cells

The effect of host cell factors on infectivity of human immunodeficiency virus type 1 (HIV-1) was studied by infecting a monoblastoid cell line (U937) or a T-cell line (MOLT-4) with a highly infective single clone of HIV-1 and comparing the infectivity of the produced viruses to different cell lines. Chronically infected U937 cells consistently produced viruses with minimal infectivity. This phenotypic change was host-dependent as the back-passage of the U937-produced low infective viruses into MOLT-4 cells resulted in regaining their original high infectivity. Southern and Northern blot analyses of the HIV-1 grown in U937 cells did not reveal any genomic difference between it and the virus grown it MOLT-4 cells. The radioimmunoprecipitation analysis of viral proteins showed that the HIV-1-infected U937 cells had a different pattern of envelope glycoproteins and core proteins, which well correlated with the low infectivity of the produced viruses. This experimental system using MOLT-4 and U937 cell lines would be useful to further explore host cell factor(s) which play an important role in the regulation of HIV-1 infectivity.     

Minimal Incidence of Serum Antibodies Reactive with Intact Primary Isolate Virions in Human Immunodeficiency Virus Type 1-Infected Individuals

Immunoglobulin G reactive with primary isolate virions was detected in 36% of serum samples from individuals infected with human immunodeficiency virus type 1. Of these individuals, serum samples from only 7% captured significant quantities of virus. Virion-specific antibody correlated with CD4 counts and, of more significance, primary isolate neutralization. Further dissection of this response should lead to the identification of antibodies and antigenic epitopes for vaccine purposes.     

Cleavage of Human Immunodeficiency Virus Type 1 Proteinase from the N-Terminally Adjacent p6* Protein Is Essential for Efficient Gag Polyprotein Processing and Viral Infectivity

Maturation of infectious human immunodeficiency virus (HIV) particles requires proteolytic cleavage of the structural polyproteins by the viral proteinase (PR), which is itself encoded as part of the Gag-Pol polyprotein. Expression of truncated PR-containing sequences in heterologous systems has mostly led to the autocatalytic release of an 11-kDa species of PR which is capable of processing all known cleavage sites on the viral precursor proteins. Relatively little is known about cleavages within the nascent virus particle, on the other hand, and controversial results concerning the active PR species inside the virion and the relative activities of extended PR species have been reported. Here, we report that HIV type 1 (HIV-1) particles of four different strains obtained from different cell lines contain an 11-kDa PR, with no extended PR proteins detectable. Furthermore, mutation of the N-terminal PR cleavage site leading to production of an N-terminally extended 17-kDa PR species caused a severe defect in Gag polyprotein processing and a complete loss of viral infectivity. We conclude that N-terminal release of PR from the HIV-1 polyprotein is essential for viral replication and suggest that extended versions of PR may have a transient function in the proteolytic cascade.     

Mutations in the Leucine Zipper-Like Heptad Repeat Sequence of Human Immunodeficiency Virus Type 1 gp41 Dominantly Interfere with Wild-Type Virus Infectivity

It has been previously shown that a proline substitution for any of the conserved leucine or isoleucine residues located in the leucine zipper-like heptad repeat sequence of human immunodeficiency virus type 1 (HIV-1) gp41 renders viruses noninfectious and envelope (Env) protein unable to mediate membrane fusion (S. S.-L. Chen, C.-N. Lee, W.-R. Lee, K. McIntosh, and T.-M. Lee, J. Virol. 67:3615–3619, 1993; S. S.-L. Chen, J. Virol. 68:2002–2010, 1994). To understand whether these variants could act as trans-dominant inhibitory mutants, the ability of these mutants to inhibit wild-type (wt) virus infectivity was examined. Comparable amounts of cell- and virion-associated gag gene products as well as virion-associated gp41 were found in transfection with wt or mutant HIV-1 provirus. Viruses obtained from coexpression of wt provirus with mutant 566 or 580 provirus inhibited more potently the production of infectious virus than did viruses generated from cotransfection of wt provirus with other mutant proviruses. Nevertheless, all viruses produced from mixed transfection showed decreased infectivity compared with that of the wt virus when a multinuclear-activation β-galactosidase induction assay was performed. The ability of wt Env to induce cytopathic effects was inhibited by coexpression with mutant Env. Coexpression of mutants inhibited the ability of the wt protein to mediate virus-to-cell transmission, as demonstrated by an env trans-complementation assay with a defective HIV-1 proviral vector. These observations indicated that mutant Env, per se, interferes with wt Env function. Moreover, cotransfection of wt and mutant proviruses produced amounts of cell- and virion-associated gag gene products comparable to those produced by transfection of wt provirus. Similar amounts of gp41 were also found in virions generated from wt-mutant cotransfection as well as from wt transfection alone. These results indicated that the inhibitory effect conferred by mutants on the wt virus infectivity does not involve the late steps of Gag protein assembly and budding, but they suggest that the wt and mutant Env proteins form a dysfunctional hetero-oligomer which is impaired in an early step of the virus replication cycle. Our study demonstrates that mutations in the HIV-1 gp41 leucine zipper-like heptad repeat sequence dominantly inhibit infectious virus production.     

Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

Human immunodeficiency virus type 1 (HIV-1) Gag and the cellular protein cyclophilin A form an essential complex in the virion core: virions produced by proviruses encoding Gag mutants with decreased cyclophilin A affinity exhibit attenuated infectivity, as do virions produced in the presence of the competitive inhibitor cyclosporine. The A224E Gag mutant has no effect on cyclophilin A affinity but renders HIV-1 replication cyclosporine resistant in Jurkat T cells. In contrast, A224E mutant virus is dead in H9 T cells, although replication is rescued by cyclosporine or by expression in cis of a Gag mutant that decreases cyclophilin A-affinity. The observation that disruption of the Gag-cyclophilin A interaction rescues A224E mutant replication in H9 cells prompted experiments which revealed that, relative to Jurkat cells, H9 cells express greater quantities of cyclophilin A. The resulting larger quantity of cyclophilin A shown to be packaged into virions produced by H9 cells is presumably disruptive to the A224E mutant virion core. Further evidence that increased cyclophilin A expression in H9 cells is of functional relevance was provided by the finding that Gag mutants with decreased cyclophilin A affinity are dead in Jurkat cells but capable of replication in H9 cells. Similarly, cyclosporine concentrations which inhibit wild-type HIV-1 replication in Jurkat cells stimulate HIV-1 replication in H9 cells. These results suggest that HIV-1 virion infectivity imposes narrow constraints upon cyclophilin A stoichiometry in virions and that infectivity is finely tuned by host cyclophilin A expression levels.     

The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles

The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [ortho-32P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.     

Formation of Syncytia Is Repressed by Tetraspanins in Human Immunodeficiency Virus Type 1-Producing Cells

In vitro propagation studies have established that human immunodeficiency virus type 1 (HIV-1) is most efficiently transmitted at the virological synapse that forms between producer and target cells. Despite the presence of the viral envelope glycoprotein (Env) and CD4 and chemokine receptors at the respective surfaces, producer and target cells usually do not fuse with each other but disengage after the viral particles have been delivered, consistent with the idea that syncytia, at least in vitro, are not required for HIV-1 spread. Here, we tested whether tetraspanins, which are well known regulators of cellular membrane fusion processes that are enriched at HIV-1 exit sites, regulate syncytium formation. We found that overexpression of tetraspanins in producer cells leads to reduced syncytium formation, while downregulation has the opposite effect. Further, we document that repression of Env-induced cell-cell fusion by tetraspanins depends on the presence of viral Gag, and we demonstrate that fusion repression requires the recruitment of Env by Gag to tetraspanin-enriched microdomains (TEMs). However, sensitivity to fusion repression by tetraspanins varied for different viral strains, despite comparable recruitment of their Envs to TEMs. Overall, these data establish tetraspanins as negative regulators of HIV-1-induced cell-cell fusion, and they start delineating the requirements for this regulation.The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is incorporated into released virus particles and enables the virus to attach to and fuse with target cells in order to initiate the infectious cycle. Before Env mediates the fusion of viral and cellular membranes, i.e., while it is still incorporated in the plasma membrane of the infected cell, it drives the adhesion between virus producer cell and target cells, which gives rise to the formation of the so-called virological synapse (VS) (21, 24, 35, 36). The VS shares certain characteristics with the immunological synapse, including an accumulation of specific cellular membrane proteins and lipids (see, e.g., reference 5), and it provides efficient and secure transfer of virus particles from infected to uninfected cells (8). Importantly, the two adhering cells, like the pre- and postsynaptic cells that form an immunological synapse, typically do not fuse during such cell-to-cell transfer events. At first glance this seems surprising, as HIV-1 Env, unlike many other viral envelope proteins, can induce membrane fusion at physiological pH. Also, adhesion of producer and target cell, which can be initiated when the uropod of the infected cell contacts the uninfected cell (8), followed by reorganization of the cytoskeleton (25) and formation of full-fledged synapses, can extend over minutes (see, e.g., reference 20). This process should allow enough time to trigger cell-cell fusion. However, it is now well established that newly synthesized Env is efficiently internalized upon its arrival at the host cell plasma membrane, unless it is recruited into budding structures by viral Gag (see, e.g., reference 11; also discussed in references 3 and 6). Further, and likely also contributing to the prevention of producer-target cell fusion, immature Gag at the host cell plasma membrane represses Env-driven fusion, and this repression is lost only once Gag is processed in released virions (9, 22, 23, 31, 50). Finally, because syncytia are clearly not required for the transmission of virus from cell to cell in vitro and are possibly detrimental to virus spread in vivo, we hypothesize that HIV-1 cooperates with cellular membrane proteins to prevent cell-cell fusion.Members of a group of cellular proteins known as tetraspanins play an important role as regulators of cellular fusion processes, including myotube formation and fertilization (28, 30, 44; reviewed in, e.g., reference 17). As membrane organizers, these proteins homo- and heteromultimerize and associate with other cellular proteins to form variably sized but discrete microdomains, the so-called tetraspanin-enriched microdomains (TEMs) (29) (also called TERMs [1] or TEAs [12]). Knowledge of the molecular mechanisms through which tetraspanins regulate the fusion of cellular membranes is still lacking, though the available evidence strongly suggests (i) that these proteins are not themselves fusogens but rather that they coordinate the fusion activity of other cellular proteins and (ii) that they can act both as positive and negative regulators of cellular fusion processes. For instance, several in vivo studies unequivocally showed that CD9 expression in oocytes is essential for sperm-egg fusion (27, 28, 30), but CD9 and CD81 ablation in monocytes enhances the formation of multinucleated phagocytes that are involved in immune defense against certain microbes (45). Interestingly, the same two tetraspanins are also known to regulate virus-induced fusion processes. CD9 is involved in regulating cell-cell fusion driven by canine distemper virus, as the anti-CD9 antibody K41 inhibits syncytium formation by this virus (42), and CD81 is a necessary cofactor for infection of cells by hepatitis C virus (see, e.g., references 2 and 52). Finally, tetraspanins on uninfected (target) cells inhibit HIV-1-induced cell-cell fusion (14). This fusion regulation is likely due to interactions of CD9 and CD81 with CD4 and coreceptors at the surface of target cells, though the tetraspanin CD63 has also been implicated in the trafficking of CXCR4 to the plasma membrane (51).Because tetraspanins in HIV-1-producing cells are enriched at budding sites (4, 10, 13, 15, 33, 46, 49) and at the VS (26), we hypothesized that they regulate Env-driven fusion at the VS. Here, we document that tetraspanins in HIV-1-producing cells can indeed restrict syncytium formation. We also define some of the requirements for this fusion inhibition, thus laying the necessary groundwork for future mechanistic analyses. In addition, the characterization of cell-cell fusion regulation parameters in this study will allow the fusion-inhibitory activities to be distinguished from other regulatory functions exerted by tetraspanins, such as the modulation of virion infectivity and the regulation of cell-to-cell transmission of HIV-1.