Functional Antagonism of Rhesus Macaque and Chimpanzee BST-2 by HIV-1 Vpu Is Mediated by Cytoplasmic Domain Interactions

Human immunodeficiency virus type 1 (HIV-1) Vpu enhances the release of viral particles from infected cells by interfering with the function of BST-2/tetherin, a cellular protein inhibiting virus release. The Vpu protein encoded by NL4-3, a widely used HIV-1 laboratory strain, antagonizes human BST-2 but not monkey or murine BST-2, leading to the conclusion that BST-2 antagonism by Vpu is species specific. In contrast, we recently identified several primary Vpu isolates, such as Vpu of HIV-1_DH12, capable of antagonizing both human and rhesus BST-2. Here we report that while Vpu interacts with human BST-2 primarily through their respective transmembrane domains, antagonism of rhesus BST-2 by Vpu involved an interaction of their cytoplasmic domains. Importantly, a Vpu mutant carrying two mutations in its transmembrane domain (A₁₄L and W₂₂A), rendering it incompetent for interaction with human BST-2, was able to interact with human BST-2 carrying the rhesus BST-2 cytoplasmic domain and partially neutralized the ability of this BST-2 variant to inhibit viral release. Bimolecular fluorescence complementation analysis to detect Vpu–BST-2 interactions suggested that the physical interaction of Vpu with rhesus or chimpanzee BST-2 involves a 5-residue motif in the cytoplasmic domain of BST-2 previously identified as important for the antagonism of monkey and great ape BST-2 by simian immunodeficiency virus (SIV) Nef. Thus, our study identifies a novel mechanism of antagonism of monkey and great ape BST-2 by Vpu that targets the same motif in BST-2 used by SIV Nef and might explain the expanded host range observed for Vpu isolates in our previous study.     

Polyvalent envelope glycoprotein vaccine elicits a broader neutralizing antibody response but is unable to provide sterilizing protection against heterologous Simian/human immunodeficiency virus infection in pigtailed macaques

The great difficulty in eliciting broadly cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) isolates has been attributed to several intrinsic properties of their viral envelope glycoprotein, including its complex quaternary structure, extensive glycosylation, and marked genetic variability. Most previously evaluated vaccine candidates have utilized envelope glycoprotein from a single virus isolate. Here we compare the breadth of NAb and protective immune response following vaccination of pigtailed macaques with envelope protein(s) derived from either single or multiple viral isolates. Animals were challenged with Simian/human immunodeficiency virus strain DH12 (SHIV(DH12)) following priming with recombinant vaccinia virus(es) expressing gp160(s) and boosting with gp120 protein(s) from (i) LAI, RF, 89.6, AD8, and Bal (Polyvalent); (ii) LAI, RF, 89.6, AD8, Bal, and DH12 (Polyvalent-DH12); (iii) 89.6 (Monovalent-89.6); and (iv) DH12 (Monovalent-DH12). Animals in the two polyvalent vaccine groups developed NAbs against more HIV-1 isolates than those in the two monovalent vaccine groups (P = 0.0054). However, the increased breadth of response was directed almost entirely against the vaccine strains. Resistance to SHIV(DH12) strongly correlated with the level of NAbs directed against the virus on the day of challenge (P = 0.0008). Accordingly, the animals in the Monovalent-DH12 and Polyvalent-DH12 vaccine groups were more resistant to the SHIV(DH12) challenge than the macaques immunized with preparations lacking a DH12 component (viz. Polyvalent and Monovalent-89.6) (P = 0.039). Despite the absence of any detectable NAb, animals in the Polyvalent vaccine group, but not those immunized with Monovalent-89.6, exhibited markedly lower levels of plasma virus than those in the control group, suggesting a superior cell-mediated immune response induced by the polyvalent vaccine.     

Induction of CD8+ cells able to suppress CCR5-tropic simian immunodeficiency virus SIVmac239 replication by controlled infection of CXCR4-tropic simian-human immunodeficiency virus in vaccinated rhesus macaques

Recent recombinant viral vector-based AIDS vaccine trials inducing cellular immune responses have shown control of CXCR4-tropic simian-human immunodeficiency virus (SHIV) replication but difficulty in containment of pathogenic CCR5-tropic simian immunodeficiency virus (SIV) in rhesus macaques. In contrast, controlled infection of live attenuated SIV/SHIV can confer the ability to contain SIV superchallenge in macaques. The specific immune responses responsible for this control may be induced by live virus infection but not consistently by viral vector vaccination, although those responses have not been determined. Here, we have examined in vitro anti-SIV efficacy of CD8+ cells in rhesus macaques that showed prophylactic viral vector vaccine-based control of CXCR4-tropic SHIV89.6PD replication. Analysis of the effect of CD8+ cells obtained at several time points from these macaques on CCR5-tropic SIVmac239 replication in vitro revealed that CD8+ cells in the chronic phase after SHIV challenge suppressed SIV replication more efficiently than those before challenge. SIVmac239 superchallenge of two of these macaques at 3 or 4 years post-SHIV challenge was contained, and the following anti-CD8 antibody administration resulted in transient CD8+ T-cell depletion and appearance of plasma SIVmac239 viremia in both of them. Our results indicate that CD8+ cells acquired the ability to efficiently suppress SIV replication by controlled SHIV infection, suggesting the contribution of CD8+ cell responses induced by controlled live virus infection to containment of HIV/SIV superinfection.     

Enhanced infectivity of an R5-tropic simian/human immunodeficiency virus carrying human immunodeficiency virus type 1 subtype C envelope after serial passages in pig-tailed macaques (Macaca nemestrina)

The increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating strategies against the transmission of the virus. A chimeric simian/human immunodeficiency virus (SHIV), SHIV(CHN19), was generated with a primary, non-syncytium-inducing HIV-1 subtype C envelope from a Chinese strain in the background of SHIV(33). Unlike R5-tropic SHIV(162), SHIV(CHN19) was not found to replicate in rhesus CD4(+) T lymphocytes. SHIV(CHN19) does, however, replicate in CD4(+) T lymphocytes of pig-tailed macaques (Macaca nemestrina). The observed replication competence of SHIV(CHN19) requires the full tat/rev genes and partial gp41 region derived from SHIV(33). To evaluate in vivo infectivity, SHIV(CHN19) was intravenously inoculated, at first, into two pig-tailed and two rhesus macaques. Although all four animals became infected, the virus replicated preferentially in pig-tailed macaques with an earlier plasma viral peak and a faster seroconversion. To determine whether in vivo adaptation would enhance the infectivity of SHIV(CHN19), passages were carried out serially in three groups of two pig-tailed macaques each, via intravenous blood-bone marrow transfusion. The passages greatly enhanced the infectivity of the virus as shown by the increasingly elevated viral loads during acute infection in animals with each passage. Moreover, the doubling time of plasma virus during acute infection became much shorter in passage 4 (P4) animals (0.2 day) in comparison to P1 animals (1 to 2 days). P2 to P4 animals all became seropositive around 2 to 3 weeks postinoculation and had a decline in CD4/CD8 T-cell ratio during the early phase of infection. In P4 animals, a profound depletion of CD4 T cells in the lamina propria of the jejunum was observed. Persistent plasma viremia has been found in most of the infected animals with sustained viral loads ranging from 10(3) to 10(5) per ml up to 6 months postinfection. Serial passages did not change the viral phenotype as confirmed by the persistence of the R5 tropism of SHIV(CHN19) isolated from P4 animals. In addition, the infectivity of SHIV(CHN19) in rhesus peripheral blood mononuclear cells was also increased after in vivo passages. Our data indicate that SHIV(CHN19) has adapted well to grow in macaque cells. This established R5-tropic SHIV(CHN19)/macaque model would be very useful for HIV-1 subtype C vaccine and pathogenesis studies.     

CD8+ and CD20+ lymphocytes cooperate to control acute simian immunodeficiency virus/human immunodeficiency virus chimeric virus infections in rhesus monkeys: modulation by major histocompatibility complex genotype

We have previously described two isogenic molecularly cloned simian immunodeficiency virus/human immunodeficiency virus chimeric viruses (SHIVs) that differ from one another by 9 amino acids and direct distinct clinical outcomes in inoculated rhesus monkeys. SHIV(DH12R-Clone 7), like other highly pathogenic CXCR4-tropic SHIVs, induces rapid and complete depletions of CD4+ T lymphocytes and immunodeficiency in infected animals. In contrast, macaques inoculated with SHIV(DH12R-Clone 8) experience only partial and transient losses of CD4+ T cells, show prompt control of their viremia, and remain healthy for periods of time extending for up to 4 years. The contributions of CD8+ and CD20+ lymphocytes in suppressing the replication of the attenuated SHIV(DH12R-Clone 8) and maintaining a prolonged asymptomatic clinical course was assessed by treating animals with monoclonal antibodies that deplete each lymphocyte subset at the time of virus inoculation. The absence of either CD8+ or CD20+ cells during the SHIV(DH12R-Clone 8) acute infection resulted in the rapid, complete, and irreversible loss of CD4+ T cells; sustained high levels of postpeak plasma viremia; and symptomatic disease in Mamu-A*01-negative Indian rhesus monkeys. In Mamu-A*01-positive animals, however, the aggressive, highly pathogenic phenotype was observed only in macaques depleted of CD8+ cells; SHIV(DH12R-Clone 8) was effectively controlled in Mamu-A*01-positive monkeys in the absence of B lymphocytes. Taken together, these results indicate that both CD8+ and CD20+ B cells contribute to the control of primate lentiviral infection in Mamu-A*01-negative macaques. Furthermore, the major histocompatibility complex genotype of an infected animal, as exemplified by the Mamu-A*01 allele in this study, has the additional capacity to shift the balance of the composite immune response.     

Infectious Simian/Human Immunodeficiency Virus with Human Immunodeficiency Virus Type 1 Subtype C from an African Isolate: Rhesus Macaque Model

Human immunodeficiency virus type 1 (HIV-1) subtype C is responsible for more than 56% of all infections in the HIV and AIDS pandemic. It is the predominant subtype in the rapidly expanding epidemic in southern Africa. To develop a relevant model that would facilitate studies of transmission, pathogenesis, and vaccine development for this subtype, we generated SHIV(MJ4), a simian/human immunodeficiency virus (SHIV) chimera based on HIV-1 subtype C. SHIV(MJ4) contains the majority of env, the entire second exon of tat, and a partial sequence of the second exon of rev, all derived from a CCR5-tropic, primary isolate envelope clone from southern Africa. SHIV(MJ4) replicated efficiently in human, rhesus, and pig-tailed macaque peripheral blood mononuclear cells (PBMCs) in vitro but not in CEMx174 cells. To assess in vivo infectivity, SHIV(MJ4) was intravenously inoculated into four rhesus macaques (Macaca mulatta). All four animals became infected as determined through virus isolation, PCR analysis, and viral loads of 10(7) to 10(8) copies of viral RNA per ml of plasma during the primary infection phase. We have established a CCR5-tropic SHIV(MJ4)/rhesus macaque model that may be useful in the studies of HIV-1 subtype C immunology and biology and may also facilitate the evaluation of vaccines to control the spread of HIV-1 subtype C in southern Africa and elsewhere.     

Vaginal transmission of chimeric simian/human immunodeficiency viruses in rhesus macaques.

Chimeric simian/human immunodeficiency viruses (SHIVs) that express the env genes derived from distinct HIV type 1 (HIV-1) isolates were tested for the ability to infect rhesus macaques following intravaginal inoculation. SHIVs containing either the HIV-1 HXBc2 or the HIV-1 89.6 envelope glycoproteins were capable of replicating in intravenously inoculated rhesus macaques. However, intravaginal inoculation of animals with these two SHIVs resulted in infection only with the SHIV containing the HIV-1 89.6 glycoprotein. Thus, properties conferred by the envelope glycoproteins in the chimeric virus affect the ability of particular SHIVs to initiate a systemic infection following vaginal inoculation. These results provide indirect support for the hypothesis that the selection of specific viral variants occurs in the genital tracts of individuals exposed to HIV by sexual contact.     

The envelope glycoprotein of human immunodeficiency virus type 2 enhances viral particle release: a Vpu-like factor?

The Vpu protein is a human immunodeficiency virus type 1 (HIV-1)-specific accessory protein that is required for the efficient release of viral particles from infected cells. Even though HIV-2 does not encode Vpu, we found that this virus is nevertheless capable of efficiently releasing virus particles. In fact, the rate of virus release from HeLa cells transfected with a full-length molecular clone of HIV-2, ROD10, was comparable to that observed for the vpu+ HIV-1 NL4-3 isolate and was not further enhanced by expression of Vpu in trans. However, consistent with previous observations showing that HIV-2 particle release is Vpu responsive in the context of HIV-1/HIV-2 chimeric constructs; exchanging the gag-pol region of NL4-3 with the corresponding region from pROD10 rendered the resulting chimeric virus Vpu responsive. Our finding that the responsiveness of HIV-2 particle release to Vpu is context dependent suggested the presence of a Vpu-like factor(s) encoded by HIV-2. Using chimeric proviruses encoding HIV-2 gag and pol in the context of the HIV-1 provirus that were coexpressed with subgenomic HIV-2 constructs, we found that the HIV-2 envelope glycoprotein had the ability to enhance HIV-2 particle release with an efficiency comparable to that of the HIV-1 Vpu protein. Conversely, inactivation of the HIV-2 env gene in the original ROD10 clone resulted in a decrease in the rate of viral particle release to a level that was comparable to that of Vpu-deficient HIV-1 isolates. Providing the wild-type envelope in trans rescued the particle release defect of the ROD10 envelope mutant. Thus, unlike HIV-1, which encodes two separate proteins to regulate virus release or to mediate viral entry, the HIV-2 Env protein has evolved to perform both functions.     

Short- and long-term clinical outcomes in rhesus monkeys inoculated with a highly pathogenic chimeric simian/human immunodeficiency virus

A highly pathogenic simian/human immunodeficiency virus (SHIV), SHIV(DH12R), isolated from a rhesus macaque that had been treated with anti-human CD8 monoclonal antibody at the time of primary infection with the nonpathogenic, molecularly cloned SHIV(DH12), induced marked and rapid CD4(+) T cell loss in all rhesus macaques intravenously inoculated with 1.0 50% tissue culture infective dose (TCID(50)) to 4.1 x 10(5) TCID(50)s of virus. Animals inoculated with 650 TCID(50)s of SHIV(DH12R) or more experienced irreversible CD4(+) T lymphocyte depletion and developed clinical disease requiring euthanasia between weeks 12 and 23 postinfection. In contrast, the CD4(+) T-cell numbers in four of five monkeys receiving 25 TCID(50)s of SHIV(DH12R) or less stabilized at low levels, and these surviving animals produced antibodies capable of neutralizing SHIV(DH12R). In the fifth monkey, no recovery from the CD4(+) T cell decline occurred, and the animal had to be euthanized. Viral RNA levels, subsequent to the initial peak of infection but not at peak viremia, correlated with the virus inoculum size and the eventual clinical course. Both initial infection rate constants, k, and decay constants, d, were determined, but only the latter were statistically correlated to clinical outcome. The attenuating effects of reduced inoculum size were also observed when virus was inoculated by the mucosal route. Because the uncloned SHIV(DH12R) stock possessed the genetic properties of a lentivirus quasispecies, we were able to assess the evolution of the input virus swarm in animals surviving the acute infection by monitoring the emergence of neutralization escape viral variants.     

Vaccination of rhesus macaques with recombinant Mycobacterium bovis bacillus Calmette-Guérin Env V3 elicits neutralizing antibody-mediated protection against simian-human immunodeficiency virus with a homologous but not a heterologous V3 motif

Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations.     

Characterization of primary isolate-like variants of simian-human immunodeficiency virus

Several different strains of simian-human immunodeficiency virus (SHIV) that contain the envelope glycoproteins of either T-cell-line-adapted (TCLA) strains or primary isolates of human immunodeficiency virus type 1 (HIV-1) are now available. One of the advantages of these chimeric viruses is their application to studies of HIV-1-specific neutralizing antibodies in preclinical AIDS vaccine studies in nonhuman primates. In this regard, an important consideration is the spectrum of antigenic properties exhibited by the different envelope glycoproteins used for SHIV construction. The antigenic properties of six SHIV variants were characterized here in neutralization assays with recombinant soluble CD4 (rsCD4), monoclonal antibodies, and serum samples from SHIV-infected macaques and HIV-1-infected individuals. Neutralization of SHIV variants HXBc2, KU2, 89.6, and 89.6P by autologous and heterologous sera from SHIV-infected macaques was restricted to an extent that these viruses may be considered heterologous to one another in their major neutralization determinants. Little or no variation was seen in the neutralization determinants on SHIV variants 89.6P, 89.6PD, and SHIV-KB9. Neutralization of SHIV HXBc2 by sera from HXBc2-infected macaques could be blocked with autologous V3-loop peptide; this was less true in the case of SHIV 89.6 and sera from SHIV 89.6-infected macaques. The poorly immunogenic but highly conserved epitope for monoclonal antibody IgG1b12 was a target for neutralization on SHIV variants HXBc2, KU2, and 89.6 but not on 89.6P and KB9. The 2G12 epitope was a target for neutralization on all five SHIV variants. SHIV variants KU2, 89.6, 89.6P, 89.6PD, and KB9 exhibited antigenic properties characteristic of primary isolates by being relatively insensitive to neutralization in peripheral blood mononuclear cells with serum samples from HIV-1-infected individuals and 12-fold to 38-fold less sensitive to inhibition with recombinant soluble CD4 than TCLA strains of HIV-1. The utility of nonhuman primate models in AIDS vaccine development is strengthened by the availability of SHIV variants that are heterologous in their neutralization determinants and exhibit antigenic properties shared with primary isolates.     

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.     

HIV-1 Vpu antagonizes BST-2 by interfering mainly with the trafficking of newly synthesized BST-2 to the cell surface

Bone marrow stromal cell antigen-2 (BST-2) inhibits human immunodeficiency virus type 1 (HIV-1) release by cross-linking nascent virions on infected cell surface. HIV-1 Vpu is thought to antagonize BST-2 by downregulating its surface levels via a mechanism that involves intracellular sequestration and lysosomal degradation. Here, we investigated the functional importance of cell-surface BST-2 downregulation and the BST-2 pools targeted by Vpu using an inducible proviral expression system. Vpu established a surface BST-2 equilibrium at ～60% of its initial levels within 6 h, a condition that coincided with detection of viral release. Analysis of BST-2 post-endocytic trafficking revealed that the protein is engaged in a late endosomal pathway independent of Vpu. While Vpu moderately enhanced cell-surface BST-2 clearance, it strongly affected the protein resupply to the plasma membrane via newly synthesized proteins. Noticeably, Vpu affected clearance of surface BST-2 more substantially in Jurkat T cells than in HeLa cells, suggesting a cell-dependent impact of Vpu on the pool of surface BST-2. Collectively, our data reveal that Vpu imposes a new BST-2 equilibrium, incompatible with efficient restriction of HIV-1 release, by combining an acceleration of surface BST-2 natural clearance, whose degree might be cell-type dependent, to a severe impairment of the protein resupply to the plasma membrane.     

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.     

Modulation by morphine of viral set point in rhesus macaques infected with simian immunodeficiency virus and simian-human immunodeficiency virus

Six rhesus macaques were adapted to morphine dependence by injecting three doses of morphine (5 mg/kg of body weight) for a total of 20 weeks. These animals along with six control macaques were infected intravenously with mixture of simian-human immunodeficiency virus KU-1B (SHIV(KU-1B)), SHIV(89.6P), and simian immunodeficiency virus 17E-Fr. Levels of circulating CD4(+) T cells and viral loads in the plasma and the cerebrospinal fluid were monitored in these macaques for a period of 12 weeks. Both morphine and control groups showed precipitous loss of CD4(+) T cells. However this loss was more prominent in the morphine group at week 2 (P = 0.04). Again both morphine and control groups showed comparable peak plasma viral load at week 2, but the viral set points were higher in the morphine group than that in the control group. Likewise, the extent of virus replication in the cerebral compartment was more pronounced in the morphine group. These results provide a definitive evidence for a positive correlation between morphine and levels of viral replication.     

Induction of simian AIDS in infant rhesus macaques infected with CCR5- or CXCR4-utilizing simian-human immunodeficiency viruses is associated with distinct lesions of the thymus

Newborn rhesus macaques were infected with two chimeric simian-human immunodeficiency virus (SHIV) strains which contain unique human immunodeficiency virus type 1 (HIV-1) env genes and exhibit distinct phenotypes. Infection with either the CCR5-specific SHIV(SF162P3) or the CXCR4-utilizing SHIV(SF33A) resulted in clinical manifestations consistent with simian AIDS. Most prominent in this study was the detection of severe thymic involution in all SHIV(SF33A)-infected infants, which is very similar to HIV-1-induced thymic dysfunction in children who exhibit a rapid pattern of disease progression. In contrast, SHIV(SF162P3) induced only a minor disruption in thymic morphology. Consistent with the distribution of the coreceptors CXCR4 and CCR5 within the thymus, the expression of SHIV(SF162P3) was restricted to the thymic medulla, whereas SHIV(SF33A) was preferentially detected in the cortex. This dichotomy of tissue tropism is similar to the differential tropism of HIV-1 isolates observed in the reconstituted human thymus in SCID-hu mice. Accordingly, our results show that the SHIV-monkey model can be used for the molecular dissection of cell and tissue tropisms controlled by the HIV-1 env gene and for the analysis of mechanisms of viral immunopathogenesis in AIDS. Furthermore, these findings could help explain the rapid progression of disease observed in some HIV-1-infected children.     

Antibody responses elicited in macaques immunized with human immunodeficiency virus type 1 (HIV-1) SF162-derived gp140 envelope immunogens: comparison with those elicited during homologous simian/human immunodeficiency virus SHIVSF162P4 and heterologous HIV-1 infection

The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIV(SF162P4), and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, DeltaV2gp140 (lacking the crown of the V2 loop), DeltaV3gp140 (lacking the crown of the V3 loop), and DeltaV2DeltaV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and DeltaV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and DeltaV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by DeltaV3gp140 or DeltaV2DeltaV3gp140. In contrast, the SHIV(SF162P4)-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.     

V3 loop-determined coreceptor preference dictates the dynamics of CD4+-T-cell loss in simian-human immunodeficiency virus-infected macaques

We used experimental infection of rhesus macaques with envelope gp120 V3 loop isogenic simian-human immunodeficiency virus (SHIV) molecular clones to more clearly define the impact of human immunodeficiency virus type 1 coreceptor usage in target cell selectivity and the rates of CD4+-T-cell depletion. Functional assays demonstrate that substitution of the V3 loop of the pathogenic CXCR4-tropic (X4) SHIV(SF33A2) molecular clone with the corresponding sequences from the CCR5-tropic (R5) SHIV(SF162P3) isolate resulted in a switch of coreceptor usage from CXCR4 to CCR5. The resultant R5 clone, designated SHIV(SF33A2(V3)), is replication competent in vivo, infecting two of two macaques by intravenous inoculation with peak viremia that is comparable to that seen in monkeys infected with X4-SHIV(SF33A2). But while primary infection with the X4 clone was accompanied by rapid and significant loss of peripheral and secondary lymphoid CD4+ T lymphocytes, infection with R5-SHIV(SF33A2(V3)) led to only a modest and transient loss. However, substantial depletion of intestinal CD4+ T cells was observed in R5-SHIV(SF33A2(V3))-infected macaques. Moreover, na?ve T cells that expressed high levels of CXCR4 were rapidly depleted in X4-SHIV(SF33A2)-infected macaques, whereas R5-SHIV(SF33A2(V3)) infection mainly affected memory T cells that expressed CCR5. These findings in a unique isogenic system illustrate that coreceptor usage is the principal determinant of tissue and target cell specificity of the virus in vivo and dictates the dynamics of CD4+-T-cell depletion during SHIV infection.     

The ESCRT-0 component HRS is required for HIV-1 Vpu-mediated BST-2/tetherin down-regulation

The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, a highly conserved set of four hetero-oligomeric protein complexes, is required for multivesicular body formation, sorting ubiquitinylated membrane proteins for lysosomal degradation, cytokinesis and the final stages of assembly of a number of enveloped viruses, including the human immunodeficiency viruses. Here, we show an additional role for the ESCRT machinery in HIV-1 release. BST-2/tetherin is a restriction factor that impedes HIV release by tethering mature virus particles to the plasma membrane. We found that HRS, a key component of the ESCRT-0 complex, promotes efficient release of HIV-1 and that siRNA-mediated HRS depletion induces a BST-2/tetherin phenotype. This activity is related to the ability of the HIV-1 Vpu protein to down-regulate BST-2/tetherin. We found that BST-2/tetherin undergoes constitutive ESCRT-dependent sorting for lysosomal degradation and that this degradation is enhanced by Vpu expression. We demonstrate that Vpu-mediated BST-2/tetherin down-modulation and degradation require HRS (ESCRT-0) function and that knock down of HRS increases cellular levels of BST-2/tetherin and restricts virus release. Furthermore, HRS co-precipitates with Vpu and BST-2. Our results provide further insight into the mechanism by which Vpu counteracts BST-2/tetherin and promotes HIV-1 dissemination, and they highlight an additional role for the ESCRT machinery in virus release.     

Outcome of simian-human immunodeficiency virus strain 89.6p challenge following vaccination of rhesus macaques with human immunodeficiency virus Tat protein

The regulatory proteins Nef, Rev, and Tat of human immunodeficiency virus type 1 (HIV-1) are attractive targets for vaccine development, since induction of effective immune responses targeting these early proteins may best control virus replication. Here we investigated whether vaccination with biologically active Tat or inactive Tat toxoid derived from HIV-1(IIIB) and simian-human immunodeficiency virus (SHIV) strain 89.6p would induce protective immunity in rhesus macaques. Vaccination induced high titers of anti-Tat immunoglobulin G in all immunized animals by week 7, but titers were somewhat lower in the 89.6p Tat group. Dominant B-cell epitopes mapped to the amino terminus, the basic domain, and the carboxy-terminal region. Tat-specific T-helper responses were detected in 50% of immunized animals. T-cell epitopes appeared to map within amino acids (aa) 1 to 24 and aa 37 to 66. In addition, Tat-specific gamma interferon responses were detected in CD4+ and/or CD8+ T lymphocytes in 11 of 16 immunized animals on the day of challenge. However, all animals became infected upon intravenous challenge with 30 50% minimal infective doses of SHIV 89.6p, and there were no significant differences in viral loads or CD4+ T-cell counts between immunized and control animals. Thus, vaccination with HIV-1(IIIB) or SHIV 89.6p Tat or with Tat toxoid preparations failed to confer protection against SHIV 89.6p infection despite robust Tat-specific humoral and cellular immune responses in some animals. Given its apparent immunogenicity, Tat may be more effective as a component of a cocktail vaccine in combination with other regulatory and/or structural proteins of HIV-1.