Recruitment of the adaptor protein 2 complex by the human immunodeficiency virus type 2 envelope protein is necessary for high levels of virus release

The envelope (Env) protein of human immunodeficiency virus type 2 (HIV-2) and the HIV-1 Vpu protein stimulate the release of retroviral particles from human cells that restrict virus production, an activity that we call the enhancement of virus release (EVR). We have previously shown that two separate domains in the HIV-2 envelope protein are required for this activity: a glycine-tyrosine-x-x-hydrophobic (GYxxtheta) motif in the cytoplasmic tail and an unmapped region in the ectodomain of the protein. We here report that the cellular partner of the GYxxtheta motif is the adaptor protein complex AP-2. The mutation of this motif or the depletion of AP-2 by RNA interference abrogated EVR activity and changed the cellular distribution of the Env from a predominantly punctate pattern to a more diffuse distribution. Since the L domain of equine infectious anemia virus (EIAV) contains a Yxxtheta motif that interacts with AP-2, we used both wild-type and L domain-defective particles of HIV-1 and EIAV to examine whether the HIV-2 Env EVR function was analogous to L domain activity. We observed that the production of all particles was stimulated by HIV-2 Env or Vpu, suggesting that the L domain and EVR activities play independent roles in the release of retroviruses. Interestingly, we found that the cytoplasmic tail of the murine leukemia virus (MLV) Env could functionally substitute for the HIV-2 Env tail, but it did so in a manner that did not require a Yxxtheta motif or AP-2. The cellular distribution of the chimeric HIV-2/MLV Env was significantly less punctate than the wild-type Env, although confocal analysis revealed an overlap in the steady-state locations of the two proteins. Taken together, these data suggest that the essential GYxxtheta motif in the HIV-2 Env tail recruits AP-2 in order to direct Env to a cellular pathway or location that is necessary for its ability to enhance virus release but that an alternate mechanism provided by the MLV Env tail can functionally substitute.     

The human immunodeficiency virus (HIV) type 2 envelope protein is a functional complement to HIV type 1 Vpu that enhances particle release of heterologous retroviruses.

We have recently shown that the envelope glycoprotein of the ROD10 isolate of human immunodeficiency virus type 2 (HIV-2) has the ability to positively regulate HIV-2 viral particle release. The activity provided by the ROD10 Env was remarkably similar to that of the HIV-1 Vpu protein, thus raising the possibility that the two proteins act in a related fashion. We now show that the ROD10 Env can functionally replace Vpu to enhance the rate of HIV-1 particle release. When provided in trans, both Vpu and the ROD10 Env restored wild-type levels of particle release in a Vpu-deficient mutant of the NL4-3 molecular clone with indistinguishable efficiencies. This effect was independent of the presence of the HIV-1 envelope protein. The ROD10 Env also enhanced HIV-1 particle release in the context of HIV-2 chimeric viruses containing the HIV-1 gag-pol, indicating a lack of need for additional HIV-1 products in this process. In addition, we show for the first time that HIV-1 Vpu, as well as ROD10 Env, has the ability to enhance simian immunodeficiency virus (SIV) particle release. The effects of Vpu and ROD10 Env on SIV particle release were indistinguishable and were observed in the context of full-length SIVmac239 and simian-human immunodeficiency virus chimeras. These results further demonstrate that ROD10 Env can functionally complement Vpu with respect to virus release. In contrast, we found no evidence of a destabilizing activity of ROD10 Env on the CD4 molecule. HIV-1 and HIV-2 thus appear to have evolved genetically distinct but functionally similar strategies to resolve the common problem of efficient release of progeny virus from infected cells.     

Vpu Downmodulates Two Distinct Targets,Tetherin and Gibbon Ape Leukemia Virus Envelope,through Shared Features in the Vpu Cytoplasmic Tail

During human immunodeficiency virus-1 (HIV-1) assembly, the host proteins CD4 (the HIV-1 receptor) and tetherin (an interferon stimulated anti-viral protein) both reduce viral fitness. The HIV-1 accessory gene Vpu counteracts both of these proteins, but it is thought to do so through two distinct mechanisms. Modulation of CD4 likely occurs through proteasomal degradation from the endoplasmic reticulum. The exact mechanism of tetherin modulation is less clear, with possible roles for degradation and alteration of protein transport to the plasma membrane. Most investigations of Vpu function have used different assays for CD4 and tetherin. In addition, many of these investigations used exogenously expressed Vpu, which could result in variable expression levels. Thus, few studies have investigated these two Vpu functions in parallel assays, making direct comparisons difficult. Here, we present results from a rapid assay used to simultaneously investigate Vpu-targeting of both tetherin and a viral glycoprotein, gibbon ape leukemia virus envelope (GaLV Env). We previously reported that Vpu modulates GaLV Env and prevents its incorporation into HIV-1 particles through a recognition motif similar to that found in CD4. Using this assay, we performed a comprehensive mutagenic scan of Vpu in its native proviral context to identify features required for both types of activity. We observed considerable overlap in the Vpu sequences required to modulate tetherin and GaLV Env. We found that features in the cytoplasmic tail of Vpu, specifically within the cytoplasmic tail hinge region, were required for modulation of both tetherin and GaLV Env. Interestingly, these same regions features have been determined to be critical for CD4 downmodulation. We also observed a role for the transmembrane domain in the restriction of tetherin, as previously reported, but not of GaLV Env. We propose that Vpu may target both proteins in a mechanistically similar manner, albeit in different cellular locations.     

Sequences in the cytoplasmic tail of the gibbon ape leukemia virus envelope protein that prevent its incorporation into lentivirus vectors

Pseudotyping retrovirus and lentivirus vectors with different viral fusion proteins is a useful strategy to alter the host range of the vectors. Although lentivirus vectors are efficiently pseudotyped by Env proteins from several different subtypes of murine leukemia virus (MuLV), the related protein from gibbon ape leukemia virus (GaLV) does not form functional pseudotypes. We have determined that this arises because of an inability of GaLV Env to be incorporated into lentivirus vector particles. By exploiting the homology between the GaLV and MuLV Env proteins, we have mapped the determinants of incompatibility in the GaLV Env. Three modifications that allowed GaLV Env to pseudotype human immunodeficiency virus type 1 particles were identified: removal of the R peptide (C-terminal half of the cytoplasmic domain), replacement of the whole cytoplasmic tail with the corresponding MuLV region, and mutation of two residues upstream of the R peptide cleavage site. In addition, we have previously proposed that removal of the R peptide from MuLV Env proteins enhances their fusogenicity by transmitting a conformational change to the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392-5398, 1998). Our analysis of chimeric MuLV/GaLV Env proteins provides further evidence in support of this model and suggests that proper Env function involves both interactions within the cytoplasmic tail and more long-range interactions between the cytoplasmic tail, the membrane-spanning region, and the ectodomain of the protein.     

Sequences in gibbon ape leukemia virus envelope that confer sensitivity to HIV-1 accessory protein Vpu

HIV-1 efficiently forms pseudotyped particles with many gammaretrovirus glycoproteins, such as Friend murine leukemia virus (F-MLV) Env, but not with the related gibbon ape leukemia virus (GaLV) Env or with a chimeric F-MLV Env with a GaLV cytoplasmic tail domain (CTD). This incompatibility is modulated by the HIV-1 accessory protein Vpu. Because the GaLV Env CTD does not resemble tetherin or CD4, the well-studied targets of Vpu, we sought to characterize the modular sequence in the GaLV Env CTD required for this restriction in the presence of Vpu. Using a systematic mutagenesis scan, we determined that the motif that makes GaLV Env sensitive to Vpu is INxxIxxVKxxVxRxK. This region in the CTD of GaLV Env is predicted to form a helix. Mutations in the CTD that would break this helix abolish sensitivity to Vpu. Although many of these positions can be replaced with amino acids with similar biophysical properties without disrupting the Vpu sensitivity, the final lysine residue is required. This Vpu sensitivity sequence appears to be modular, as the unrelated Rous sarcoma virus (RSV) Env can be made Vpu sensitive by replacing its CTD with the GaLV Env CTD. In addition, F-MLV Env can be made Vpu sensitive by mutating two amino acids in its cytoplasmic tail to make it resemble more closely the Vpu sensitivity motif. Surprisingly, the core components of this Vpu sensitivity sequence are also present in the host surface protein CD4, which is also targeted by Vpu through its CTD.     

Human immunodeficiency virus type 1 envelope protein endocytosis mediated by a highly conserved intrinsic internalization signal in the cytoplasmic domain of gp41 is suppressed in the presence of the Pr55gag precursor protein.

The mechanisms involved in the incorporation of viral glycoproteins into virions are incompletely understood. For retroviruses, incorporation may involve interactions between the Gag proteins of these viruses and the cytoplasmic domains of the relevant envelope (Env) glycoproteins. Recent studies have identified within the cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) Env protein a tyrosine-containing internalization motif similar to those found in the cytoplasmic domains of certain cell surface proteins that undergo rapid constitutive endocytosis in clathrin-coated pits. Given that surface expression of the HIV-1 Env protein is essential for the production of infectious virus, the presence of this internalization motif is surprising. We show here that in contrast to the rapid rate of Env protein internalization observed in cells expressing the Env protein in the absence of other HIV-1 proteins, the rate of internalization of Env protein from the surfaces of HIV-1-infected cells is extremely slow. The presence of the Pr55gag precursor protein is necessary and sufficient for inhibition of Env protein internalization, while a mutant Pr55-gag that is incapable of mediating Env incorporation into virions is also unable to inhibit endocytosis of the Env protein. The failure of the Env protein to undergo endocytosis from the surface of an HIV-1-infected cell may reflect the fact that the proposed interaction of the matrix domain of the Gag protein with Env during assembly prevents the interaction of Env with host adaptin molecules that recruit plasma membrane molecules such as the transferrin receptor into clathrin-coated pits. When the normal ratio of Gag and Env proteins in the infected cells is altered by overexpression of Env protein, this mechanism allows removal of excess Env protein from the cell surface. Taken together, these results suggest that a highly conserved system to reduce surface levels of the Env protein functions to remove Env protein that is not associated with Gag and that is therefore not destined for incorporation into virions. This mechanism for the regulation of surface levels of Env protein may protect infected cells from Env-dependent cytopathic effects or Env-specific immune responses.     

Functional Complementation of a Model Target to Study Vpu Sensitivity

HIV-1 forms infectious particles with Murine Leukemia virus (MLV) Env, but not with the closely related Gibbon ape Leukemia Virus (GaLV) Env. We have determined that the incompatibility between HIV-1 and GaLV Env is primarily caused by the HIV-1 accessory protein Vpu, which prevents GaLV Env from being incorporated into particles. We have characterized the ‘Vpu sensitivity sequence’ in the cytoplasmic tail domain (CTD) of GaLV Env using a chimeric MLV Env with the GaLV Env CTD (MLV/GaLV Env). Vpu sensitivity is dependent on an alpha helix with a positively charged face containing at least one Lysine. In the present study, we utilized functional complementation to address whether all the three helices in the CTD of an Env trimer have to contain the Vpu sensitivity motif for the trimer to be modulated by Vpu. Taking advantage of the functional complementation of the binding defective (D84K) and fusion defective (L493V) MLV and MLV/GaLV Env mutants, we were able to assay the activity of mixed trimers containing both MLV and GaLV CTDs. Mixed trimers containing both MLV and GaLV CTDs were functionally active and remained sensitive to Vpu. However, trimers containing an Env with the GaLV CTD and an Env with no CTD remained functional but were resistant to Vpu. Together these data suggest that the presence of at least one GaLV CTD is sufficient to make an Env trimer sensitive to Vpu, but only if it is part of a trimeric CTD complex.     

Surface stability and immunogenicity of the human immunodeficiency virus envelope glycoprotein: role of the cytoplasmic domain

The effects of two functional domains, the membrane-proximal YXXPhi motif and the membrane-distal inhibitory sequence in the long cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env), on immunogenicity of the envelope protein were investigated. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env and a truncated Env with 50 amino acids in the cytoplasmic domain to delete the membrane distal inhibitory sequence for surface expression. Additional genes were generated in which the tyrosine residue in the YXXPhi motif was changed into a serine. Pulse-chase radioactive labeling and immunoprecipitation studies indicated that both domains can mediate endocytosis of the HIV Env, and removal of both domains is required to enhance HIV Env protein surface stability. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the mutant Env exhibiting enhanced surface stability induced significantly higher levels of antibody responses against the HIV Env protein. Our results suggest that the HIV Env cytoplasmic domain may play important roles in virus infection and pathogenesis by modulating its immunogenicity.     

Genetic evidence for an interaction between human immunodeficiency virus type 1 matrix and alpha-helix 2 of the gp41 cytoplasmic tail

The incorporation of envelope (Env) glycoproteins into virions is an essential step in the retroviral replication cycle. Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), encode Env glycoproteins with unusually long cytoplasmic tails, the functions of which have not been fully elucidated. In this study, we examine the effects on virus replication of a number of mutations in a helical motif (alpha-helix 2) located near the center of the HIV-1 gp41 cytoplasmic tail. We find that, in T-cell lines, small deletions in this domain disrupt the incorporation of Env glycoproteins into virions and markedly impair virus infectivity. Through the analysis of viral revertants, we demonstrate that a single amino acid change (34VI) in the matrix domain of Gag reverses the Env incorporation and infectivity defect imposed by a small deletion near the C terminus of alpha-helix 2. These results provide genetic evidence, in the context of infected T cells, for an interaction between HIV-1 matrix and the gp41 cytoplasmic tail and identify domains of both proteins involved in this putative interaction.     

Rescue of human immunodeficiency virus type 1 matrix protein mutants by envelope glycoproteins with short cytoplasmic domains.

The matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) forms the outer protein shell directly underneath the lipid envelope of the virion. The MA protein has a key role in different aspects of virus assembly, including the incorporation of the HIV-1 Env protein complex, which contains a transmembrane glycoprotein with an unusually long cytoplasmic tail. In this study, we compared the abilities of HIV-1 MA mutants to incorporate Env protein complexes with long and short cytoplasmic tails. While the mutant particles failed to incorporate the authentic HIV-1 Env protein complex, they retained the ability to efficiently and functionally incorporate the amphotropic murine leukemia virus Env protein complex, which has a short cytoplasmic tail. Moreover, incorporation of the autologous Env protein complex could be restored by a second-site mutation that resulted in the truncation of the cytoplasmic tail of the HIV-1 transmembrane glycoprotein. Remarkably, the second-site mutation also restored the ability of MA mutants to replicate in MT-4 cells. These results imply that the long cytoplasmic tail of the transmembrane glycoprotein is responsible for the exclusion of the HIV-1 Env protein complex from MA mutant particles.     

Effect of extension of the cytoplasmic domain of human immunodeficiency type 1 virus transmembrane protein gp41 on virus replication

The biological significance of the presence of a long cytoplasmic domain in the envelope (Env) transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1) is still not fully understood. Here we examined the effects of cytoplasmic tail elongation on virus replication and characterized the role of the C-terminal cytoplasmic tail in interactions with the Gag protein. Extensions with six and nine His residues but not with fewer than six His residues were found to severely inhibit virus replication through decreased Env electrophoretic mobility and reduced Env incorporation compared to the wild-type virus. These two mutants also exhibited distinct N glycosylation and reduced cell surface expression. An extension of six other residues had no deleterious effect on infectivity, even though some mutants showed reduced Env incorporation into the virus and/or decreased cell surface expression. We further show that these elongated cytoplasmic tails in a format of the glutathione S-transferase fusion protein still interacted effectively with the Gag protein. In addition, the immediate C terminus of the cytoplasmic tail was not directly involved in interactions with Gag, but the region containing the last 13 to 43 residues from the C terminus was critical for Env-Gag interactions. Taken together, our results demonstrate that HIV-1 Env can tolerate extension at its C terminus to a certain degree without loss of virus infectivity and Env-Gag interactions. However, extended elongation in the cytoplasmic tail may impair virus infectivity, Env cell surface expression, and Env incorporation into the virus.     

Contribution of Vpu, Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to superinfection

Human immunodeficiency virus type 1 (HIV-1) utilizes Vpu, Env, and Nef to down-modulate its primary CD4 receptor from the cell surface, and this function seems to be critical for the pathogenesis of AIDS. The physiological relevance of CD4 down-modulation, however, is currently not well understood. In the present study, we analyzed the kinetics of CD4 down-modulation and the susceptibility of HIV-1-infected T cells to superinfection using proviral HIV-1 constructs containing individual and combined defects in vpu, env, and nef and expressing red or green fluorescent proteins. T cells infected with HIV-1 mutants containing functional nef genes expressed low surface levels of CD4 from the first moment that viral gene expression became detectable. In comparison, Vpu and Env had only minor to moderate effects on CD4 during later stages of infection. Consistent with these quantitative differences, Nef inhibited superinfection more efficiently than Vpu and Env. Notably, nef alleles from AIDS patients were more effective in preventing superinfection than those derived from a nonprogressor of HIV-1 infection. Our data suggest that protection against X4-tropic HIV-1 superinfection involves both CD4-independent and CD4-dependent mechanisms of HIV-1 Nef. X4 was effectively down-regulated by simian immunodeficiency virus and HIV-2 but not by HIV-1 Nef proteins. Thus, maximal protection seems to involve an as-yet-unknown mechanism that is independent of CD4 or coreceptor down-modulation. Finally, we demonstrate that superinfected primary T cells show enhanced levels of apoptosis. Accordingly, one reason that HIV-1 inhibits CD4 surface expression and superinfection is to prevent premature cell death in order to expand the period of effective virus production.     

Regulation of Virus Release by the Macrophage-Tropic Human Immunodeficiency Virus Type 1 AD8 Isolate Is Redundant and Can Be Controlled by either Vpu or Env

The human immunodeficiency virus type 1 (HIV-1) Vpu and Env proteins are expressed from a bicistronic mRNA. To address the biological significance of the coordinated expression of vpu and env, we compared the relative effects on particle release of HIV-1 isolates containing an intact vpu gene or carrying point mutations in its initiation codon or internal deletions, respectively. We found that the primary AD8 isolate, which is unable to express vpu due to a mutation in its translation initiation codon, was able to replicate in primary macrophages and peripheral blood mononuclear cells with efficiency similar to that of an isogenic variant expressing Vpu. Interestingly, AD8 lacking a vpu initiation codon produced higher levels of Env protein than its Vpu-expressing isogenic variant. In contrast, disabling Vpu without removing the vpu initiation codon did not alter Env expression but significantly reduced virus production. AD8 Env when provided in trans was capable of enhancing release not only of AD8 particles but also of viruses of the T-cell-tropic NL4-3 isolate. We conclude that AD8 Env encodes a Vpu-like activity similar to that previously reported for HIV-2 Env proteins and is thus able to augment virus secretion. When expressed at elevated levels, i.e., following mutation of the vpu initiation codon, AD8 Env was able to compensate for the lack of Vpu and thereby ensure efficient virus release. Thus, the ability to regulate virus release is redundant in AD8 and can be controlled by either Vpu or Env. Since Vpu controls several independent functions, including CD4 degradation, our results suggest that some HIV-1 isolates may have evolved a mechanism to regulate Vpu activity without compromising their ability to efficiently replicate in the host cells.     

Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vpu Transmembrane Domain That Promotes the Enhanced Release of Virus-Like Particles from the Plasma Membrane of Mammalian Cells

Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.     

The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is an integral membrane phosphoprotein that induces CD4 degradation in the endoplasmic reticulum and enhances virus release from the cell surface. CD4 degradation is specific, requires phosphorylation of Vpu, and involves the interaction between Vpu and the CD4 cytoplasmic domain. In contrast, regulation of virus release is less specific and not restricted to HIV-1 and may be mechanistically-distinct from CD4 degradation. We show here that a mutant of Vpu, Vpu35, lacking most of its cytoplasmic domain has residual biological activity for virus release but is unable to induce CD4 degradation. This finding suggests that the N terminus of Vpu encoding the transmembrane (TM) anchor represents an active domain important for the regulation of virus release but not CD4 degradation. To better define the functions of Vpu's TM anchor and cytoplasmic domain, we designed a mutant, VpuRD, containing a scrambled TM sequence with a conserved amino acid composition and alpha-helical structure. The resulting protein was integrated normally into membranes, was able to form homo-oligomers, and exhibited expression levels, protein stability, and subcellular localization similar to those of wild-type Vpu. Moreover, VpuRD was capable of binding to CD4 and to induce CD4 degradation with wild-type efficiency, confirming proper membrane topology and indicating that the alteration of the Vpu TM domain did not interfere with this function of Vpu. However, VpuRD was unable to enhance the release of virus particles from infected or transfected cells, and virus encoding VpuRD had replication characteristics in T cells indistinguishable from those of a Vpu-deficient HIV-1 isolate. Mutation of the phosphorylation sites in VpuRD resulted in a protein which was unable to perform either function of Vpu. The results of our experiments suggest that the two biological activities of Vpu operate via two distinct molecular mechanisms and involve two different structural domains of the Vpu protein.     

Efficient incorporation of HLA class II onto human immunodeficiency virus type 1 requires envelope glycoprotein packaging

HLA class II DR is one of the most abundant cell surface proteins incorporated onto human immunodeficiency virus type 1 (HIV-1) during budding. The mechanism for HLA class II protein incorporation is not known and may involve a viral protein. To determine whether Env affects HLA class II protein incorporation, HIV-1 virions, either with or without Env on their surface, were produced from HLA class II-expressing cells and analyzed by whole-virus immunoprecipitation with antisera against HLA class II proteins. HLA class II proteins were detected on virions only when wild-type Env was incorporated, while similar experiments showed that HLA class I proteins were incorporated independent of Env packaging. Therefore, the packaging of HIV-1 Env protein is required for the efficient incorporation of HLA class II but not class I proteins into the virion. Analysis of two Env mutants revealed that the presence of a 43-amino-acid sequence between amino acids 708 and 750 in the gp41(TM) cytoplasmic tail was required for efficient incorporation of HLA class II proteins. These data show that HIV-1 actively incorporates HLA class II proteins in a process that, either directly or indirectly, requires Env.     

Wild-type-like viral replication potential of human immunodeficiency virus type 1 envelope mutants lacking palmitoylation signals

Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.     

Identification of calcium-modulating cyclophilin ligand as a human host restriction to HIV-1 release overcome by Vpu

The HIV-1 Vpu protein is required for efficient viral release from human cells. For HIV-2, the envelope (Env) protein replaces the role of Vpu. Both Vpu and HIV-2 Env enhance virus release by counteracting an innate host-cell block within human cells that is absent in African green monkey (AGM) cells. Here we identify calcium-modulating cyclophilin ligand (CAML) as a Vpu-interacting host factor that restricts HIV-1 release. Expression of human CAML (encoded by CAMLG) in AGM cells conferred a strong restriction of virus release that was reversed by Vpu and HIV-2 Env, suggesting that CAML is the mechanistic link between these two viral regulators. Depletion of CAML in human cells eliminated the need for Vpu in enhancing HIV-1 and murine leukemia virus release. These results point to CAML as a Vpu-sensitive host restriction factor that inhibits HIV release from human cells. The ability of CAML to inhibit virus release should illuminate new therapeutic strategies against HIV.     

Both linear and discontinuous ribosome scanning are used for translation initiation from bicistronic human immunodeficiency virus type 1 env mRNAs

Human immunodeficiency virus type 1 (HIV-1) generates 16 alternatively spliced isoforms of env mRNA that contain the same overlapping open reading frames for Vpu and Env proteins but differ in their 5' untranslated regions (UTR). A subset of env mRNAs carry the extra upstream Rev initiation codon in the 5' UTR. We explored the effect of the alternative UTR on the translation of Vpu and Env proteins from authentic env mRNAs expressed from cDNA constructs. Vpu expression from the subset of env mRNA isoforms with exons containing an upstream Rev AUG codon was minimal. However, every env mRNA isoform expressed similar levels of Env protein. Mutations that removed, altered the strength of, or introduced upstream AUG codons dramatically altered Vpu expression but had little impact on the consistent expression of Env. These data show that the different isoforms of env mRNA are not redundant but instead regulate Vpu production in HIV-1-infected cells. Furthermore, while the initiation of Vpu translation conforms to the leaky ribosome-scanning model, the consistent Env synthesis infers a novel, discontinuous ribosome-scanning mechanism to translate Env.     

Human immunodeficiency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domains of CD4: role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic reticulum.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is a transmembrane phosphoprotein which induces rapid degradation of CD4 in the endoplasmic reticulum (ER). To identify sequences in CD4 for Vpu-induced degradation, we generated four chimeric envelope glycoproteins having the ectodomain of HIV-1 gp160, the anchor domain of CD4, and 38, 25, 24, and 18 amino acids (aa) of the CD4 cytoplasmic domain. Using the vaccinia virus-T7 RNA polymerase expression system, we analyzed the expression of chimeric proteins in the presence and absence of Vpu. In singly transfected cells, the chimeric envelope glycoproteins having 38, 24, and 18 aa of the CD4 cytoplasmic domain were endoproteolytically cleaved and biologically active in the fusion of HeLa CD4+ cells. However, one of the chimeras having 25 aa of the CD4 cytoplasmic tail was retained in the ER using the transmembrane ER retention signal and was defective in membrane fusion. Furthermore, biochemical analyses of the coexpressing cells revealed that the Vpu protein induced degradation of the envelope glycoproteins having 38, 25, and 24 aa of the CD4 cytoplasmic tail and degradation occurred in the ER. Consequently, the fusion-competent glycoproteins did not induce the formation of syncytia in HeLa CD4+ cells expressing Vpu. However, the HIV-1 gp160 and chimeric envelope glycoprotein having the membrane-proximal 18 aa of the CD4 cytoplasmic tail were stable and fusion competent in cells expressing Vpu. In addition, we examined the stability of CD4 molecules in the presence of Vpu. Coexpression analyses revealed that the Vpu protein induced degradation of CD4 whereas mutant CD4 having the membrane-proximal 18 aa of the cytoplasmic domain was relatively stable in the presence of Vpu. Taken together, these studies have elucidated that the Vpu protein requires sequences or sequence determinants in the cytoplasmic domain of CD4 to induce degradation of the glycoproteins in the cell.