Membrane raft association of the Vpu protein of human immunodeficiency virus type 1 correlates with enhanced virus release.

The Vpu protein of human immunodeficiency virus type 1 (HIV-1) is known to enhance virion release from certain cell types. To accomplish this function, Vpu interacts with the restriction factor known as bone marrow stromal cell antigen 2 (BST-2)/tetherin. In this study, we analyzed whether the Vpu protein is associated with microdomains known as lipid or membrane rafts. Our results indicate that Vpu partially partitions into detergent-resistant membrane (DRM) fractions when expressed alone or in the context of simian-human immunodeficiency virus (SHIV) infection. The ability to be partitioned into rafts was observed with both subtype B and C Vpu proteins. The use of cholesterol lowering lovastatin/M-ß-cyclodextrin and co-patching experiments confirmed that Vpu can be detected in cholesterol rich regions of membranes. Finally, we present data showing that raft association-defective transmembrane mutants of Vpu have impaired enhanced virus release function, but still maintain the ability to down-regulate CD4.

BST-2 mediated restriction of simian-human immunodeficiency virus.

Pathogenic simian-human immunodeficiency viruses (SHIV) contain HIV-1 Vpu and SIV Nef, both shown to counteract BST-2 (HM1.24; CD317; tetherin) inhibition of virus release in a species-specific manner. We show that human and pig-tailed BST-2 (ptBST-2) restrict SHIV. We found that sequential "humanization" of the transmembrane domain (TMD) of the pig-tailed BST-2 (ptBST-2) protein resulted in a fluctuation in sensitivity to HIV-1 Vpu. Our results also show that the length of the TMD in human and ptBST-2 proteins is important for BST-2 restriction and susceptibility to Vpu. Taken together, our results emphasize the importance of tertiary structure in BST-2 antagonism and suggests that the HIV-1 Vpu transmembrane domain may have additional functions in vivo unrelated to BST-2 antagonism.

Comparison of the replication and persistence of simian-human immunodeficiency viruses expressing Vif proteins with mutation of the SLQYLA or HCCH domains in macaques.

The Vif protein of primate lentiviruses interacts with APOBEC3 proteins, which results in shunting of the APOBEC3-Vif complex to the proteosome for degradation. Using the simian-human immunodeficiency virus (SHIV)/macaque model, we compared the replication and pathogenicity of SHIVs that express a Vif protein in which the entire SLQYLA (SHIV(Vif5A)) or HCCH (SHIV(VifHCCH(-))) domains were substituted with alanine residues. Each virus was inoculated into three macaques and various viral and immunological parameters followed for 6 months. All macaques maintained stable circulating CD4+ T cells, developed low viral loads, maintained the engineered mutations, yielded no histological lesions, and developed immunoprecipitating antibodies early post-inoculation. Sequence analysis of nef and vpu from three lymphoid tissues revealed a high percentage of G-to-A-substitutions. Our results show that while the presence of HCCH and SLQYLA domains are critical in vivo, there may exist APOBEC3 negative reservoirs that allow for low levels of viral replication and persistence but not disease.

Identification of amino acids within the second alpha helical domain of the human immunodeficiency virus type 1 Vpu that are critical for preventing CD4 cell surface expression.

Human immunodeficiency virus type 1 (HIV-1) encodes for a Vpu protein, which interacts with CD4 resulting in its degradation. In this study, we examined the role of the 10 amino acids within the predicted second alpha-helical domain of the subtype B Vpu cytoplasmic tail in CD4 down-modulation using a VpuEGFP reporter system. Our findings indicate that the invariant leucine at position 63 and, to a lesser extent, the valine at position 68 were required for CD4 down-modulation. Mutation of analogous L63 in Vpu proteins subtypes A2, B(YU-2), C, D, and H also abolished CD4 down-modulation from the cell surface. Co-immunoprecipitation analysis revealed that L63A and V68A mutants were capable of binding CD4 and still retained the ability to interact with h-beta-TrCP1. Taken together, these results indicate that amino acid substitutions in the second alpha-helical domain that retain the predicted structure and binding to h-beta-TrCP1 can influence Vpu-mediated CD4 degradation.

Mutations in the highly conserved SLQYLA motif of Vif in a simian-human immunodeficiency virus result in a less pathogenic virus and are associated with G-to-A mutations in the viral genome.

The simian-human immunodeficiency virus (SHIV)/macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of accessory genes in lentiviral pathogenesis. In this study, we introduced two amino acid changes in the highly conserved SLQYLA domain (to AAQYLA) of the SIV Vif protein. The resulting virus, SHIV(VifAAQYLA), was used to infect three macaques, which were followed for over six months. Plasma viral loads and circulating CD4(+) T cell levels were assessed during the course of infection. The three macaques inoculated with SHIV(VifAAQYLA) did not develop significant CD4(+) T cell loss over the course of their infection, had plasma viral RNA loads that were over 100-fold lower than macaques inoculated with parental SHIV(KU-1bMC33), and developed no histological lesions in lymphoid tissues. DNA and RT-PCR analysis revealed that only a select number of tissues were infected with this virus. Sequence analysis indicates that the site-directed changes were stable during the first three weeks after inoculation but thereafter the S147A amino acid substitution changed to a threonine in two of three macaques. The L148A substitution remained stable in the vif amplified from the PBMC of all three macaques. Sequence analysis of vif, vpu, env and nef genes revealed G-to-A mutations in the genes amplified from macaques inoculated with SHIV(VifAAQYLA), which were higher than in a macaque inoculated with parental SHIV(KU-1bMC33). We found that the majority (>85%) of the G-to-A mutations were in the context of 5'-TC (minus strand) and not 5'-CC, suggestive that one or more of the rhesus APOBEC3 proteins may be responsible for the observed mutational patterns. The data also suggest that rhesus APOBEC3G probably accounted for a minority of the mutations since its GG-to-AG mutational pattern was infrequently detected. Finally, macaques inoculated with SHIV(VifAAQYLA) developed immunoprecipitating antibody responses against the virus. The results from this study provide the first in vivo evidence of the importance of the SLQYLA domain in viral pathogenesis and show that targeted mutations in vif can lead to a persistent infection with G-to-A changes accumulating in the viral genome.

Requirements of the membrane proximal tyrosine and dileucine-based sorting signals for efficient transport of the subtype C Vpu protein to the plasma membrane and in virus release.

Previously, we showed that the Vpu protein from HIV-1 subtype C is more efficiently transported to the cell surface than the well studied subtype B Vpu (Pacyniak et al., 2005) and that a SHIV expressing the subtype C Vpu exhibited a decreased rate of CD4+ T cell loss following inoculation in macaques (Hill et al., 2008). In this study, we examined the role of overlapping tyrosine-based (YXXPhi) and dileucine-based ([D/E]XXXL[L/I]) motifs in the membrane proximal region of the subtype C Vpu (EYRKLL) in Vpu intracellular transport, CD4 surface expression and virus release from the cell surface. We constructed three site-directed mutants of the subtype C vpu and fused these genes to the gene for enhanced green fluorescent protein (EGFP). The first mutation made altered the tyrosine (EARKLL; VpuSCEGFPY35A), the second altered the dileucine motif (EYRKLG; VpuSCEGFPL39G), and the third contained both amino acid substitutions (EARKLG; VpuSCEGFPYL35,39AG) in this region of the Vpu protein. The VpuSCEGFPY35A protein was transported to the cell surface similar to the unmodified VpuSCEGFP1 while VpuSCEGFPL39G was expressed at the cell surface at significantly reduced levels. The VpuSCEGFPYL35,39AG was found to have an intermediate level of cell surface expression. All three mutant Vpu proteins were analyzed for the ability to prevent cell surface expression of CD4. We found that both single mutants did not significantly effect CD4 surface expression while the double mutant (VpuSCEGFPYL35,39AG) was significantly less efficient at preventing cell surface CD4 expression. Chimeric simian human immunodeficiency viruses were constructed with these mutations in vpu (SHIVSCVpuY35A, SHIVSCVpuL39G and SHIVSCVpuYL35,39AG). Our results indicate that SHIVSCVpuL39G replicated much more efficiently and was much more cytopathic than SHIVSCVpu. In contrast, SHIVSCVpuY35A and SHIVSCVpuYL35,39AG replicated less efficiently when compared to the parental SHIVSCVpu. Taken together, these results show for the first time that the membrane proximal tyrosine-based sorting motif in the cytoplasmic domain of Vpu is essential for efficient virus release. These results also indicate that the dileucine-based sorting motif affects the intracellular trafficking of subtype C Vpu proteins, virus replication, and release.

Modulation of the severe CD4+ T-cell loss caused by a pathogenic simian-human immunodeficiency virus by replacement of the subtype B vpu with the vpu from a subtype C HIV-1 clinical isolate.

Previously, we showed that the Vpu protein from subtype C human immunodeficiency virus type 1 (HIV-1) was efficiently targeted to the cell surface, suggesting that this protein has biological properties that differ from the well-studied subtype B Vpu protein. In this study, we have further analyzed the biological properties of the subtype C Vpu protein. Flow cytometric analysis revealed that the subtype B Vpu (strain HXB2) was more efficient at down-regulating CD4 surface expression than the Vpu proteins from four subtype C clinical isolates. We constructed a simian-human immunodeficiency virus virus, designated as SHIV(SCVpu), in which the subtype B vpu gene from the pathogenic SHIV(KU-1bMC33) was substituted with the vpu from a clinical isolate of subtype C HIV-1 (strain C.96.BW16B01). Cell culture studies revealed that SHIV(SCVpu) replicated with slightly reduced kinetics when compared with the parental SHIV(KU-1bMC33) and that the viral Env and Gag precursor proteins were synthesized and processed similarly compared to the parental SHIV(KU-1bMC33). To determine if substitution of the subtype C Vpu protein affected the pathogenesis of the virus, three pig-tailed macaques were inoculated with SHIV(SCVpu) and circulating CD4+ T-cell levels and viral loads were monitored for up to 44 weeks. Our results show that SHIV(SCVpu) caused a more gradual decline in the rate of CD4+ T cells in pig-tailed macaques compared to those inoculated with parental subtype B SHIV(KU-1bMC33). These results show for the first time that different Vpu proteins of HIV-1 can influence the rate at which CD4+ T-cell loss occurs in the SHIV/pig-tailed macaque model.

APOBEC3G expression is restricted to epithelial cells of the proximal convoluted tubules and is not expressed in the glomeruli of macaques.

The Vif protein of human immunodeficiency virus-1 (HIV-1) interacts with members of the APOBEC family of cytidine deaminases. In this study, we isolated RNA from renal cortex as well as from isolated glomeruli and tubulointerstitial fractions from two pigtailed macaques that were exsanguinated and perfused with saline. RT-PCR results indicate that APOBEC3G was detected in the tubule fractions but not in the glomerular fractions. Immunoblot analysis using lysates prepared from these same fractions and a monoclonal antibody to APOBEC3G confirmed the RT-PCR findings. To determine which cell types express APOBEC3G, immunohistochemical studies were performed using this monoclonal antibody on renal cortical sections. Our results clearly show that the glomeruli do not express APOBEC3G but that select tubules within the cortex express APOBEC3G at high levels. To further differentiate the distribution of APOBEC3G expression, serial sections were stained with the lectins Dolichos biflorus agglutinin (DBA) and Phaseolus vulgaris erythroagglutinin (PHA-E), which differentially bind to epithelial cells of the tubules and glomeruli. Our results indicate that APOBEC3G expression was restricted to PHA-E-staining tubules and not DBA-staining tubules, suggesting that APOBEC3G expression was restricted to proximal convoluted tubules. These findings suggest that infection of epithelial cells of proximal renal tubules could suppress Vif-defective HIV-1 replication, whereas infection of cells of the glomeruli, a major target of HIV-associated nephropathy, could act as a reservoir for the replication of Vif-defective HIV-1.

APOBEC3G expression is restricted to neurons in the brains of pigtailed macaques.

The Vif protein of human immunodeficiency virus-1 (HIV-1) has been shown to interact with members of the APOBEC family of cytidine deaminases, particularly APOBEC3G/F. In this study, we isolated RNA from 12 regions of the brain from two pigtailed macaques that were exsanguinated and perfused with saline. Our results indicate that APOBEC3G was detected in all regions of the brain analyzed. Immunoblot analysis using lysates prepared from these same regions of the brain and a monoclonal antibody to APOBEC3G confirmed the RT-PCR findings. To determine which cell types express APOBEC3G, immunohistochemical studies were performed using this monoclonal antibody on whole brain sections. Our results clearly show that the pyramidal neurons within the gray matter of cerebral and cerebellar cortices express APOBEC3G. However, APOBEC3G expression in the pyramidal neurons appeared to be nuclear or associated with nuclei. In contrast to our findings in the cerebral cortex, immunohistochemical analysis of the spleen and kidney tissues revealed that APOBEC3G expression in the cells of these tissues was predominantly cytoplasmic. We further investigated the expression of APOBEC3G in astrocytes. Immunohistochemical staining of serial sections was performed using antibodies to glial fibrillary acidic protein (GFAP) and APOBEC3G. As expected, the cortical and cerebellar white matter showed extensive immunostaining of astrocytes with the antibody against GFAP but a lack of reactivity to the antibody to APOBEC3G. Additionally, Immunoblot analysis of lysates prepared from primary human fetal astrocytes revealed a lack of APOBEC3G expression. Taken together, these results indicate that APOBEC3G expression is restricted to neurons in the brain and that astrocytes and microglia probably do not express this protein or express it at levels undetectable by immunohistochemistry. These finding have implications for the brain as a potential reservoir for Vif-defective viruses.

A single amino acid substitution within the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein renders simian-human immunodeficiency virus (SHIV(KU-1bMC33)) susceptible to rimantadine.

Previous studies from our laboratory have shown that the transmembrane domain (TM) of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) contributes to the pathogenesis of SHIV(KU-1bMC33) in macaques and that the TM domain of Vpu could be replaced with the M2 protein viroporin from influenza A virus. Recently, we showed that the replacement of the TM domain of Vpu with that of the M2 protein of influenza A virus resulted in a virus (SHIV(M2)) that was sensitive to rimantadine [Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M., Powers, M.F., Wong, S.W., Stephens, E.B., 2006. Substitution of the transmembrane domain of Vpu in simian human immunodeficiency virus (SHIV(KU-1bMC33)) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques. Virology 344, 541-558]. Based on previous studies of the M2 protein which have shown that the His-X-X-X-Trp motif within the M2 is essential to the function of the M2 proton channel, we have constructed a novel SHIV in which the alanine at position 19 of the TM domain was replaced with a histidine residue resulting in the motif His-Ile-Leu-Val-Trp. The SHIV(VpuA19H) replicated with similar kinetics as the parental SHIV(KU-1bMC33) and pulse-chase analysis revealed that the processing of viral proteins was similar to SHIV(KU-1bMC33). This SHIV(VpuA19H) virus was found to be more sensitive to the M2 ion channel blocker rimantadine than SHIV(M2). Electron microscopic examination of SHIV(VpuA19H)-infected cells treated with rimantadine revealed an accumulation of viral particles at the cell surface and within intracellular vesicles, which was similar to that previously observed to SHIV(M2)-infected cells treated with rimantadine. These data indicate that the Vpu protein of HIV-1 can be converted into a rimantadine-sensitive ion channel with the alteration of one amino acid and provide additional evidence that drugs targeting the Vpu TM/ion channel can be effective anti-HIV-1 drugs.

Substitution of the transmembrane domain of Vpu in simian-human immunodeficiency virus (SHIVKU1bMC33) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques.

The Vpu protein of human immunodeficiency virus type 1 has been shown to shunt the CD4 receptor molecule to the proteasome for degradation and to enhance virus release from infected cells. The exact mechanism by which the Vpu protein enhances virus release is currently unknown but some investigators have shown that this function is associated with the transmembrane domain and potential ion channel properties. In this study, we determined if the transmembrane domain of Vpu could be functionally substituted with that of the prototypical viroporin, the M2 protein of influenza A virus. We constructed chimeric vpu gene in which the transmembrane domain of Vpu was replaced with that of the M2 protein of influenza. This chimeric vpu gene was substituted for the vpu gene in the genome of a pathogenic simian human immunodeficiency virus, SHIVKU-1bMC33. The resulting virus, SHIVM2, synthesized a Vpu protein that had a slightly different Mr compared to the parental SHIVKU-1bMC33, reflecting the different sizes of the two Vpu proteins. The SHIVM2 was shown to replicate with slightly reduced kinetics when compared to the parental SHIVKU-1bMC33 but electron microscopy revealed that the site of maturation was similar to the parental virus SHIVKU1bMC33. We show that the replication and spread of SHIVM2 could be blocked with the antiviral drug rimantadine, which is known to target the M2 ion channel. Our results indicate a dose dependent inhibition of SHIVM2 with 100 microM rimantadine resulting in a >95% decrease in p27 released into the culture medium. Rimantadine did not affect the replication of the parental SHIVKU-1bMC33. Examination of SHIVM2-infected cells treated with 50 microM rimantadine revealed numerous viral particles associated with the cell plasma membrane and within intracytoplasmic vesicles, which is similar to HIV-1 mutants lacking a functional vpu. To determine if SHIVM2 was as pathogenic as the parental SHIVKU-1bMC33 virus, two pig-tailed macaques were inoculated and followed for up to 8 months. Both pig-tailed macaques developed severe CD4+ T cell loss within 1 month of inoculation, high viral loads, and histological lesions consistent with lymphoid depletion similar to the parental SHIVKU-1bMC33. Taken together, these results indicate for the first time that the TM domain of the Vpu protein can be functionally substituted with the TM of M2 of influenza A virus, and shows that compounds that target the TM domain of Vpu protein of HIV-1 could serve as novel anti-HIV-1 drugs.