CD63 is not required for production of infectious human immunodeficiency virus type 1 in human macrophages

During the assembly of human immunodeficiency virus type 1 (HIV-1) particles, the tetraspanin CD63 can be incorporated into the viral membrane. Indeed, cell surface tetraspanin microdomains that include CD63 have been proposed as sites for virus release. In addition, antibodies against CD63 can inhibit HIV infection of macrophages. In this cell type, HIV assembles into intracellularly sequestered plasma membrane domains that contain several other tetraspanins, including CD81, CD9, and CD53. CD63 is recruited to this domain following HIV infection. Together, these observations suggest that CD63 may have some function in the assembly of infectious virus particles and/or the infectivity of assembled virions. Here we have used RNA interference to knock down CD63 expression in monocyte-derived primary macrophages. We show that in the absence of CD63, HIV assembly is quantitatively comparable to that seen in CD63-expressing macrophages and that virus assembly occurs on compartments positive for CD81, CD9, and CD53. Moreover, the infectivity of macrophage-derived virus is unaffected by the loss of CD63. Together, our results indicate that at least in tissue culture, CD63 expression is not required for either the production or the infectivity of HIV-1.     

Endocytic entry of HIV-1

Enveloped viruses enter target cells by membrane fusion or endocytosis. In the latter case, fusion of the viral envelope is induced by the acidic pH of the endocytic vesicle [1]. As with most other retroviruses, entry of the human immunodeficiency virus (HIV) is thought to be exclusively by pH-independent membrane fusion after interaction of its envelope with CD4 and a chemokine co-receptor on the target cell [2,3]. Expression of CD4 on the virus-producing cell impairs the release and infectivity of HIV-1(NL4-3) particles [4-6]. In sharp contrast, we found that the infectivity of another HIV isolate, HIV-1SF2, was enhanced by expression of CD4 on the producer cells, which correlated with significantly increased amounts of viral proteins in the vesicular fraction of target cells. Endocytic inhibitors decreased infectivity of HIV-1SF2 but enhanced that of HIV-1 NL4-3. Expression of CD4 in the producer cell did not remove gp41 from HIV-1SF2 virions. With these cells, the formation of syncytia could be induced by acidic medium. Thus, HIV-1SF2 can enter the cytoplasm by an endocytic route after activation of gp41 by the acidic pH of endocytic vesicles. Endocytic entry might expand the range of cells that HIV could infect and should be considered in antiviral strategies against AIDS.     

Recombinant extracellular domains of tetraspanin proteins are potent inhibitors of the infection of macrophages by human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) infection of human macrophages can be inhibited by antibodies which bind to the tetraspanin protein CD63, but not by antibodies that bind to other members of the tetraspanin family. This inhibitory response was limited to CCR5 (R5)-tropic virus and was only observed using macrophages, but not T cells. Here, we show that recombinant soluble forms of the large extracellular domain (EC2) of human tetraspanins CD9, CD63, CD81, and CD151 produced as fusion proteins with glutathione S-transferase (GST) can all potently and completely inhibit R5 HIV-1 infection of macrophages with 50% inhibitory concentration values of 0.11 to 1.2 nM. Infection of peripheral blood mononuclear cells could also be partly inhibited, although higher concentrations of EC2 proteins were required. Inhibition was largely coreceptor independent, as macrophage infections by virions pseudotyped with CXCR4 (X4)-tropic HIV-1 or vesicular stomatitis virus (VSV)-G glycoproteins were also inhibited, but was time dependent, since addition prior to or during, but not after, virus inoculation resulted in potent inhibition. Incubation with tetraspanins did not decrease CD4 or HIV-1 coreceptor expression but did block virion uptake. Colocalization of fluorescently labeled tetraspanin EC2 proteins and HIV-1 virions within, and with CD4 and CXCR4 at the cell surfaces of, macrophages could be detected, and internalized tetraspanin EC2 proteins were directed to vesicular compartments that contained internalized dextran and transferrin. Collectively, the data suggest that the mechanism of inhibition of HIV-1 infection by tetraspanins is at the step of virus entry, perhaps via interference with binding and/or the formation of CD4-coreceptor complexes within microdomains that are required for membrane fusion events.     

Tetraspanins CD9 and CD81 modulate HIV-1-induced membrane fusion

Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.     

Role for human immunodeficiency virus type 1 membrane cholesterol in viral internalization

The membrane of human immunodeficiency virus type 1 (HIV-1) virions contains high levels of cholesterol and sphingomyelin, an enrichment that is explained by the preferential budding of the virus through raft microdomains of the plasma membrane. Upon depletion of cholesterol from HIV-1 virions with methyl-beta-cyclodextrin, infectivity was almost completely abolished. In contrast, this treatment had only a mild effect on the infectiousness of particles pseudotyped with the G envelope of vesicular stomatitis virus. The cholesterol-chelating compound nystatin had a similar effect. Cholesterol-depleted HIV-1 virions exhibited wild-type patterns of viral proteins and contained normal levels of cyclophilin A and glycosylphosphatidylinositol-anchored proteins. Nevertheless, and although they could still bind target cells, these virions were markedly defective for internalization. These results indicate that the cholesterol present in the HIV-1 membrane plays a prominent role in the fusion process that is key to viral entry and suggest that drugs capable of disturbing the lipid composition of virions could serve as a basis for the development of microbicides.     

Cell-surface expression of CD4 reduces HIV-1 infectivity by blocking Env incorporation in a Nef- and Vpu-inhibitable manner.

BACKGROUND: Human immunodeficiency virus-1 (HIV-1) infection decreases the cell-surface expression of its cellular receptor, CD4, through the combined actions of Nef, Env and Vpu. Such functional convergence strongly suggests that CD4 downregulation is critical for optimal viral replication, yet the significance of this phenomenon has so far remained a puzzle. RESULTS: We show that high levels of CD4 on the surface of HIV-infected cells induce a dramatic reduction in the infectivity of released virions by the sequestering of the viral envelope by CD4. CD4 is able to accumulate in viral particles while at the same time blocking incorporation of Env into the virion. Nef and Vpu, through their ability to downregulate CD4, counteract this effect. CONCLUSIONS: The CD4-mediated 'envelope interference' described here probably explains the plurality of mechanisms developed by HIV to downregulate the cell-surface expression of its receptor.     

Presence of host ICAM-1 in human immunodeficiency virus type 1 virions increases productive infection of CD4+ T lymphocytes by favoring cytosolic delivery of viral material

Although there is now convincing evidence that the infectivity of human immunodeficiency virus type 1 (HIV-1) is increased by incorporation of host intercellular adhesion molecule 1 (ICAM-1) in budding virions, the exact mechanism(s) through which ICAM-1 can so significantly affect HIV-1 biology remains obscure. To address this question, we focused our attention on the most proximal events in the virus life cycle. We made comparative analyses to estimate attachment and internalization of isogenic HIV-1 particles either lacking or bearing host-derived ICAM-1. Using attachment-and-entry assays and confocal fluorescence microscopy, we found that virus binding and uptake were both markedly enhanced by insertion of ICAM-1 within the virus envelope when PM1 lymphoid cells and primary human cells (i.e., peripheral blood lymphocytes and purified CD4(+) T cells) were used as targets. Moreover, ICAM-1-bearing virions entered cells with faster uptake kinetics than viruses devoid of ICAM-1. Experiments conducted with fully competent viruses further confirmed the positive effect of virion-anchored host ICAM-1 on HIV-1 replication. Interestingly, subcellular-fractionation assays revealed that ICAM-1 incorporation modifies the HIV-1 entry route by increasing the level of viral material released in the cytosol, a process of internalization known to be mediated mainly by pH-independent membrane fusion and to result in productive infection. A virion-based fusion assay confirmed that the acquisition of ICAM-1 increases the efficiency of productive HIV-1 entry in primary CD4(+) T lymphocytes. These observations provide new insights into how interactions other than those with gp120 and CD4-coreceptor complex can modulate the process of productive HIV-1 infection in CD4(+) T lymphocytes, a cell target highly relevant to HIV-1 pathogenesis.     

Cell surface CD4 interferes with the infectivity of HIV-1 particles released from T cells.

The CD4 protein is required for the entry of human immunodeficiency virus (HIV) into target cells. Upon expression of the viral genome, three HIV-1 gene products participate in the removal of the primary viral receptor from the cell surface. To investigate the role of surface-CD4 in HIV replication, we have created a set of Jurkat cell lines which constitutively express surface levels of CD4 comparable to those found in peripheral blood lymphocytes and monocytes. Expression of low levels of CD4 on the surface of producer cells exerted an inhibitory effect on the infectivity of HIV-1 particles, whereas no differences in the amount of cell-free p24 antigen were observed. Higher levels of cell surface CD4 exerted a stronger inhibitory effect on infectivity, and also affected the release of free virus in experiments where the viral genomes were delivered by electrotransfection. The CD4-mediated inhibition of HIV-1 infectivity was not observed in experiments where the vesicular stomatitis virus G protein was used to pseudotype viruses, suggesting that an interaction between CD4 and gp120 is required for interference. In contrast, inhibition of particle release by high levels of cell-surface CD4 was not overcome by pseudotyping HIV-1 with foreign envelope proteins. Protein analysis of viral particles released from HIV-infected Jurkat-T cells revealed a CD4-dependent reduction in the incorporation of gp120. These results demonstrate that physiological levels of cell-surface CD4 interfere with HIV-1 replication in T cells by a mechanism that inhibits envelope incorporation into viral membranes, and therefore provide an explanation for the need to down-modulate the viral receptor in infected cells. Our findings have important implications for the spread of HIV in vivo and suggest that the CD4 down-modulation function may be an alternative target for therapeutic intervention.     

Polyclonal immunoglobulin G from patients neutralizes human immunodeficiency virus type 1 primary isolates by binding free virions, but without interfering with an initial CD4-independent attachment of the virus to primary blood mononuclear cells

We investigated the relationship between human immunodeficiency virus type 1 (HIV-1) primary isolate (PI) antibody-mediated neutralization and attachment to primary blood mononuclear cells (PBMC). Incubation of PIs with immunoglobulin G (IgG) purified from infected patients did not inhibit attachment of the viruses with PBMC, but partial to complete neutralization was achieved. Neutralization of PIs already fixed on the cells was achieved by some IgG samples only and was of limited intensity compared to the former neutralization protocol. On the contrary, the binding of IgG to free virions was shown to be sufficient to reach potent neutralization, as the infectivity of IgG-PI complexes purified from the bulk of antibodies before addition to PBMC was strongly diminished compared to mock-treated controls. Monoclonal antibodies to the CDR2 domain of CD4 completely inhibited the infection of PBMC without interfering with the attachment of PIs to the cells, suggesting that, under these experimental conditions, the initial attachment of viruses to PBMC involves alternative cellular receptors. This initial interaction may also involve other components of the viral envelope than gp120, as partial depletion of the surface glycoproteins of primary viral particles that resulted in an almost complete loss of infectivity did not impair attachment to PBMC. A limited inhibition of attachment was observed when interfering with putative interactions with cellular heparan sulfate, whereas no effect was observed for cellular CD147 or nucleolin or for virion-incorporated cyclophilin A. Altogether, our results favor a mechanism of neutralization of HIV-1 PIs by polyclonal IgG where antibodies predominantly bind free virions and neutralize without interfering with the attachment to PBMC, which, in this model, is mainly CD4 independent.     

Nef can enhance the infectivity of receptor-pseudotyped human immunodeficiency virus type 1 particles

Nef is an accessory protein of human immunodeficiency virus type 1 (HIV-1) that enhances the infectivity of progeny virions when expressed in virus-producing cells. The requirement for Nef for optimal infectivity is, at least in part, determined by the envelope (Env) glycoprotein, because it can be eliminated by pseudotyping HIV-1 particles with pH-dependent Env proteins. To investigate the role of Env in the function of Nef, we have examined the effect of Nef on the infectivity of Env-deficient HIV-1 particles pseudotyped with viral receptors for cells expressing cognate Env proteins. We found that Nef significantly enhances the infectivity of CD4-chemokine receptor pseudotypes for cells expressing HIV-1 Env. Nef also increased the infectivity of HIV-1 particles pseudotyped with Tva, the receptor for subgroup A Rous sarcoma virus (RSV-A), even though Nef had no effect if the pH-dependent Env protein of RSV-A was used for pseudotyping. However, Nef does not always enhance viral infectivity if the normal orientation of the Env-receptor interaction is reversed, because the entry of Env-deficient HIV-1 into cells expressing the vesicular stomatitis virus G protein was unaffected by Nef. Together, our results demonstrate that the presence of a viral Env protein during virus production is not required for the ability of Nef to increase viral infectivity. Furthermore, since the infectivity of Tva pseudotypes was blocked by inhibitors of endosomal acidification, we conclude that low-pH-dependent entry does not always bypass the requirement for Nef.     

Nef stimulates human immunodeficiency virus type 1 replication in primary T cells by enhancing virion-associated gp120 levels: coreceptor-dependent requirement for Nef in viral replication

The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) replication through an unknown mechanism. We and others have previously reported that efficient HIV-1 replication in activated primary CD4(+) T cells depends on the ability of Nef to downregulate CD4 from the cell surface. Here we demonstrate that Nef greatly enhances the infectivity of HIV-1 particles produced in primary T cells. Nef-defective HIV-1 particles contained significantly reduced quantities of gp120 on their surface; however, Nef did not affect the levels of virion-associated gp41, indicating that Nef indirectly stabilizes the association of gp120 with gp41. Surprisingly, Nef was not required for efficient replication of viruses that use CCR5 for entry, nor did Nef influence the infectivity or gp120 content of these virions. Nef also inhibited the incorporation of CD4 into HIV-1 particles released from primary T cells. We propose that Nef, by downregulating cell surface CD4, enhances HIV-1 replication by inhibiting CD4-induced dissociation of gp120 from gp41. The preferential requirement for Nef in the replication of X4-tropic HIV-1 suggests that the ability of Nef to downregulate CD4 may be most important at later stages of disease when X4-tropic viruses emerge.     

Cellular and viral specificities of human immunodeficiency virus type 1 vif protein

The vif gene of human immunodeficiency virus type 1 (HIV-1) greatly enhances the infectivity of HIV-1 virions that are released from cells classified as nonpermissive (e.g., lymphocytes, macrophages, and H9 leukemic T cells) but is irrelevant in permissive cells (e.g., HeLa or COS cells). Recently, it was reported that vif expression in nonpermissive cells dramatically increases infectivity not only of HIV-1 but also of other enveloped viruses, including murine leukemia viruses (MLVs). This was surprising in part because MLVs and other murine retroviruses lack vif genes yet replicate efficiently in T lymphocytes. To investigate these issues, we first developed improved methods for producing substantial quantities of HIV-1 virions with vif deletions from healthy H9 cells. These virions had approximately the same amounts of major core proteins and envelope glycoproteins as the control wild-type virions but were only approximately 1% as infectious. We then produced H9 cells that contained wild-type or vif deletion HIV-gpt proviruses, which lack a functional env gene. After superinfection with either xenotropic or amphotropic MLVs, these cells released HIV-gpt virions pseudotyped with an MLV envelope plus replication-competent MLV. Interestingly, the pseudotyped HIV-gpt (vif deletion) virions were noninfectious, whereas the MLV virions simultaneously released from the same H9 cells were fully infectious. These results strongly suggest that the Vif protein functions in a manner that is both cell specific and at least substantially specific for HIV-1 and related lentiviruses. In addition, these results confirm that vif deletion HIV-1 virions from nonpermissive cells are blocked at a postpenetration stage of the infection pathway.     

Human immunodeficiency virus infection of CD4-bearing cells occurs by a pH-independent mechanism.

The effect of weak bases (NH4Cl and amantadine) and carboxylic ionophores (monensin) on the infection of CD4 (T4) positive human cell lines by HIV-1 is examined. These reagents, which raise the pH of acidic intracellular organelles, fail to inhibit HIV-1 entry and the events leading to viral protein synthesis at concentrations inhibitory for low pH-dependent fusogenic enveloped viruses. The infectivity of VSV (HIV-1) pseudotypes is unaffected by weak bases at concentrations causing 95% plaque reduction of VSV in its own envelope. HIV-1 dependent cell--cell fusion (syncytium formation) occurs in medium maintained at pH 7.4-7.6, and virions are not irreversibly inactivated by incubation in acid medium. Our results show that HIV-1 entry and membrane fusion do not require exposure to low pH. The production of infectious HIV-1 particles, however, is inhibited in cells treated with NH4Cl.     

Insertion of host-derived costimulatory molecules CD80 (B7.1) and CD86 (B7.2) into human immunodeficiency virus type 1 affects the virus life cycle

Human immunodeficiency virus type 1 (HIV-1) carries virus-encoded and host-derived proteins. Recent advances in the functional characterization of host molecules inserted into mature virus particles have revealed that HIV-1 biology is influenced by the acquisition of host cell membrane components. The CD28/B7 receptor/ligand system is considered one of the fundamental elements of the normal immune response. Two major cell types that harbor HIV-1 in vivo, i.e., monocytes/macrophages and CD4+ T cells, express the costimulatory molecules CD80 (B7.1) and CD86 (B7.2). We investigated whether CD80 and CD86 are efficiently acquired by HIV-1, and if so, whether these host-encoded molecules can contribute to the virus life cycle. Here we provide the first evidence that the insertion of CD80 and CD86 into HIV-1 increases virus infectivity by facilitating the attachment and entry process due to interactions with their two natural ligands, CD28 and CTLA-4. Moreover, we demonstrate that NF-kappaB is induced by CD80- and CD86-bearing virions when they are combined with the engagement of the T-cell receptor/CD3 complex, an event that is inhibited upon surface expression of CTLA-4. Finally, both CD80 and CD86 were found to be efficiently incorporated into R5- and X4-tropic field strains of HIV-1 expanded in cytokine-treated macrophages. Thus, besides direct interactions between the virus envelope glycoproteins and cell surface constituents, such as CD4 and some specific chemokine coreceptors, HIV-1 may attach to target cells via interactions between cell-derived molecules incorporated into virions and their natural ligands. These findings support the theory that HIV-1-associated host proteins alter virus-host dynamics.     

Specific reactions between purified HIV-1 particles and CD4+ cell membrane fragments in a cell-free system of virus fusion or entry

The initial step of human immunodeficiency virus type 1 (HIV-1) infection has been studied by Env-mediated fusion or entry assays with appropriate cells expressing CD4 or CXCR4/CCR5 receptors in cultures, where many factors underlying cellular activities likely regulate the fusion/entry efficiency. Here we attempted to develop a more simplified in vitro cell-free fusion/entry reaction that mimics HIV-1 infection in cultures. Membrane fragments of target cells and intact infectious HIV-1 particles were purified, mixed and incubated. The core p24 protein was released from the purified virions and detected by ELISA without detergents in the supernatant of the reaction mixtures. This release reaction proceeded temperature-dependently and in a dose-dependent manner between the virion and membrane fractions, and was specific for HIV-1 Env and CD4. Env-deleted or VSV-G-pseudotyped HIV-1 released little p24, if any. Pretreatment of the membrane fragments with anti-CD4 antibodies inhibited the p24 induction from both X4-tropic and R5-tropic HIV-1. Furthermore, X4 but not R5 HIV-1 reacted with the membrane prepared from intrinsically CXCR4-positive HeLa-CD4 cells, whereas both viruses reacted with that prepared from CCR5-transduced HeLa-CD4 cells, indicating that this cell-free reaction mimics coreceptor usage of HIV-1 infection. Therefore, a potent entry inhibitor of X4 HIV-1, SDF-1alpha, blocked the release from X4 but not R5 HIV-1. Inversely, a weak entry inhibitor of R5 HIV-1, MIP-1beta, partially affected only the release from R5 HIV-1. These results suggest that this cell-free reaction system provides a useful tool to study biochemical fusion/entry mechanisms of HIV-1 and its inhibitors.     

Infectious HIV-1 assembles in late endosomes in primary macrophages

Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1-infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.     

Human macrophages accumulate HIV-1 particles in MHC II compartments

Macrophages are important targets for HIV-1 infection and harbor the virions in an as yet unidentified organelle. To determine the location of HIV-1 in these cells, an extensive analysis of primary human macrophages infected in vitro with HIV-1 was carried out by immuno-electron microscopy. Virus particles were found to accumulate in intracellular multivesicular compartments which were enriched in major histocompatibility complex class II molecules and CD63. These features are characteristics of major histocompatibility complex class II compartments where maturing class II molecules acquire their peptide cargo. The membrane-delimited, electron-dense virus particles of 100–110 nm diameter labeled strongly for HIV-1 p24 antigen, major histocompatibility complex class II molecules, CD63 and, to a lesser extent for HIV-1 gp120 envelope protein and Lamp 1. Our data suggest that virus particles may access the lumen of the major histocompatibility complex class II compartment by budding from the limiting membrane, thus acquiring proteins of this membrane such as class II and CD63. Viral assembly and budding would therefore occur in macrophages by a process similar to the formation of the internal vesicles in multivesicular bodies and at the same location. This could account for the particular content in lipids and proteins previously found in the membrane wrapping HIV particles. Our observations also suggest direct fusion of the virus containing major histocompatibility complex class II compartment with the plasma membrane, leading to massive release of viral particles into the extracellular medium.     

Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A.

Human immunodeficiency virus type 1 (HIV-1) normally enters cells by direct fusion with the plasma membrane. In this report, HIV-1 particles capable of infecting cells through an endocytic pathway are described. Chimeric viruses composed of the HIV-1 core and the envelope glycoprotein of vesicular stomatitis virus (VSV-G) were constructed and are herein termed HIV-1(VSV) pseudotypes. HIV-1(VSV) pseudotypes were 20- to 130-fold more infectious than nonpseudotyped HIV-1. Infection by HIV-1(VSV) pseudotypes was markedly diminished by ammonium chloride and concanamycin A, a selective inhibitor of vacuolar H+ ATPases, demonstrating that these viruses require endosomal acidification to achieve productive infection. HIV-1 is thus capable of performing all of the viral functions necessary for infection when entry is targeted to an endocytic route. Maximal HIV-1 infectivity requires the presence of the viral Nef protein and the cellular protein cyclophilin A (CyPA) during virus assembly. Pseudotyping by VSV-G markedly suppressed the requirement for Nef. HIV-1(VSV) particles were also resistant to inhibition by cyclosporin A; however, the deleterious effect of a gag mutation inhibiting CyPA incorporation was not relieved by VSV-G. These results suggest that Nef acts at a step of the HIV-1 life cycle that is either circumvented or facilitated by targeting virus entry to an endocytic pathway. The findings also support the hypothesis that Nef and CyPA enhance HIV-1 infectivity through independent processes and demonstrate a mechanistic difference between reduction of HIV-1 infectivity by cyclosporin A and gag mutations that decrease HIV-1 incorporation of CyPA.     

Expression of the human immunodeficiency virus type 1 (HIV-1) nef gene during HIV-1 production increases progeny particle infectivity independently of gp160 or viral entry.

The nef gene product of human immunodeficiency virus type 1 (HIV-1) promotes more-rapid kinetics of viral replication in primary peripheral blood mononuclear cells. We have previously shown that these enhancing effects of Nef on HIV-1 replication reflect an increase in viral infectivity detectable both in limiting dilution assays and through a single-cycle infection of the HeLa-CD4-long terminal repeat-beta-galactosidase indicator cell line. We now demonstrate that nef-defective HIV-1 can be rescued to near wild-type levels of infectivity by coexpressing Nef in trans in the cell line producing the virus. This observation indicates that HIV-1 virions produced in the presence of Nef are intrinsically different. However, we show that the major viral structural proteins are quantitatively similar in purified viral preparations. We also demonstrate the functional equivalence of the gp120-gp41 envelope glycoprotein complexes of Nef+ and Nef- HIV-1 through an assay for viral entry. Finally, we show that env-defective Nef+ HIV-1 pseudotyped with an amphotropic envelope is also more infectious than similarly pseudotyped Nef- HIV-1. Thus, the production of HIV-1 in the presence of Nef results in viral particles that are more infectious, and this increased infectivity is manifested at a stage after viral entry but prior to or coincident with HIV-1 gene expression.     

Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions

The envelope glycoprotein (Env) of the Human Immunodeficiency Virus Type-1 (HIV-1) is a critical determinant of viral infectivity, tropism and is the main target for humoral immunity; however, little is known about the cellular machinery that directs Env trafficking and its incorporation into nascent virions. Here we identify the mammalian retromer complex as a novel and important cellular factor regulating Env trafficking. Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport. Consistent with this function, inactivating retromer using RNAi targeting the cargo selective trimer complex inhibited retrograde trafficking of endocytosed Env to the Golgi. Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity. Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26. Taken together these results provide novel insight into regulation of HIV-1 Env trafficking and infectious HIV-1 morphogenesis and show for the first time a role for retromer in the late-steps of viral replication and assembly of a virus.