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Reports the results of the establishment of stable lymphoid cell lines expressing the human immunodeficiency virus type 1 (HIV-1) `nef' gene product, p 27. The presence of p27 in lymphoid cells suppressed some strains of HIV-1, although it was unsuccessful in the lymphoid cell lines affected by advanced stages of HIV-1.

Examines the interrelation of animal retroviruses with AIDS. Association of mutations in CCR5-coding sequences with SIV carrier status in African primates; Effects of soluble CD4 on simian immunodeficiency virus infection of CD4-positive and CD4-negative cells; Induction of AIDS rhesus monkeys.

Abstract SIV or SHIV infection of nonhuman primates (NHP) has been used to investigate the impact of coreceptor usage on the composition and dynamics of the CD4+ T cell compartment, mechanisms of disease induction and development of clinical syndrome. As the entire course of infection can be followed, with frequent access to tissue compartments, infection of rhesus macaques with CCR5-tropic SHIVs further allows for study of HIV-1 coreceptor switch after intravenous and mucosal inoculation, with longitudinal and systemic analysis to determine the timing, anatomical sites and cause for the change in envelope glycoprotein and coreceptor preference. Here, we review our current understanding of coreceptor use in NHPs and their impact on the pathobiological characteristics of the infection, and discuss recent advances in NHP studies to uncover the underlying selective pressures for the change in coreceptor preference in vivo.

Abstract Background Mucosally transmissible and pathogenic CCR5 (R5)-tropic simian-human immunodeficiency virus (SHIV) molecular clones are useful reagents to identity neutralization escape in HIV-1 vaccine experiments and to study the envelope evolutionary process and mechanistic basis for coreceptor switch during the course of natural infection. Results We observed progression to AIDS in rhesus macaques infected intrarectally with molecular clones of the pathogenic R5 SHIVSF162P3N isolate. Expansion to CXCR4 usage was documented in one diseased macaque that mounted a neutralizing antibody response and in another that failed to do so, with the latter displaying a rapid progressor phenotype. V3 loop envelop glycoprotein gp120 sequence changes that are predictive of a CXCR4 (X4)-using phenotype in HIV-1 subtype B primary isolates, specifically basic amino acid substations at positions 11 (S11R), 24 (G24R) and 25 (D25K) of the loop were detected in the two infected macaques. Functional assays showed that envelopes with V3 S11R or D25K mutation were dual-tropic, infecting CD4+ target cells that expressed either the CCR5 or CXCR4 coreceptor. And, consistent with findings of coreceptor switching in macaques infected with the pathogenic isolate, CXCR4-using variant was first detected in the lymph node of the chronically infected rhesus monkey several weeks prior to its presence in peripheral blood. Moreover, X4 emergence in this macaque coincided with persistent peripheral CD4+ T cell loss and a decline in neutralizing antibody titer that are suggestive of immune deterioration, with macrophages as the major virus-producing cells at the end-stage of disease. Conclusions The data showed that molecular clones derived from the R5 SHIVSF162P3N isolate are mucosally transmissible and induced disease in a manner similar to that observed in HIV-1 infected individuals, providing a relevant and useful animal infection model for in-depth analyses of host selection pressures and the env evolutionary changes that influence disease outcome, coreceptor switching and vaccine escape.

Abstract Background We previously reported that adoption of an “open” envelope glycoprotein (Env) to expose the CD4 binding site for efficient receptor binding and infection of cell targets such as macrophages that express low levels of the receptor represents an early event in the process of coreceptor switch in two rapidly progressing (RP) R5 SHIVSF162P3N-infected rhesus macaques, releasing or reducing Env structural constraints that have been suggested to limit the pathways available for a change in coreceptor preference. Here we extended these studies to two additional RP monkeys with coreceptor switch and three without to confirm and identify additional factors that facilitated the process of phenotypic conversion. Results We found that regardless of coreceptor switching, R5 viruses in SHIVSF162P3N-infected RP macaques evolved over time to infect macrophages more efficiently; this was accompanied by increased sCD4 sensitivity, with structural changes in the CD4 binding site, the V3 loop and/or the fusion domain of their Envs that are suggestive of better CD4 contact, CCR5 usage and/or virus fusion. However, sCD4-sensitive variants with improved CD4 binding were observed only in RPs with coreceptor switch. Furthermore, cumulative viral load was higher in RPs with than in those without phenotypic switch, with the latter maintaining a longer period of seroconversion. Conclusions Our data suggest that the increased virus replication in the RPs with R5-to-X4 conversion increased the rate of virus evolution and reduction in the availability of target cells with optimal CD4 expression heightened the competition for binding to the receptor. In the absence of immunological restrictions, variants that adopt an “open” Env to expose the CD4 binding site for better CD4 use are selected, allowing structural changes that confer CXCR4-use to be manifested. Viral load, change in target cell population during the course of infection and host immune response therefore are interdependent variables that influence R5 virus evolution and coreceptor switch in SHIVSF162P3N-infected rhesus macaques. Because an "open" Env conformation also renders the virus more susceptible to antibody neutralization, our findings help to explain the infrequent and late appearance of X4 virus in HIV-1 infection when the immune system deteriorates.

CCR5-tropic simian/human immunodeficiency viruses (SHIV) with clade C transmitted/founder envelopes represent a critical tool for the investigation of HIV experimental vaccines and microbicides in nonhuman primates, although many such isolates lead to spontaneous viral control post infection. Here, we generated a high-titer stock of pathogenic SHIV-C109p5 by serial passage in two rhesus macaques (RM) and tested its virulence in aged monkeys. The co-receptor usage was confirmed before infecting five geriatric rhesus macaques (four female and one male). Plasma viral loads were monitored by reverse transcriptase-quantitative PCR (RT-qPCR), cytokines by multiplex analysis, and biomarkers of gastrointestinal damage by enzyme-linked immunosorbent assay. Antibodies and cell-mediated responses were also measured. Viral dissemination into tissues was determined by RNAscope. Intravenous SHIV-C109p5 infection of aged RMs leads to high plasma viremia and rapid disease progression; rapid decrease in CD4 + T cells, CD4 + CD8 + T cells, and plasmacytoid dendritic cells; and wasting necessitating euthanasia between 3 and 12 weeks post infection. Virus-specific cellular immune responses were detected only in the two monkeys that survived 4 weeks post infection. These were Gag-specific TNFα+CD8+, MIP1β+CD4+, Env-specific IFN-γ+CD4+, and CD107a+ T cell responses. Four out of five monkeys had elevated intestinal fatty acid binding protein levels at the viral peak, while regenerating islet-derived protein 3α showed marked increases at later time points in the three animals surviving the longest, suggesting gut antimicrobial peptide production in response to microbial translocation post infection. Plasma levels of monocyte chemoattractant protein-1, interleukin-15, and interleukin-12/23 were also elevated. Viral replication in gut and secondary lymphoid tissues was extensive.IMPORTANCESimian/human immunodeficiency viruses (SHIV) are important reagents to study prevention of virus acquisition in nonhuman primate models of HIV infection, especially those representing transmitted/founder (T/F) viruses. However, many R5-tropic SHIV have limited fitness in vivo leading to many monkeys spontaneously controlling the virus post acute infection. Here, we report the generation of a pathogenic SHIV clade C T/F stock by in vivo passage leading to sustained viral load set points, a necessity to study pathogenicity. Unexpectedly, administration of this SHIV to elderly rhesus macaques led to extensive viral replication and fast disease progression, despite maintenance of a strict R5 tropism. Such age-dependent rapid disease progression had previously been reported for simian immunodeficiency virus but not for R5-tropic SHIV infections., Competing Interests: The authors declare no conflict of interest.

Macrophage (mac)-tropic human immnunodeficiency virus type 1 (HIV-1) and simian immnunodeficiency virus (SIV) in brain are associated with neurological disease. Mac-tropic HIV-1 evolves enhanced CD4 interactions that enable macrophage infection via CD4, which is in low abundance. In contrast, mac-tropic SIV is associated with CD4-independent infection via direct CCR5 binding. Recently, mac-tropic simian-human immunodeficiency virus (SHIV) from macaque brain was also reported to infect cells via CCR5 without CD4. Since SHIV envelope proteins (Envs) are derived from HIV-1, we tested more than 100 HIV-1 clade B Envs for infection of CD4-negative, CCR5 + Cf2Th/CCR5 cells. However, no infection was detected. Our data suggest that there are differences in the evolution of mac-tropism in SIV and SHIV compared to HIV-1 clade B due to enhanced interactions with CCR5 and CD4, respectively.

Simian-human immunodeficiency virus (SHIV) infection provides a relevant animal model to study HIV-1 neutralization breadth. With previously identified SHIV SF162P3N infected rhesus macaques that did or did not develop neutralization breadth, we characterized the transmitted/founder viruses and initial autologous/homologous neutralizing antibodies in these animals. The plasma viral load and blood CD4 count did not distinguish macaques with and without breadth, and only one tested homologous envelope clone revealed a trend for macaques with breadth to favor an early homologous response. In two macaques with breadth, GB40 and FF69, infected with uncloned SHIV SF162P3N , multiple viral variants were transmitted, and the transmitted variants were not equal in neutralization sensitivity. The targets of initial autologous neutralizing antibodies, arising between 10 and 20 weeks post infection, were mapped to N462 glycan and G460a in gp120 V5 in GB40 and FF69, respectively. Although it is unclear whether these targets are related to later neutralization breadth development, the G460a target but not N462 glycan appeared more common in macaques with breadth than those without. Longitudinal plasmas revealed 2⁻3 sequential waves of neutralizing antibodies in macaques with breadth, implicating that 3 sequential envelope variants, if not more, may be required for the broadening of HIV-1 neutralizing antibodies.

Background: HIV-1 is known to adapt to the local environment in its usage of receptors, and it can become CD4 independent in the brain where the receptor is scarce. This adaptation is through amino acid variations, but the patterns of such variation are not yet well understood. Given that infection of long-lived CD4-low and CD4-negative cells in anatomical compartments such as the brain expands cell tropism in vivo and may serve as potential viral reservoirs that pose challenge for HIV eradication, understanding the evolution to CD4 independence and envelope conformation associated with infection in the absence of CD4 will not only broaden our insights into HIV pathogenesis but may guide functional cure strategies as well., Methods: We characterize, by site-directed mutagenesis, neutralization assay, and structural analysis, a pair of CD4-dependent (cl2) and CD4-independent (cl20) envelopes concurrently isolated from the cerebral spinal fluid of an SHIV-infected macaque with neurological AIDS and with minimum sequence differences., Results: Residues different between cl2 and cl20 are mapped to the V1V2 and surrounding regions. Mutations of these residues in cl2 increased its CD4 independence in infection, and the effects are cumulative and likely structural., Conclusions: Our data suggested that the determinants of CD4 independence in vivo mapped principally to V1V2 of gp120 that can destabilize the apex of the envelope spike, with an additional change in V4 that abrogated a potential N-linked glycan to facilitate movement of the V1V2 domain and further expose the coreceptor-binding site.

Lentiviruses are able to establish persistent infection in their respective hosts despite a potent type-I interferon (IFN-I) response following transmission. A number of IFN-I-induced host factors that are able to inhibit lentiviral replication in vitro have been identified, and these studies suggest a role for IFN-induced factors as barriers to cross-species transmission. However, the ability of these factors to inhibit viral replication in vivo has not been well characterized, nor have the viral determinants that contribute to evasion or antagonism of the host IFN-I response. In this study, we hypothesized that the host IFN-I response serves as a strong selective pressure in the context of SIV/HIV chimeric virus (SHIV) infection of macaques and sought to identify the viral determinants that contribute to IFN-I resistance. We assessed the ability of SHIVs encoding HIV-1 sequences adapted by serial passage in macaques versus SHIVs encoding HIV sequences isolated directly from infected individuals to replicate in the presence of IFNα in macaque lymphocytes. We demonstrate that passage in macaques selects for IFNα resistant viruses that have higher replication kinetics and increased envelope content. SHIVs that encode HIV-1 sequences derived directly from infected humans were sensitive to IFNα -induced inhibition whereas SHIVs obtained after passage in macaques were not. This evolutionary process was directly observed in viruses that were serially passaged during the first few months of infection-a time when the IFNα response is high. Differences in IFNα sensitivity mapped to HIV-1 envelope and were associated with increased envelope levels despite similar mRNA expression, suggesting a post-transcriptional mechanism. These studies highlight critical differences in IFNα sensitivity between HIV-1 sequences in infected people and those used in SHIV models.

Unlabelled: To improve our understanding of the similarities and differences between neutralizing antibodies elicited by simian-human immunodeficiency virus (SHIV)-infected rhesus macaques and human immunodeficiency virus type 1 (HIV-1)-infected humans, we examined the plasma of 13 viremic macaques infected with SHIVSF162P3Nand 85 HIV-1-infected humans with known times of infection. We identified 5 macaques (38%) from 1 to 2 years postinfection (p.i.) with broadly neutralizing antibodies (bnAbs) against tier 2 HIV-1. In comparison, only 2 out of 42 (5%) human plasma samples collected in a similar time frame of 1 to 3 years p.i. exhibited comparable neutralizing breadths and potencies, with the number increasing to 7 out of 21 (30%) after 3 years p.i. Plasma mapping with monomeric gp120 identified only 2 out of 9 humans and 2 out of 4 macaques that contained gp120-reactive neutralizing antibodies, indicating distinct specificities in these plasma samples, with most of them recognizing the envelope trimer (including gp41) rather than the gp120 monomer. Indeed, a total of 20 gp120-directed monoclonal antibodies (MAbs) isolated from a human subject (AD358) and a Chinese rhesus macaque (GB40) displayed no or limited neutralizing activity against tier 2 strains. These isolated MAbs, mapped to the CD4-binding site, the V3 loop, the inner domain, and the C5 region of gp120, revealed genetic similarity between the human and macaque immunoglobulin genes used to encode some V3-directed MAbs. These results also support the use of envelope trimer probes for efficient isolation of HIV-1 bnAbs., Importance: HIV-1 vaccine research can benefit from understanding the development of broadly neutralizing antibodies (bnAbs) in rhesus macaques, commonly used to assess vaccine immunogenicity and efficacy. Here, we examined 85 HIV-1-infected humans and 13 SHIVSF162P3N-infected macaques for bnAbs and found that, similar to HIV-1-infected humans, bnAbs in SHIV-infected macaques are also rare, but their development might have been faster in some of the studied macaques. Plasma mapping with monomeric gp120 indicated that most bnAbs bind to the envelope trimer rather than the gp120 monomer. In support of this, none of the isolated gp120-reactive monoclonal antibodies (MAbs) displayed the neutralization breadth observed in the corresponding plasma. However, the MAb sequences revealed similarity between human and macaque genes used to encode some V3-directed MAbs. Our study sheds light on the timing and development of bnAbs in SHIV-infected macaques in comparison to HIV-1-infected humans and highlights the use of envelope trimer probes for efficient recovery of bnAbs., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objective: Gender differences in immune response and the rate of disease progression in HIV-infected individuals have been reported but the underlying mechanism remains unclear, in part because of the lack of relevant animal models. Here, we report a novel nonhuman primate model for investigation of sex disparity in HIV disease progression., Design/methods: Viral load and rate of disease progression were evaluated in rhesus macaques infected intrarectally with lineage-related subtype C R5 simian HIVs. Cytokine/chemokine levels in rectal swab eluates, and bacterial species adherent to the swabs and in the feces were determined., Results: Simian HIV-infected female rhesus macaques progressed faster to AIDS than male macaques, recapitulating the sex bias in HIV-1 disease in humans. There were no significant differences in the levels of soluble immune mediators in the rectal mucosa of naive female and male macaques. However, an exploratory longitudinal study in six infected macaques indicates that the female macaques mounted an earlier and more robust proinflammatory skewed rectal immune response to infection. Moreover, expansion of Proteobacteria that increase in other intestinal inflammatory disorders was significantly higher in the rectal mucosa of female than male macaques during acute infection., Conclusion: These findings suggest that sex differences in local innate immune activation and compositional shifts in the gut microbiota could be the drivers of increased disease susceptibility in female macaques. Further studies with this novel nonhuman primate model of HIV infection could lead to innovative research on gender differences, which may have important therapeutic implications for controlling disease in infected men as well as women.

Long-acting GSK1265744 (GSK744 LA) is a strand transfer inhibitor of the HIV/SIV (simian immunodeficiency virus) integrase and was shown to be an effective preexposure prophylaxis (PrEP) agent in a low-dose intrarectal SHIV (simian-human immunodeficiency virus) rhesus macaque challenge model. We examined the pharmacokinetics and efficacy of GSK744 LA as PrEP against repeat high-dose intravaginal SHIV challenge in female rhesus macaques treated with Depo-Provera (depot medroxyprogesterone acetate), which promotes viral transmission vaginally. When Depo-Provera-treated female rhesus macaques were dosed with GSK744 LA (50 mg/kg) monthly, systemic and tissue drug concentrations were lower than previously observed in male rhesus macaques. GSK744 concentrations were fivefold lower on average in cervical tissues than in rectal tissues. Eight female rhesus macaques were treated with GSK744 LA at week 0, and four female rhesus macaques served as controls. All animals received a high-dose challenge of SHIV162P3 at week 1. No infection was detected in GSK744 LA-treated rhesus macaques, whereas viremia was detected 1 to 2 weeks after SHIV challenge in all control animals. The GSK744 LA-treated rhesus macaques were given a second administration of drug at week 4 and further challenged at weeks 5 and 7. GSK744 LA treatment protected six of eight female rhesus macaques against three high-dose SHIV challenges, whereas all control animals became infected after the first challenge (P = 0.0003, log-rank test). These results support further clinical development of GSK744 LA for PrEP., (Copyright © 2015, American Association for the Advancement of Science.)

Unlabelled: Human immunodeficiency virus type 1 (HIV-1) infection in the central nervous system (CNS) is characterized by replication in macrophages or brain microglia that express low levels of the CD4 receptor and is the cause of HIV-associated dementia and related cognitive and motor disorders that affect 20 to 30% of treatment-naive patients with AIDS. Independent viral envelope evolution in the brain has been reported, with the need for robust replication in resident CD4(low) cells, as well as CD4-negative cells, such as astrocytes, proposed as a major selective pressure. We previously reported giant-cell encephalitis in subtype B and C R5 simian-human immunodeficiency virus (SHIV)-infected macaques (SHIV-induced encephalitis [SHIVE]) that experienced very high chronic viral loads and progressed rapidly to AIDS, with varying degrees of macrophage or microglia infection and activation of these immune cells, as well as astrocytes, in the CNS. In this study, we characterized envelopes (Env) amplified from the brains of subtype B and C R5 SHIVE macaques. We obtained data in support of an association between severe neuropathological changes, robust macrophage and microglia infection, and evolution to CD4 independence. Moreover, the degree of Env CD4 independence appeared to correlate with the extent of astrocyte infection in vivo. These findings further our knowledge of the CNS viral population phenotypes that are associated with the severity of HIV/SHIV-induced neurological injury and improve our understanding of the mechanism of HIV-1 cellular tropism and persistence in the brain., Importance: Human immunodeficiency virus type 1 (HIV-1) infection of astrocytes in the brain has been suggested to be important in HIV persistence and neuropathogenesis but has not been definitively demonstrated in an animal model of HIV-induced encephalitis (HIVE). Here, we describe a new nonhuman primate (NHP) model of R5 simian-human immunodeficiency virus (SHIV)-induced encephalitis (SHIVE) with several classical HIVE features that include astrocyte infection. We further show an association between severe neuropathological changes, robust resident microglia infection, and evolution to CD4 independence of viruses in the central nervous system (CNS), with expansion to infection of truly CD4-negative cells in vivo. These findings support the use of the R5 SHIVE models to study the contribution of the HIV envelope and viral clades to neurovirulence and residual virus replication in the CNS, providing information that should guide efforts to eradicate HIV from the body., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Severe genetic bottleneck occurs during HIV-1 sexual transmission whereby most infections are initiated by a single transmitted/founder (T/F) virus. Similar observations had been made in nonhuman primates exposed mucosally to SIV/SHIV. We previously reported variable clinical outcome in rhesus macaques inoculated intravaginally (ivg) with a high dose of R5 SHIVSF162P3N. Given the potential contributions of viral diversity to HIV-1 persistence and AIDS pathogenesis and recombination between retroviral genomes increases the genetic diversity, we tested the hypothesis that transmission of multiple variants contributes to heightened levels of virus replication and faster disease progression in the SHIVSF162P3N ivg-infected monkeys., Results: We found that the differences in viral replication and disease progression between the transiently viremic (TV; n = 2), chronically-infected (CP; n = 8) and rapid progressor (RP; n = 4) ivg-infected macaques cannot be explained by which variant in the inoculum was infecting the animal. Rather, transmission of a single variant was observed in both TV rhesus, with 1-2 T/F viruses found in the CPs and 2-4 in all four RP macaques. Moreover, the genetic relatedness of the T/F viruses in the CP monkeys with multivariant transmission was greater than that seen in the RPs. Biological characterization of a subset of T/F envelopes from chronic and rapid progressors revealed differences in their ability to mediate entry into monocyte-derived macrophages, with enhanced macrophage tropism observed in the former as compared to the latter., Conclusion: Our study supports the tenet that sequence diversity of the infecting virus contributes to higher steady-state levels of HIV-1 virus replication and faster disease progression and highlights the role of macrophage tropism in HIV-1 transmission and persistence.

GSK1265744 (GSK744) is an integrase strand-transfer inhibitor that has been formulated as a long-acting (LA) injectable suitable for monthly to quarterly clinical administration. GSK744 LA was administered at two time points 4 weeks apart beginning 1 week before virus administration, and macaques were challenged weekly for 8 weeks. GSK744 LA, at plasma concentrations achievable with quarterly injections in humans, protected all animals against repeated low-dose challenges. In a second experiment, macaques were given GSK744 LA 1 week before virus administration and challenged repeatedly until infection occurred. Protection decreased over time and correlated with the plasma drug levels. With a quarterly dosing schedule in humans, our results suggest that GSK744 LA could potentially decrease adherence problems associated with daily preexposure prophylaxis (PrEP).

Neurocognitive disorders such as dementia and cognitive/motor impairments are among the most significant complications associated with human immunodeficiency virus (HIV) infection, especially in aging populations, yet the pathogenesis remains poorly understood. Activated macrophages and microglia in white matter along with the hallmark multinucleated giant cells are prominent features of HIV encephalitis (HIVE) and of several simian immunodeficiency virus (SIV) models. While infected microglia have been demonstrated in HIVE, this feature is not routinely seen in experimental infections in rhesus macaques using SIV or chimeric simian/HIV (SHIV) strains, limiting utility in HIV-1 pathogenesis and treatment studies. Here, 50 rhesus macaques were inoculated with the CCR5 (R5)-tropic SHIVSF162P3N virus by one of three routes: intravenously (n = 9), intrarectally (n = 17), or intravaginally (n = 24). Forty-three monkeys became viremic, 26 developed AIDS, and 7 (7/26, 27 %) developed giant cell SIV encephalitis (SIVE). Rapid progressor phenotype was evident in five of seven (71 %) macaques with SIVE, and expansion to utilize the CXCR4 coreceptor (X4 coreceptor switch) was observed in four out of seven (57 %). SIVE lesions were present in gray and white matter in the cerebrum, cerebellum, thalamus, and brain stem of affected animals. Lesions were composed of virally infected CD68(+), CD163(+), and HLA-DR(+) macrophages accompanied by white matter damage, necrosis, and astroglial and microglial activation. Importantly, microglial infection was observed, which makes R5 SHIVSF162P3N infection of macaques an attractive animal model not only to study transmission and HIVE pathogenesis but also to conduct preclinical evaluation of therapeutic interventions aimed at eradicating HIV-1 from the central nervous system (CNS).

Most human immunodeficiency virus (HIV) transmissions are initiated with CCR5 (R5)-using viruses across mucosal surfaces, with the majority in regions where HIV type 1 (HIV-1) clade C predominates. Mucosally transmissible, highly replication competent, pathogenic R5 simian-human immunodeficiency viruses (SHIVs) encoding biologically relevant clade C envelopes are therefore needed as challenge viruses in vaccine efficacy studies with nonhuman primates. Here we describe the generation of three lineage-related subtype C SHIVs through four successive rapid transfers in rhesus macaques of SHIVC109F.PB4, a molecular clone expressing the soluble-CD4 (sCD4)-sensitive CCR5-tropic clade C envelope of a recently infected subject in Zambia. The viruses differed in their monkey passage histories and neutralization sensitivities but remained R5 tropic. SHIVC109P3 and SHIVC109P3N were recovered from a passage-3 rapid-progressor animal during chronic infection (24 weeks postinfection [wpi]) and at end-stage disease (34 wpi), respectively, and are classified as tier 1B strains, whereas SHIVC109P4 was recovered from a passage-4 normal-progressor macaque at 22 wpi and is a tier 2 virus, more difficult to neutralize. All three viruses were transmitted efficiently via intrarectal inoculation, reaching peak viral loads of 10(7) to 10(9) RNA copies/ml plasma and establishing viremia at various set points. Notably, one of seven (GC98) and two of six (CL31, FI08) SHIVC109P3- and SHIVC109P3N-infected macaques, respectively, progressed to AIDS, with neuropathologies observed in GC98 and FI08, as well as coreceptor switching in the latter. These findings support the use of these new SHIVC109F.PB4-derived viruses to study the immunopathology of HIV-1 clade C infection and to evaluate envelope-based AIDS vaccines in nonhuman primates.

Background: Infection of rhesus macaques (RMs) of Indian origin with simian immunodeficiency virus or simian-HIV (SHIV) provided powerful tools to study HIV-1 transmission and disease and for testing the efficacy of novel drugs, vaccines, and prevention strategies. In developing alternative nonhuman primate AIDS models for the CCR5 (R5)-tropic SHIVSF162P3N, we characterized virus transmission and infection in Chinese-origin RMs., Methods: Virologic, immunologic, and pathogenic evaluations of R5 SHIVSF162P3N infection in Chinese RMs challenged intrarectally (ir) or intravaginally were performed and compared with those previously observed in Indian-origin rhesus exposed to the same inoculum dose and via similar route., Results: R5 SHIVSF162P3N transmits efficiently across mucosal surfaces in Chinese RMs. The magnitude and kinetics of early virus dissemination after ir inoculation in the Chinese macaques were similar to those observed in Indian rhesus, but a trend toward increased SHIVSF162P3N vaginal infectivity and rapid virus spread was seen in the Chinese macaques compared with the Indian-origin animals. Once infected, however, set point viremia in the ir- and intravaginal-infected Chinese rhesus was significantly lower and the animals survived longer compared with infected Indian rhesus., Conclusions: The R5 SHIVSF162P3N/Chinese RM infection model is suitable for studies of mucosal HIV-1 transmission and protection, but the high frequency of spontaneous control of chronic viremia and reduced virulence with SHIVSF162P3N in this macaque subspecies may limit its utility in studying HIV-1 pathogenesis and in evaluating vaccines and antiretrovirals that rely on reduction in chronic viral load or AIDS development as an experimental end point.

We previously reported efficient transmission of the pathogenic R5 simian-human immunodeficiency virus SHIV(SF162P3N) isolate in Indian rhesus macaques by intravenous and intrarectal inoculations, with a switch to CXCR4 coreceptor usage in ∼50% of infected animals that progressed rapidly to disease. Since women continue to be disproportionately affected by HIV, we developed an animal model based on the intravaginal challenge of female rhesus monkeys with SHIV(SF162P3N) and sought to validate the utility of this model to study relevant aspects of HIV transmission and pathogenesis. The effect of viral dose on infection outcome was evaluated to determine the optimal conditions for the evaluation of HIV-1 preventive and therapeutic strategies. We found that the virus can successfully cross the vaginal mucosal surface to establish infection and induce disease with coreceptor switch, but with lower efficiencies compared to intravenous and rectal transmissions. In contrast to intrarectal infection, peak and cumulative viral load over a 1 year-infection period were significantly greater in macaques exposed intravaginally to lower rather than higher inoculum doses. Moreover, low and transient viremia was observed only in macaques that were challenged intravaginally twice within the same day with a high dose of virus, which can be seen as doubling the dose. Taken together, these results show that SHIV(SF162P3N) can successfully transmit across the genital mucosa, undergo coreceptor switch, and induce disease. However, the administered dose appears to impact SHIV(SF162P3N) vaginal infection outcome in an unexpected manner.

Objective: Development of an effective vaccine or topical compound to prevent HIV transmission remains a major goal for control of the AIDS pandemic. Using a nonhuman primate model of heterosexual HIV-1 transmission, we tested whether a topical microbicide that reduces viral infectivity can potentiate the efficacy of a T-cell-based HIV vaccine., Design: A DNA prime and rAd5 virus boost vaccination strategy was employed, and a topical microbicide against the HIV nucleocapsid protein was used. To rigorously test the combination hypothesis, the vaccine constructs contained only two transgenes and the topical microbicide inhibitor was used at a suboptimal dose. Vaccinees were exposed in the absence and presence of the topical microbicide to repeated vaginal R5 simian human immunodeficiency virus (SHIV)(SF162P3) challenge at an escalating dose to more closely mimic high-risk exposure of women to HIV., Methods: Infection status was determined by PCR. Antiviral immune responses were evaluated by gp120 ELISA and intracellular cytokine staining., Results: A significant delay in SHIV acquisition (log-rank test; P = 0.0416) was seen only in vaccinated macaques that were repeatedly challenged in the presence of the topical microbicide. Peak acute viremia was lower (Mann-Whitney test; P = 0.0387) and viral burden was also reduced (Mann-Whitney test; P = 0.0252) in the combination-treated animals., Conclusion: The combined use of a topical microbicide to lower the initial viral seeding/spread and a T-cell-based vaccine to immunologically contain the early virological events of mucosal transmission holds promise as a preventive approach to control the spread of the AIDS epidemic.

We recently described a coreceptor switch in rapid progressor (RP) R5 simian-human immunodeficiency virus SF162P3N (SHIV(SF162P3N))-infected rhesus macaques that had high virus replication and undetectable or weak and transient antiviral antibody response (S. H. Ho et al., J. Virol. 81:8621-8633, 2007; S. H. Ho, N. Trunova, A. Gettie, J. Blanchard, and C. Cheng-Mayer, J. Virol. 82:5653-5656, 2008; and W. Ren et al., J. Virol. 84:340-351, 2010). The lack of antibody selective pressure, together with the observation that the emerging X4 variants were neutralization sensitive, suggested that the absence or weakening of the virus-specific humoral immune response could be an environmental factor fostering coreceptor switching in vivo. To test this possibility, we treated four macaques with 50 mg/kg of body weight of the anti-CD20 antibody rituximab every 2 to 3 weeks starting from the week prior to intravenous infection with SHIV(SF162P3N) for a total of six infusions. Rituximab treatment successfully depleted peripheral and lymphoid CD20(+) cells for up to 25 weeks according to flow cytometry and immunohistochemical staining, with partial to full recovery in two of the four treated monkeys thereafter. Three of the four treated macaques failed to mount a detectable anti-SHIV antibody response, while the response was delayed in the remaining animal. The three seronegative macaques progressed to disease, but in none of them could the presence of X4 variants be demonstrated by V3 sequence and tropism analyses. Furthermore, viruses did not evolve early in these diseased macaques to be more soluble CD4 sensitive. These results demonstrate that the absence or diminution of humoral immune responses by itself is insufficient to drive the R5-to-X4 switch and the neutralization susceptibility of the evolving viruses.

A change in coreceptor preference from CCR5 to CXCR4 towards the end stage disease in some HIV-1 infected individuals has been well documented, but the reasons and mechanisms for this tropism switch remain elusive. It has been suggested that envelope structural constraints in accommodating amino acid changes required for CXCR4 usage is an obstacle to tropism switch, limiting the rate and pathways available for HIV-1 coreceptor switching. The present study was initiated in two R5 SHIV(SF162P3N)-infected rapid progressor macaques with coreceptor switch to test the hypothesis that an early step in the evolution of tropism switch is the adoption of a less constrained and more "open" envelope conformation for better CD4 usage, allowing greater structural flexibility to accommodate further mutational changes that confer CXCR4 utilization. We show that, prior to the time of coreceptor switch, R5 viruses in both macaques evolved to become increasingly sCD4-sensitive, suggestive of enhanced exposure of the CD4 binding site and an "open" envelope conformation, and this correlated with better gp120 binding to CD4 and with more efficient infection of CD4(low) cells such as primary macrophages. Moreover, significant changes in neutralization sensitivity to agents and antibodies directed against functional domains of gp120 and gp41 were seen for R5 viruses close to the time of X4 emergence, consistent with global changes in envelope configuration and structural plasticity. These observations in a simian model of R5-to-X4 evolution provide a mechanistic basis for the HIV-1 coreceptor switch.

Fitness disadvantage of the transitional intermediates compared to the initial R5 viruses has been suggested to constitute one of the blockades to coreceptor switching, explaining the late appearance of X4 viruses. Using a simian model for human immunodeficiency virus type 1 (HIV-1) coreceptor switching, we demonstrate in this study that similar molecular evolutionary pathways to coreceptor switch occur in more than one R5 simian/human immunodeficiency virus (SHIV)(SF162P3N)-infected macaque. In infected animals where multiple pathways for expansion or switch to CXCR4 coexist, fitness of the transitional intermediates in coreceptor usage efficiency influences their outgrowth and representation in the infecting virus population. Dualtropic and X4 viruses appear at different disease stages, but they have lower entry efficiency than the coexisting R5 strains, which may explain why they do not outcompete the R5 viruses. Similar observations were made in two infected macaques with coreceptor switch, providing in vivo evidence that fitness disadvantage is an obstacle to X4 emergence and expansion.

We previously reported coreceptor switch in rhesus macaques inoculated intravenously with R5 simian-human immunodeficiency virus SF162P3N (SHIV(SF162P3N)). Whether R5-to-X4 virus evolution occurs in mucosally infected animals and in which anatomic site the switch occurs, however, were not addressed. We herein report a change in coreceptor preference in macaques infected intrarectally with SHIV(SF162P3N). The switch occurred in infected animals with high levels of virus replication and undetectable antiviral antibody response and required sequence changes in the V3 loop of the gp120 envelope protein. X4 virus emergence was associated with an accelerated drop in peripheral CD4(+) T-cell count but followed rather than preceded the onset of CD4(+) T-cell loss. The conditions, genotypic requirements, and patterns of coreceptor switch in intrarectally infected animals were thus remarkably consistent with those found in macaques infected intravenously. They also overlapped with those reported for humans, suggestive of a common mechanism for coreceptor switch in the two hosts. Furthermore, two independent R5-to-X4 evolutionary pathways were identified in one infected animal, giving rise to dual-tropic and X4 viruses which differed in switch kinetics and tissue localization. The dual-tropic switch event predominated early, and the virus established infection in multiple tissues sites. In contrast, the switch to X4 virus occurred later, initiating and expanding mainly in peripheral lymph nodes. These findings help define R5 SHIV(SF162P3N) infection of rhesus macaques as a model to study the mechanistic basis, dynamics, and sites of HIV-1 coreceptor switch.

Here, we report that the S-acyl-2-mercaptobenzamide thioester (SAMT) class of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NCp7) inhibitors was able to prevent transmission of HIV-1 from infected cells, including primary cells. Furthermore, when SAMTs were introduced during an HIV-1 challenge of cervical explant tissue, inhibition of dissemination of infectious virus by cells emigrating from the tissue explants was observed. Preliminary studies using a rhesus macaque vaginal challenge model with mixed R5 and X4 simian-human immunodeficiency virus infection found that five of six monkeys were completely protected, with the remaining animal being partially protected, infected only by the R5 virus. These data suggest that SAMTs may be promising new drug candidates for further development in anti-HIV-1 topical microbicide applications.

The human immunodeficiency virus (HIV) enters target cells via interaction of the viral glycoprotein with the cellular receptor CD4 and two principal coreceptors, CCR5 (R5 viruses) and CXCR4 (X4 viruses). Most HIV-1 transmissions result in a predominantly R5 virus infection. With time, X4 variants arise and coexist with R5 virus variants in approximately 50% of subtype B infected individuals. The underlying basis for virus coreceptor switch late in infection remains an enigma, but will be important to understand given that the appearance of X4 virus in HIV-1 infected patients inevitably heralds an unfavorable clinical outcome. Recently, emergence of X4 viruses was observed in rhesus macaques experimentally infected with a CCR5-tropic simian-human immunodeficiency virus (SHIV) with progression to disease, providing some insights into the process of coreceptor switching in vivo. Further studies in this animal model should enhance our understanding of the mechanistic basis for, and obstacles to, coreceptor switch.

To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIV(SF162P3N)-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIV(SF162P3N) and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.

We report here a second case of coreceptor switch in R5 simian-human immunodeficiency virus SF162P3N (SHIV(SF162P3N))-infected macaque CA28, supporting the use of this experimental system to examine factors that drive the change in coreceptor preference in vivo. Virus recovered from CA28 plasma (SHIV(CA28NP)) used both CCR5 and CXCR4 for entry, but the virus recovered from lymph node (SHIV(CA28NL)) used CXCR4 almost exclusively. Sequence and functional analyses showed that mutations in the V3 loop that conferred CXCR4 usage in macaque CA28 differed from those described in the previously reported case, demonstrating divergent mutational pathways for change in the coreceptor preference of the R5 SHIV(SF162P3N) isolate in vivo.

Intravaginal inoculation of rhesus macaques with varying doses of the CXCR4 (X4)-tropic SHIV(SF33A) isolate revealed a threshold inoculum for establishment of systemic virus infection and a dose dependency in overall viral burden and CD4+ T cell depletion. While exposure to inoculum size of 1000 or greater 50% tissue infectious dose (TCID(50)) resulted in high viremia and precipitous CD4+ T cell loss, occult infection was observed in seven of eight macaques exposed to 500 TCID(50) of the same virus. The latter was characterized by intermittent detection of low level virus with no evidence of seroconversion or CD4+ T cell decline, but with signs of an ongoing antiviral T cell immune response. Upon vaginal re-challenge with the same limiting dose 11-12 weeks after the first, classic pathogenic X4 SHIV(SF33A) infection was established in four of the seven previously exposed seronegative macaques, implying enhanced susceptibility to systemic infection with prior exposure. Pre-existing peripheral SIV gag-specific CD4+ T cells were more readily demonstrable in macaques that became systemically infected following re-exposure than those that were not. In contrast, early presence of circulating polyfunctional cytokine secreting CD8+ T cells or strong virus-specific proliferative responses in draining lymph nodes and in the gut associated lymphoid tissue (GALT) following the first exposure was associated with protection from systemic re-infection. These studies identify the gut and lymphoid tissues proximal to the genital tract as sites of robust CD8 T lymphocyte responses that contribute to containment of virus spread following vaginal transmission.

The basis for the switch from CCR5 to CXCR4 coreceptor usage seen in approximately 50% of human immunodeficiency virus type 1 (HIV-1) subtype B-infected individuals as disease advances is not well understood. Among the reasons proposed are target cell limitation and better immune recognition of the CXCR4 (X4)-tropic compared to the CCR5 (R5)-tropic virus. We document here X4 virus emergence in a rhesus macaque (RM) infected with R5-tropic simian/human immunodeficiency virus, demonstrating that coreceptor switch can happen in a nonhuman primate model of HIV/AIDS. The switch to CXCR4 usage in RM requires envelope sequence changes in the V3 loop that are similar to those found in humans, suggesting that the R5-to-X4 evolution pathways in the two hosts overlap. Interestingly, compared to the inoculating R5 virus, the emerging CXCR4-using virus is highly neutralization sensitive. This finding, coupled with the observation of X4 evolution and appearance in an animal with undetectable circulating virus-specific antibody and low cellular immune responses, lends further support to an inhibitory role of antiviral immunity in HIV-1 coreceptor switch.

We examined the effect of inoculum dose on SHIV transmission and infection. We found that repeated low-dose intravaginal exposure with either R5-SHIV(SF162P3) or X4-SHIV(SF33A) results in infections that are blunted and rapidly controlled. Interestingly, although the transmission rate after all repeated exposures is comparable for the two viruses, the probability of low-dose vaginal transmission is greater for the X4 than R5 virus. Furthermore, X4-SHIV(SF33A) replication predominates in low-dose dually-exposed macaques, suggesting that it is better at establishing a systemic infection following transmission. However, X4-SHIV(SF33A) advantage in transmission and infection is not observed in macaques inoculated intravenously with low-dose mixed inoculum. The finding that although matched in tissue culture infectious dose, the X4 inoculum is more complex leads us to hypothesize that the greater genetic heterogeneity of the X4 virus population may have rendered it less susceptible to the severe bottleneck effects imposed by IVAG inoculation with small doses, allowing for greater probability of transmission and establishment of a generalized infection. These data have implications for HIV-1 transmission and infection in humans.

Nine rhesus macaques in groups of three received a single dose of the injectable progestin-based contraceptive Depo-Provera 5 weeks prior to challenge intravaginally with varying doses of a mixture of the pathogenic CXCR4 (X4)-SHIV(SF33A) and CCR5 (R5)-SHIV(SF162P3) isolates. As controls, seven Depo-naive animals were inoculated once with a high-dose of the mixed inoculum. Irrespective of inoculum dose, acute viremia was higher in the Depo-treated than in the Depo-naive animals. Further, genetic complexity of the replicating virus was greater and replication of the X4 virus was favored in dually infected animals treated with Depo-Provera. Analysis of cellular immune responses revealed slower response rates in virus-specific IFN-gamma production to SIV Gag in the Depo-treated macaques. The immunosuppressive effect of Depo-Provera on mounting an antiviral cellular immune response may account for the increase viral burden and diversity, and the predominance of X4 virus replication in SHIV infected macaques that were administered the progestin-based contraceptive.

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

We previously reported the derivation of a CCR5 (R5)-tropic pathogenic strain SHIVSF162P3. Here, we show that a simian-HIV (SHIV) molecular clone expressing the entire env gp160 of SHIVSF162P3, termed SHIV P3gp160, could fully recapitulate the in vivo replicative characteristics of the parental isolate. SHIV P3gp160 is mucosally transmissible, preferentially depletes memory CD4 T cells, and induced simian AIDS in 2 of 6 infected macaques. The availability of an infectious R5 SHIV molecular clone that can be transmitted mucosally and causes disease provides an important reagent for studies of lentiviral pathogenesis and AIDS vaccine research.

Objectives: To assess the safety and distribution of a cellulose acetate 1,2-benzenedicarboxylate (CAP) gel formulation in rhesus macaques as part of the development process for its use as a vaginally administered product in humans., Design: The similarities between the reproductive physiology, anatomy and vaginal microflora of human and non-human primates makes non-human primates a relevant animal model to assess the safety and distribution of candidate anti-HIV microbicides., Methods: CAP gel was instilled once or once daily for 4 days into the vaginal vault of rhesus macaques. Colposcopy and magnetic resonance imaging were performed to detect adverse effects and spread of CAP, respectively. Additionally, vaginal pH and composition of the vaginal micorflora in macaques before, during and after CAP instillations were determined, and vaginal biopsies obtained following repeated CAP exposures were examined to further document its safety., Results: CAP is safe for repeated use and exhibits a favorable distribution profile, showing no evidence of penetration into cells that line the vaginal epithelium. Further, the presence of CAP has no adverse effect on vaginal pH or the composition of the vaginal microflora, and does not induce vaginal epithelial thinning or inflammation., Conclusions: CAP gel shows minimal toxicity in vivo, supporting its use as a candidate vaginal microbicide in humans.

Objectives: To evaluate the protective efficacy of cellulose acetate 1,2-benzenedicarboxylate (CAP) formulated in a glycerol-based gel against infection with CXCR4 (X4) and CCR5 (R5) viruses in the simian/human immunodeficiency virus (SHIV)/rhesus macaque model of HIV-1 transmission., Design: Mucosal infection of non-human primates is a reasonable model for use in the investigation of HIV-1 intervention strategies., Methods: Rhesus macaques treated with Depo-Provera 5 weeks prior to challenge were inoculated intravaginally twice, over a period of 6 h with mixed inocula of pathogenic X4- and R5-SHIV in the presence or absence of CAP. Plasma viral load, peripheral and mucosal CD4 T cell counts as well as the genotype of the circulating virus were determined., Results: CAP protected seven of ten macaques against transmission of both X4- and R5-SHIV, reaching statistically significant values (P = 0.0256). Delayed and/or reduced virus replication, as well as blunting of peripheral and mucosal CD4 T cell loss was noted in the three macaques that were infected in the CAP treated group compared to those in the placebo group. Further, protection conferred by CAP appeared to be more effective against X4- than R5-SHIV infection., Conclusions: CAP is protective against highly permissive challenges with X4 and R5 viruses in vivo. Research on further development of this promising compound as a candidate microbicide for the prevention of sexual HIV-1 transmission is therefore warranted.

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, naïve 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.

Multiple mechanisms are used by the human immunodeficiency virus type 1 (HIV-1) to interfere with host-cell immune effector functions. The 27-kD Nef protein has been shown to down-modulate specific genes of the major histocompatibility complex class I (MHC-I) on the surface of infected primary T cells, facilitating their escape from lysis by cytolytic T lymphocytes. Macrophages, as the other major immune cell type targeted by the virus, also contribute to the transmission, persistence, and pathogenesis of HIV-1. Yet, whether Nef modulates MHC-I expression on HIV-infected primary macrophages remains unclear. Currently available infectious HIV-1 molecular clones, which express a reporter gene, only infect T cells and/or do not express Nef. To overcome these limitations, we generated macrophage-tropic green fluorescent protein (GFP)-tagged HIV-1 viruses, which express the complete viral genome, and used these to assess the expression of human leukocyte antigen (HLA)-A2 on the surface of productively infected macrophages. The reporter viral genomes were replication-competent and stable, as Nef, p24 antigen, and GFP expression could be detected by immunostaining of infected, monocyte-derived macrophages (MDM) after more than 2 months postinfection. Fluorescence-activated cell sorter analyses of infected macrophages and T cells revealed that although wild-type reporter virus infection induced a statistically significant decrease in the density of surface HLA-A2, down-regulation of HLA-A2 was not seen in cells infected with reporter viruses encoding a frameshift or a single point mutation in Nef at prolines 74P and P80. The impact of Nef on HLA-A2 surface expression in MDM was also confirmed by confocal microscopy. These results suggest that the mechanisms of HLA-A2 down-modulation are similar in primary T cells and macrophages.

It is well documented that removal of the V1V2 region or of the V2 loop alone from the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) increases the susceptibility of these viruses to neutralization by antibodies. The specific role of the V1 loop in defining the neutralization susceptibility of HIV is, however, not well documented. Our current studies indicate that although the V1V2 region is a global modulator of the HIV-1 neutralization susceptibility, the individual roles the V1 and V2 loops have in defining the neutralization susceptibility profile of HIV-1 differ and in some cases are opposite. While deletion of the V2 loop renders the virus more susceptible to neutralization by antibodies that recognize diverse epitopes, in particular certain ones located in the CD4 binding site and the V3 loop, deletion of the V1 loop renders the virus refractory to neutralization, especially by antibodies that recognize CD4-induced epitopes and certain CD4-site binding antibodies. Our current studies also indicate that the relative involvement of the V2 loop of the HIV-1 envelope during virus-cell entry appears to be envelope background dependent. As a result, although deletion of the V2 loop from the clade B, R5-tropic SF162 HIV-1 virus resulted in a virus that was replication competent, the same modification introduced on the background of two other R5-tropic isolates, SF128A (clade B) or SF170 (clade A), abrogated the ability of these envelopes to mediate virus-cell entry.

hTid-1, a human homolog of the Drosophila tumor suppressor l(2)Tid and a novel DnaJ protein, regulates the activity of nuclear factor kappaB (NF-kappaB), but its mechanism is not established. We report here that hTid-1 strongly associated with the cytoplasmic protein complex of NF-kappaB-IkappaB through direct interaction with IkappaBalpha/beta and the IKKalpha/beta subunits of the IkappaB kinase complex. These interactions resulted in suppression of the IKK activity in a J-domain-dependent fashion and led to the cytoplasmic retention and enhanced stability of IkappaB. Overexpression of hTid-1 by using recombinant baculovirus or adenovirus led to inhibition of cell proliferation and induction of apoptosis of human osteosarcoma cells regardless of the p53 expression status. Adherent cultured cells transduced with Ad.hTid-1 detached from the dish surface. Morphological changes consistent with apoptosis and cell death were evident 48 h after Ad.EGFP-hTid-1 transduction. In contrast, cells transduced with Ad.EGFP or Ad.EGFP-hTd-1DeltaN100, a mutant that has the N-terminal J domain deletion and that lost suppressive activity on IKK, continued to proliferate. Similar data were obtained with A375 human melanoma cells. Ad.EGFP or Ad.EGFP-hTd-1DeltaN100 ex vivo-transduced A375 cells injected subcutaneously into nude mice produced growing tumors, whereas Ad.EGFP-hTid-1-transduced cells did not. Collectively, the data suggest that hTid-1 represses the activity of NF-kappaB through physical and functional interactions with the IKK complex and IkappaB and, in doing so, it modulates cell growth and death.

The human immunodeficiency virus type 1 (HIV-1) Nef protein has been shown to accelerate viral growth kinetics in primary human T-lymphocytes and macrophages; however, the specific function(s) of Nef responsible for this phenotype in macrophages is unknown. To address this issue, mutants of a molecularly cloned macrophage-tropic isolate, HIV-1(SF162), were generated expressing single point mutations that abrogate the ability of Nef to interact with cellular kinases or mediate CD4 down-regulation. Infection of primary monocyte-derived macrophages (MDM) with these mutant viruses revealed that residues in the PXXP motif contribute to efficient replication. Interestingly, viruses expressing alleles of Nef defective in CD4 down-modulation activity retain wild-type levels of infectivity in single-round assays but exhibited delayed replication kinetics and grew to lower titres compared to the wild-type virus in MDM. These data suggest that efficient HIV-1 replication is dependent on the ability of Nef to interact with cellular kinases and remove CD4 from the surface of infected macrophages.

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.