Search

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.

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.

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 sub-optimal dose. Vaccinees were exposed in the absence and presence of the topical microbicide to repeated vaginal R5 SHIVSF162P3 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. Conclusions—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

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.

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.

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 SHIVSF33A 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 (TCID50) resulted in high viremia and precipitous CD4+ T cell loss, occult infection was observed in seven of eight macaques exposed to 500 TCID50 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 SHIVSF33A 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 ∼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-SHIVSF162P3 or X4-SHIVSF33A 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-SHIVSF33A replication predominates in low-dose dually-exposed macaques, suggesting that it is better at establishing a systemic infection following transmission. However, X4-SHIVSF33A 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.

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 glutathioneS-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.

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.

Multiple mechanisms are used by the hu- man immunodeficiency virus type 1 (HIV-1) to inter- fere 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. Cur- rently 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 greenfluorescent protein (GFP)-tagged HIV-1 viruses, which express the com- plete viral genome, and used these to assess the expres- sion of human leukocyte antigen (HLA)-A2 on the sur- face of productively infected macrophages. The re- porter 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 analy- ses 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 frame- shift or a single point mutation in Nef at prolines 74 P and P 80 . The impact of Nef on HLA-A2 surface expres- sion in MDM was also confirmed by confocal micros- copy. These results suggest that the mechanisms of HLA-A2 down-modulation are similar in primary T cells and macrophages. J. Leukoc. Biol. 78: 675-685; 2005.

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.

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-1SF162, 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.

We coinfected rhesus macaques with CXC chemokine receptor 4- and CC chemokine receptor 5-specific simian/human immunodeficiency viruses (SHIVs) to elucidate the basis for the early dominance of R5-tropic strains seen in HIV-infected humans. We found no intrinsic barrier to the transmission and dissemination of high-dose X4-SHIV in the dually infected macaques. In animals that maintained a viral set point, the R5 virus predominated. The time of appearance of R5 dominance coincided with the development of virus-specific immunity (3–6 weeks postinfection), suggestive of differential immune control of the two viruses. Indeed, after depletion of CD8 + T cells in the coinfected animals, X4 virus emerged, supporting the concept that differential CD8 + T cell-mediated immune control of X4- and R5-SHIV replication is responsible for the selective outgrowth of R5 viruses. These findings provide critical insights into a key question related to HIV pathogenesis and have important implications for the development and testing of antiviral vaccines and therapeutics.

The live, attenuated vaccine simian immunodeficiency virus SIVmac239Δnef efficiently protects rhesus macaques against infection with wild-type SIVmac but occasionally causes CD4 + T-cell depletion and progression to simian AIDS (SAIDS). Virus recovered from a vaccinated macaque (Rh1490) that progressed to SAIDS had acquired an additional deletion in the nef gene, resulting in a frameshift that restored the original nef open reading frame (R. I. Connor, D. C. Montefiori, J. M. Binley, J. P. Moore, S. Bonhoeffer, A. Gettie, E. A. Fenamore, K. E. Sheridan, D. D. Ho, P. J. Dailey, and P. A. Marx, J. Virol. 72:7501-7509, 1998). Intravenous inoculation of the Rh1490 viral isolate into four naive rhesus macaques induced CD4 + T-cell depletion and disease in three out of four animals within 2 years, indicating a restoration of virulence. A DNA fragment encompassing the truncated nef gene amplified from the Rh1490 isolate was inserted into the genetic backbone of SIVmac239. The resulting clone, SIVmac239-Δ2nef, expressed a Nef protein of approximately 23 kDa, while the original SIVmac239Δnef clone expressed a shorter protein of 8 kDa. The revertant form of Nef did not cause downregulation of CD4, CD3, or major histocompatibility complex class I. The infectivity of SIVmac239-Δ2nef was similar to that of SIVmac239Δnef in single-cycle assays using indicator cell lines. In contrast, SIVmac239-Δ2nef replicated more efficiently than SIVmac239Δnef in peripheral blood mononuclear cell (PBMC) cultures infected under unstimulated conditions. The p27 Gag antigen levels in SIVmac239-Δ2nef-infected cultures were still lower than those obtained with wild-type SIVmac239, consistent with a partial recovery of Nef function. The transcriptional activity of long terminal repeat (LTR)-luciferase constructs containing the nef deletions did not differ markedly from that of wild-type LTR. Introduction of a premature stop codon within Nef-Δ2 abolished the replicative advantage in PBMCs, demonstrating that the Nef-Δ2 protein, rather than the structure of the U3 region of the LTR, was responsible for the increase in viral replication. Taken together, these results show that SIV with a deletion in the nef gene can revert to virulence and that expression of a form of nef with multiple deletions may contribute to this process by increasing viral replication.

The potential role of dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) binding in human immunodeficiency virus transmission across the mucosal barrier was investigated by assessing the ability of simian-human immunodeficiency chimeric viruses (SHIVs) showing varying degrees of mucosal transmissibility to bind the DC-SIGN expressed on the surface of transfected cells. We found that gp120 of the highly transmissible, pathogenic CCR5-tropic SHIVSF162P3bound human and rhesus DC-SIGN with an efficiency threefold or greater than that of gp120 of the nonpathogenic, poorly transmissible parental SHIVSF162, and this increase in binding to the DC-SIGN of the SHIVSF162P3envelope gp120 translated into an enhancement of T-cell infection intrans. The presence of an additional glycan at the N-terminal base of the V2 loop of SHIVSF162P3gp120 compared to that of the parental virus was shown to be responsible for the increase in binding to DC-SIGN. Interestingly, this glycan also conferred escape from autologous neutralization, raising the possibility that the modification occurred as a result of immune selection. Our data suggest that more-efficient binding of envelope gp120 to DC-SIGN could be relevant to the enhanced mucosal transmissibility of SHIVSF162P3compared to that of parental SHIVSF162.

Infection of rhesus macaques with chimeric simian-human immunodeficiency viruses (SHIV) is an established model to study acquired immunodeficiency syndrome (AIDS) pathogenesis. Such a controlled system allows for detailedanalysis of the molecular determinants of viral pathogenesis in addition to studying host-specific immune responses that modulate disease progression. Furthermore, the use of a pathogenic molecular clone affords the opportunity to study both viral evolution within a host and to examine the generation of tissue specific variants. In this report we describe viral diversification within tissues of two rhesus macaques infected intravenously with the CXCR4-specific molecular clone SHIV S F 3 3 A 2 . Heteroduplex tracking analysis (HTA) was used to determine the complexity of viral DNA within distinct lymphoid tissues. Not surprising, heterogeneity of the proviral quasispecies in tissues obtained during the acute infection was limited. However, tissues obtained at necropsy harbored a more diverse and often different population of env variants. As the inoculating virus is a molecular clone, the variants generated are likely due to the presence of tissue specific selective forces rather than a founder's effect.

Simian-human immunodeficiency viruses (SHIVs) are chimeric viruses engineered to test the function or immunogenicity of human immunodeficiency virus (HIV) gene products in simian models. Frequently used constructs contain an HIV type 1 (HIV-1) DNA fragment containing the tat, rev, vpu, and env genes inserted in the proviral genome of SIVmac239, a molecular clone that causes simian AIDS in rhesus macaques (3, 11, 22, 28, 35, 37, 47, 49). SHIVs play a key role in the development of vaccines that are directed against the HIV-1 envelope glycoprotein and are also valuable tools in the understanding of AIDS pathogenesis (41, 42). Comparative studies of SHIVs that use CXCR4 and the CCR5 coreceptor (X4 and R5 SHIVs, respectively) have revealed the role of tropism in the rate and localization of CD4+ T-cell depletion (19). Pathogenic X4 SHIVs typically cause a severe depletion of CD4+ T lymphocytes in blood and peripheral lymph nodes within 1 month of infection (14, 21, 27, 36). Pathogenic isolates of dualtropic R5X4 SHIVs also cause rapid CD4+ T-cell depletion (29, 46, 50), while pathogenic R5 SHIVs cause a more protracted decrease of peripheral CD4+ T cells similar to that seen in SIVmac infection (18). Rhesus macaques chronically infected with pathogenic X4 or R5X4 SHIVs often maintain a high viral load in spite of an almost complete depletion of CD4+ T lymphocytes, a finding that may be explained by the productive infection of activated macrophages (25). SHIV constructs do not readily induce disease in rhesus macaques and require in vivo adaptation and often serial in vivo passages to acquire the capacity to replicate at high levels, deplete CD4+ T cells, and induce simian AIDS. Most pathogenic SHIV isolates, such as SHIVKU-1, SHIV-89.6P, and SHIV162P3, have been recovered after serial transfer of bone marrow or of blood from infected animals (19, 28, 46). Administration of an anti-CD8 monoclonal antibody (MAb) during primary infection with clone SHIVDH-12 resulted in CD4+ T-cell depletion in one rhesus macaque, indicating that immunosuppression could favor the emergence of pathogenic SHIVs (26). SHIVSF33 differs from other isolates in that it evolved into a pathogenic virus without serial in vivo passages or immunosuppressive treatment. The original virus replicated to intermediate levels in rhesus macaques and did not cause CD4+ T-cell depletion, except in one juvenile animal which developed signs of simian AIDS 2 years after inoculation. The isolate recovered from this animal, designated SHIVSF33A (A, adapted), replicated to high titers, induced a rapid and profound depletion of peripheral CD4+ T cells, and caused an AIDS-like syndrome when passaged intravenously in rhesus macaques (36, 37). In addition, SHIVSF33A could be transmitted by the vaginal route even though it used exclusively the CXCR4 coreceptor for entry (21). Comparison of original SHIV constructs with their in vivo-adapted counterparts provides a system to map the viral determinants of SHIV virulence (29, 56). Genetic analyses of pathogenic SHIV molecular clones have demonstrated that virulence is primarily modulated by adaptive changes in the two subunits of the envelope glycoprotein (2, 15, 16, 30, 33). We showed previously that, in the context of the SHIVSF33 genome, replacing the V1-to-V5 region of the env gene with corresponding sequences amplified from SHIVSF33A-infected animals was sufficient to confer pathogenicity (20). The extent of CD4+ T-cell depletion and the disease course in animals infected with recombinant molecular clone SHIVSF33A2 were similar to those caused by the SHIVSF33A isolate, demonstrating that pathogenic determinants mapped to the V1-to-V5 region of gp120. The aim of the present study was to characterize the properties conferred by the envelope glycoprotein of pathogenic SHIVSF33A molecular clones. We found that changes in gp120 were responsible for increases in fusogenicity, cytopathicity, replication capacity, and neutralization resistance of SHIVSF33A and thus conferred a set of properties that could account for the pathogenic phenotype observed in vivo. Furthermore, a conserved CD4-induced epitope that overlaps with the coreceptor binding site was spontaneously exposed at the surfaces of SHIVSF33A virions, even in the absence of CD4 binding. This finding provided a structural basis for the enhanced fusogenicity and replication capacity of pathogenic SHIVSF33A.

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.

Keeping HIV at Bay? Preexposure prophylaxis involving daily doses of drugs can, with variable success rates, interrupt HIV transmission for individuals at high risk of acquiring HIV infection. However, one reason for the variability seen in the response to such drugs is a lack of adherence to the prescribed regimen. Andrews et al. (p. 1151 ) formulated a potent integrase inhibitor as a long-acting agent that protected macaques from repeated intrarectal challenges of simian HIV. Decay of plasma levels of drug were associated with increased susceptibility to infection after virus exposure. The drug levels required for a high degree of protection could potentially be achieved with quarterly injections in humans.

Infection of rhesus macaques with chimeric simian-human immunodeficiency viruses (SHIV) is an established method to study AIDS pathogenesis and is increasingly used to assess the efficacy of vaccine and antiviral candidates. For these reasons, a detailed understanding of those molecular determinants, which confer pathogenic potential to SHIV viruses, should assist in both rational experimental design and interpretation of results. In this report, we describe the development and in vivo characterization of a pathogenic molecular clone, SHIV SF33A2 , which contains an envelope sequence derived from the CXCR4-dependent isolate, HIV-1 SF33 . Proviral DNA, amplified from a rhesus macaque infected with the pathogenic isolate SHIV SF33A , was substituted into the corresponding region of the parental, nonpathogenic SHIV SF33 genome creating the molecular clone SHIV SF33A2 . Coreceptor specificity of SHIV SF33A2 was determined to be CXCR4 specific. Naive rhesus macaques were productively infected after a single exposure to cell-free SHIV SF33A2 by either the intravenous (IV) or intravaginal (IVAG) routes. Animals infected with SHIV SF33A2 suffered a severe loss of peripheral CD4 + T cells and high acute plasma viremia with development of simian AIDS 9 months after inoculation. Sequence analysis identified 25 discreet amino acid changes within the V1-V5 regions of the envelope protein when compared with the nonpathogenic parental virus. These data indicate that domains within the HIV-1 envelope protein are sufficient to define pathogenic potential in the context of the SIVmac239 genome.

The chemokine receptor CCR5 is known to be a critical determinant of human immunodeficiency virus (HIV) transmission and pathogenesis in the human host. Towards the development of a macaque model to evaluate the efficacy of vaccines and therapeutics against infection with CCR5-specific viruses, and to delineate the pathogenic properties of such viruses, we constructed a chimeric simian human immunodeficiency virus, SHIV(SF162), containing the env, tat, rev, and vpu genes from HIV-1(SF162) (R5, MT/NSI) in the context of the molecular clone simian immunodeficiency virus, SIV(mac239). Virus generated from this molecular clone was used to intravenously infect two juvenile macaques, followed by three consecutive serial blood/bone marrow transfusions. Animals infected with parental SHIV(SF162) (P1) had detectable levels of viral replication (as determined by p27(gag) production) within days of infection; however, viral set-points fell below detection by Week 3. Late passage animals (P3 and P4) had a two-log increase in the level of plasma p27(gag) antigen. These results demonstrate that in vivo serial passage of the R5-specific SHIV(SF162) enhanced its replicative capacity.

We previously reported on the in vivo adaptation of an infectious molecular simian/human immunodeficiency virus (SHIV) clone, SHIV SF33 , into a pathogenic biologic viral variant, designated SHIV SF33A . In the present study, we show that SHIV SF33A is resistant to neutralization by human immunodeficiency virus (HIV) and SHIV antisera. Multiple amino acid substitutions accumulated over time throughout the env gene of SHIV SF33A ; some of them coincided with the acquisition of the neutralization resistance of the virus. Of interest are changes that resulted in the removal, repositioning, and addition of potential glycosylation sites within the V1, V2, and V3 regions of envelope gp120. To determine whether potential glycosylation changes within these principal neutralization domains of HIV type 1 formed the basis for the resistance to serum neutralization of SHIV SF33A , mutant viruses were generated on the backbone of parental SHIV SF33 and tested for their neutralization sensitivity. The mutations generated did not alter the in vitro replication kinetics or cytopathicity of the mutant viruses in T-cell lines. However, the removal of a potential glycosylation site in the V1 domain or the creation of such a site in the V3 domain did allow the virus to escape serum neutralization antibodies that recognized parental SHIV SF33 . The combination of the V1 and V3 mutations conferred an additive effect on neutralization resistance over that of the single mutations. Taken together, these data suggest that (i) SHIV variants with changes in the Env SU can be selected in vivo primarily by virtue of their ability to escape neutralizing antibody recognition and (ii) carbohydrates play an important role in conferring neutralization escape, possibly by altering the structure of envelope gp120 or by shielding principal neutralization sites.

Several studies have reported on the differential susceptibility to antibody-mediated neutralization between primary (PR) and laboratory-adapted (LA) human immunodeficiency virus type 1 (HIV-1) isolates (1, 2, 6, 23, 24, 32, 39, 40, 45), the latter being more susceptible than the former to neutralization. To a large extent, this difference is explained by a documented differential exposure of neutralization epitopes within oligomeric envelope structures of PR and LA isolates. Such epitopes are less exposed on PR isolates than LA isolates (1, 24, 36, 40). The identification of the regions that shield neutralization epitopes and the nature of the neutralization epitopes themselves on PR isolates should shed light on the mechanism(s) by which HIV evades neutralization by serum antibodies and greatly benefit the design of effective HIV envelope-based immunogens. It has been reported that simultaneous deletion of the V1 and V2 loops of the neutralization-sensitive LA isolate HIV-1IIIb increases even further its susceptibility to neutralization by certain monoclonal antibodies (MAbs) whose epitopes are located within the V3 loop, the CD4-binding site, and conserved gp120 regions (3). We previously reported that the individual deletion of the V1 (17 amino acids) and V2 (30 amino acids) loops of the PR, neutralization-resistant HIV-1 isolate SF162 (7, 35, 39) does not reduce the potential of the virus to replicate in peripheral blood mononuclear cells (PBMC) or alter the coreceptor function of the envelope protein (38). Immunochemical analysis of the two mutant envelopes revealed that the deleted V1 and V2 loop sequences differentially affect the exposure and conformation of specific gp120 epitopes. While deletion of the V2 loop affects the structure of certain complex epitopes which comprise elements of the CD4-binding site, deletion of the V1 loop alters the conformation and exposure of specific epitopes located in the V2 and V3 loops and the CD4-binding site. However, regardless of these specific structural changes, neither deletion alters the overall ability of the envelope to interact with CD4 or its ability to undergo conformational changes upon receptor binding (38). In this study, we examined whether individual partial deletion of the V1 or V2 loop exposes cryptic neutralization epitopes on the surface of SF162 virions. We show that the V2, but not the V1, loop deletion renders the virus, termed SF162ΔV2, highly susceptible to serum-mediated neutralization. Most importantly, SF162ΔV2 becomes neutralizable by sera obtained from patients infected not only with clade B HIV-1 isolates but also with clade A, C, D, E, and F HIV-1 isolates. The introduced V2 loop deletion does not destabilize the gp120-gp41 subunit association, and the virus remains as resistant to antibody-mediated gp120-virion dissociation as wild-type (wt) SF162. Based on these observations, we suggest that this particular V2 loop deletion unveils an envelope region rich in neutralization epitopes whose overall structure is conserved among PR isolates. Neutralization studies conducted with various anti-gp120 MAbs failed to identify the precise epitopes forming this region, but it appears that it comprises structural elements of the CD4-binding site and conserved regions of gp120.

Infection of macaques with chimeric simian/human immunodeficiency virus (SHIV) expressing the envelope protein of HIV-1 provides a model system for studying HIV-1 infection in humans. To this end, four rhesus macaques (Macaca mulatta) were given a single intravaginal (IVAG) inoculation of cell-free SHIVSF33A and longitudinal samples of peripheral blood and lymph nodes were analyzed for viremia, antigenemia, and various T-cell populations. Rhesus macaques infected IVAG with SHIVSF33A demonstrated a dramatic decrease in the CD4(+) PBMC subset in the initial weeks after viral exposure, a time that corresponded to peak in plasma viremia and antigenemia. Within 4 months of SHIVSF33A inoculation, partial to complete rebound of the CD4(+) PBMC was seen in these animals. Notably, the regeneration of the CD4(+) subset was associated with regeneration of the naive T-cell population and was concordant with clearance of plasma viremia. DNA heteroduplex tracking assays revealed transmission of minor variants within the SHIVSF33A inoculum to the IVAG-inoculated animals. The cell-free SHIVSF33A inoculum as well as virus isolated from animals early after transmission used the chemokine molecule CXCR4 as the primary cellular coreceptor, demonstrating that viruses expressing envelope glycoproteins of the syncytia inducing (SI) phenotype can be transported across the vaginal mucosa. Although none of the animals has yet to develop clinical symptoms of simian AIDS (SAIDS), infectious virus and viral nucleic acids could be persistently isolated from each animal. Furthermore, animals transfused with blood from IVAG-infected macaques drawn 2 weeks after inoculation suffered a more profound and sustained CD4(+) T-cell loss, persistent plasma viremia, and the development of SAIDS in one animal, indicating that IVAG-passaged SHIVSF33A was pathogenic. Taken together, these results establish that a pathogenic CXCR4-utilizing SHIVSF33A species crossed the cervicovaginal mucosa. Different courses of infection in the IVAG versus transfusion animals suggest that host-mediated responses elicited upon transmission across mucosal barriers may serve to limit viral replication and delay disease progression in the IVAG-infected animals.

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).

A T-cell-line-tropic, syncytium-inducing, sCD4- and serum neutralization-sensitive variant (R3H) of the macrophage-tropic, non-syncytium-inducing, sCD4- and serum neutralization-resistant molecular clone HIV-1SF162 was obtained by passage through the T-cell line HUT 78. Sequence analyses of the V1-V5 regions of envelope gp120 of the variant R3H revealed amino acid substitutions in the V3 (amino acids 307, 312) and V4 (amino acid 390) domains. Site-directed mutagenesis of the HIV-1SF162 genome showed that specific mutations in the V3 loop of this isolate can alter tropism and cytopathicity of the virus, but only moderately affect its sensitivity to sCD4 and serum neutralization. These results show that adaptation to growth in T-cell lines can render a primary-like virus sensitive to sCD4 and serum neutralization. However, the extent of T-cell line tropism does not correlate with the degree of susceptibility to sCD4 and serum neutralization. The latter appears to be dependent on the amino acid compositions of the V3 loop and other regions of envelope gp120, whereas the former is primarily determined by the V3 loop. Our findings further illustrate the importance of the V3 loop in influencing HIV-1 cell tropism and syncytium formation, and the interplay among V3 loop residues in maintaining a structure of the loop that influences biological phenotype of the virus.

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.

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.

Using recombinant and mutant viruses generated between two human immunodeficiency virus type 1 isolates that display differences in cell tropism and sensitivity to soluble CD4 neutralization, we show that these two properties of the virus are regulated by different mechanisms. Whereas there is an association between V3 loop conformation and a particular cellular tropism, soluble CD4 neutralization sensitivity appears to be determined by amino acid differences in the C2 domain of the envelope gp120 that modulate the stability of gp120-gp41 association. Our findings further illustrate the importance of functional interactions among different regions of the envelope gp120 in regulating the biological phenotypes of human immunodeficiency virus and suggest that additional probing of the V3 loop with monoclonal antibodies may identify specific structural features of this loop that determine cell tropism.

Objective: To determine whether the HIV-1 genomes that grow out in vitro from peripheral blood mononuclear cells (PBMC) better represent the in vivo quasispecies present in plasma or PBMC. Results: For one patient (9606), PBMC culture represented more accurately the plasma rather than the in vivo PBMC quasispecies distribution, because a large number of fat-defective proviruses present in PBMC in vivo were not detected in plasma nor in the PBMC cultures. For a second patient (9605), PBMC culture was representative of both in vivo PBMC and plasma tat sequences, but selection of C2-V3 env sequences was observed in PBMC cultures compared with sequences present in both plasma and PBMC in vivo. This selection consisted of the absence in vitro of genomes with certain amino-acid substitutions at or near conserved glycosylation sites of the C2 region at positions 276 and 289. Site 276 has been reported to be important for viral infectivity, and these substitutions may therefore have affected infectivity. In the third patient (10095), selection of both tat and C2-V3 sequences was observed in culture as compared to plasma and PBMC in vivo. In contrast to the first two patients, this third patient contained V3 sequences in vivo that were predicted to impart syncytium induction and enhanced replication capacity. It was these sequences that grew out preferentially in vitro. Conclusion: This study suggests that short-term PBMC culture is representative of HIV-1 genomes present in PBMC and plasma in vivo to the degree that they are infectious.

The first exon of tat was sequenced from 23 provirus genomes randomly amplified directly from an HIV-1-infected individual's peripheral blood. Twelve of the 23 sequences constituted a distinct subset of the quasi-species detected. This subset had in common two inactivating mutations in the tat gene. In addition, two of these defective genomes each had a unique mutation. This is the second instance of a defective early gene being present in a high percentage of the proviruses present in the PBMCs of an HIV-1-infected individual, (the first reported by Martins LP et al.: J Virol 1991;65:4502-4507), and suggests that genomes defective in an early gene can participate in the infectious spread of HIV-1 in vivo.

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.

HIV-1 strains, including a molecularly cloned isolate, that had been passaged through different cell types adapted to faster growth in the same cell type and displayed a different host cell tropism. The only change in viral proteins revealed by immunoblot analyses was the molecular size of the envelope glycoprotein gp120 that varied for viruses recovered from the different infected cells. The alteration in size was most likely the result of modification of gp120. Host range differences were also observed for a molecularly cloned HIV-1 strain when passed through the peripheral white blood cells from different individuals. Thus, this phenomenon could have clinical relevance in HIV pathogenesis.

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.

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.

We previously reported the derivation of a CCR5 (R5)-tropic pathogenic strain SHIV SF162P3 . Here, we show that a simian-HIV (SHIV) molecular clone expressing the entire env gp160 of SHIV SF62P3 , termed SHIV P3gp160, could fully recapitulate the in vivo replicative characteristics of the parental isolate. SHIV P3gpl60 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.

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) SHIVSF33A2molecular clone with the corresponding sequences from the CCR5-tropic (R5) SHIVSF162P3isolate resulted in a switch of coreceptor usage from CXCR4 to CCR5. The resultant R5 clone, designated SHIVSF33A2(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-SHIVSF33A2. 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-SHIVSF33A2(V3)led to only a modest and transient loss. However, substantial depletion of intestinal CD4+T cells was observed in R5-SHIVSF33A2(V3)-infected macaques. Moreover, naïve T cells that expressed high levels of CXCR4 were rapidly depleted in X4-SHIVSF33A2-infected macaques, whereas R5-SHIVSF33A2(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.

Through rapid serial transfer in vivo, the chimeric CCR5-tropic simian/human immunodeficiency virus SHIV SF162 evolved from a virus that is nonpathogenic and poorly transmissible across the vaginal mucosa to a variant that still maintains CCR5 usage but which is now pathogenic and establishes intravaginal infection efficiently. To determine whether envelope glycoprotein gp120 is responsible for increased pathogenesis and transmissibility of the variant SHIV SF162P3 , we cloned and sequenced the dominant envelope gene (encoding P3 gp120) and characterized its functions in vitro. Chimeric SHIV SF162 virus expressing P3 gp120 of the pathogenic variant, designated SHIV SF162PC , was also constructed and assessed for its pathogenicity and mucosal transmissibility in vivo. We found that, compared to wild-type SHIV SF162 gp120, P3 gp120 conferred in vitro neutralization resistance and increased entry efficiency of the virus but was compromised in its fusion-inducing capacity. In vivo, SHIV SF162PC infected two of two and two of three rhesus macaques by the intravenous and intravaginal routes, respectively. Nevertheless, although peak viremia reached 10 6 to 10 7 RNA copies per ml of plasma in some infected animals and was associated with depletion of gut-associated CD4 + lymphocytes, none of the animals maintained a viral set point that would be predictive of progression to disease. Together, the data from this study suggest a lack of correlation between entry efficiency and cytopathic properties of envelope glycoproteins with viral pathogenicity. Furthermore, whereas env gp120 contains the determinant for enhanced mucosal transmissibility of SHIV SF162P3 , the determinant(s) of its increased virulence may require additional sequence changes in env gp41 and/or maps to other viral genes.

A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV 162P4 . Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent cellular immune response is an important area for further study.

We generated DNA constructs expressing soluble truncated forms of the envelope of SF162, a neutralization-resistant primary human immunodeficiency virus type 1 isolate, and SF162AV2, a neutralization-susceptible virus derived from SF162 after the deletion of 30 amino acids from the V2 loop. The constructs express the entire gp120 subunit and the extracellular region of the gp41 subunit, with either the presence ("cleaved" forms, designated gp140C) or the absence ("fused" forms, designated gp140F) of the gp120-gp41 cleavage site. Both gp140 forms derived from SF162 and SF162deltaV2 are secreted in the cell medium and are recognized by the oligomer-specific anti-gp41 MAb T4. As is the case for the corresponding virion-associated envelope molecules, the CD4-binding region is occluded within both gp140F and gp140C forms. However, structural differences exist between these two forms. The gp140F proteins are less efficiently recognized than the gp140C proteins by antibodies present in the sera of HIV-infected patients with neutralizing activities against SF162 and SF162AV2. Also, the V3 loop is more exposed on gp140F than gp140C. As is the case for intact virions, on CD4 binding both the gp140F and gp140C proteins undergo conformational changes that result in the exposure of the epitope recognized by MAb 17b, which has been implicated in coreceptor binding. In contrast, during these structural changes the exposure of specific V3 loop epitopes is not increased on either gp140C or gp140F. Taken together, our data indicate that although these gp140 forms differ structurally from the native envelope, their similarities, in particular that of gp140C, outweigh their differences.

Infection of macaques with chimeric simian–human immunodeficiency virus (SHIV) provides an excellent in vivo model for examining the influence of envelope on HIV-1 pathogenesis. Infection with a pathogenic CCR5 (R5)–specific enveloped virus, SHIVSF162P, was compared with infection with the CXCR4 (X4)–specific SHIVSF33A.2. Despite comparable levels of viral replication, animals infected with the R5 and X4 SHIV had distinct pathogenic outcomes. SHIVSF162Pcaused a dramatic loss of CD4+intestinal T cells followed by a gradual depletion in peripheral CD4+T cells, whereas infection with SHIVSF33A.2caused a profound loss in peripheral T cells that was not paralleled in the intestine. These results suggest a critical role of co-receptor utilization in viral pathogenesis and provide a reliable in vivo model for preclinical examination of HIV-1 vaccines and therapeutic agents in the context of the HIV-1 envelope protein.

The macaque/SIV animal system is an important model for studying AIDS pathogenesis and for evaluating the efficacy of vaccines and anti-viral therapeutics. However, differences between HIV-1 and SIV envelope proteins exist that render the SIV/macaque model of limited value when examining envelope determinants of retroviral pathogenesis. To overcome this problem, we utilized a chimeric virus, SHIV(SF33), containing the env gene from HIV-1SF33 in the context of the molecular clone SIVmac239, in the macaque animal model. In this study SHIV(SF33A), a pathogenic virus that evolved in vivo from a rhesus macaque infected intravenously with the molecular clone SHIV(SF33) was used in both in vitro and in vivo studies. By using a cell culture system, we examined the biological properties of our parental and animal-adapted chimeric viruses and compared in vitro susceptibility to in vivo studies.

Two HIV-1 envelope mutant proteins were generated by introducing deletions in the first and second hypervariable gp120 regions (V1 and V2 loops, respectively) of a macrophage-tropic primary HIV-1 isolate, SF162, to study the effect of the deleted sequences on envelope structure, viral entry, and replication potentials. The first mutant lacked 17 amino acids of the V1 loop and the latter 30 amino acids of the V2 loop. A comparison of the immunochemical structure of the wild-type and mutant monomeric and virion-associated gp120 molecules revealed that the V1 and V2 loop deletions differentially altered the structure of the V3 loop, the CD4-binding site, and epitopes within conserved regions of gp120. Regardless of differences in structure, both mutated envelope proteins supported viral replication into peripheral blood mononuclear cells to levels comparable to those of the wild-type SF162 virus. However, they decreased the viral replication potential in macrophages, even though they did not alter the coreceptor usage of the viruses. These studies support and extend previous observations that a complex structural interaction between the V1, V2, and V3 loops and elements of the CD4-binding site of gp120 controls entry of virus into cells. The present studies, however, suggest that the effect of the V1 and V2 loops in viral entry is cell dependent.

Background The primate lentiviruses, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), encode a conserved accessory gene product, Nef. In vivo , Nef is important for the maintenance of high virus loads and progression to AIDS in SIV-infected adult rhesus macaques. In tissue culture cells expressing Nef, this viral protein interacts with a cellular serine kinase, designated Nef-associated kinase. Results This study identifies the Nef-associated kinase as a member of the p21-activated kinase (PAK) family of kinases and investigates the role of this Nef-associated kinase in vivo . Mutants of Nef that do not associate with the cellular kinase are unable to activate the PAK-related kinase in infected cells. To determine the role of cellular kinase association in viral pathogenesis, macaques were infected with SIV containing point-mutations in Nef that block PAK activation. Virus recovered at early time points after inoculation with mutant virus was found to have reverted to prototype Nef function and sequence. Reversion of the kinase-negative mutant to a kinase-positive genotype in macaques infected with the mutant virus preceded the induction of high virus loads and disease progression. Conclusions Nef associates with and activates a PAK-related kinase in lymphocytes infected in vitro . Moreover, the Nef-mediated activation of a PAK-related kinase correlates with the induction of high virus loads and the development of AIDS in the infected host. These findings reveal that there is a strong selective pressure in vivo for the interaction between Nef and the PAK-related kinase.

To elucidate the functions of human immunodeficiency virus type 1 (HIV-1) genes in a nonhuman primate model, we have constructed infectious recombinant viruses (chimeras) between the pathogenic molecular clone of simian immunodeficiency virus (SIV) SIVmac239 and molecular clones of HIV-1 that differ in phenotypic properties controlled by the env gene. HIV-1SF33 is a T-cell-line-tropic virus which induces syncytia, and HIV-1SF162 is a macrophage-tropic virus that does not induce syncytia. A DNA fragment encoding tat, rev, and env (gp160) of SIVmac239 has been replaced with the counterpart genetic region of HIV-1SF33 and HIV-1SF162 to derive chimeric recombinant simian/human immunodeficiency virus (SHIV) strains SHIVSF33 and SHIVSF162, respectively. In the acute infection stage, macaques inoculated with SHIVSF33 had levels of viremia similar to macaques infected with SIVmac239, whereas virus loads were 1/10th to 1/100th those in macaques infected with SHIVSF162. Of note is the relatively small amount of virus detected in lymph nodes of SHIVSF162-infected macaques. In the chronic infection stage, macaques infected with SHIVSF33 also showed higher virus loads than macaques infected with SHIVSF162. Virus persists for over 1 year, as demonstrated by PCR for amplification of viral DNA in all animals and by virus isolation in some animals. Antiviral antibodies, including antibodies to the HIV-1 env glycoprotein (gp160), were detected; titers of antiviral antibodies were higher in macaques infected with SHIVSF33 than in macaques infected with SHIVSF162. Although virus has persisted for over 1 year after inoculation, these animals have remained healthy with no signs of immunodeficiency. These findings demonstrate the utility of the SHIV/macaque model for analyzing HIV-1 env gene functions and for evaluating vaccines based on HIV-1 env antigens.

We have examined the influence of the V1/V2 region of the human immunodeficiency virus type 1 (HIV-1) gp120 on certain biologic properties of the virus. We observed that on the genomic background of the T-cell-line-tropic strain, HIV-1SF2mc, both the V1 and V2 domains of the macrophage-tropic strain, HIV-1SF162mc, in addition to the required V3 domain, are necessary to attain full macrophage tropism. Furthermore, the V2 domain modulates the sensitivity of HIV-1 to soluble CD4 neutralization. Structural studies of recombinant and mutant envelope glycoproteins suggest that the function of the V1/V2 region is to interact with the V3 domain and confer on the envelope gp120 of HIV-1SF2mc a conformation more similar to that of the macrophage-tropic strain HIV-1SF162mc. The conformation of the envelope gp120 appears to be strain specific and plays an important role in determining HIV-1 tissue tropism and sensitivity to soluble CD4 neutralization.

We investigated how amino acid changes within and outside the V3 loop of the envelope glycoprotein of human immunodeficiency virus type 1 influence the infectivity, host range, and syncytium-forming ability of the virus. Our studies show that on the genomic backgrounds of the human immunodeficiency virus type 1 strains SF2 and SF13, a reciprocal exchange of full-loop sequences does not alter the syncytium-forming ability of the viruses, indicating that a determinant(s) for this biological property maps outside the loop. However, specific amino acid substitutions, both within and outside the V3 loop, resulted in loss of infectivity, host range, and syncytium-forming potential of the virus. Furthermore, it appears that a functional interaction of the V3 loop with regions in the C2 domain of envelope gp120 plays a role in determining these biological properties. Structural studies of mutant glycoproteins show that the mutations introduced affect the proper association of gp120 with the transmembrane glycoprotein gp41. Our results suggest that mutations that alter the structure of the V3 loop can affect the overall conformation of gp120 and that, reciprocally, the structure of the V3 loop is influenced by the conformation of other regions of gp120. Since the changes in the replicative potential, host range, and fusogenic ability of the mutant viruses correlate well with the changes in gp120 conformation, as monitored by the association of gp120 with gp41, our results support a close relationship between envelope gp120 structural conformation and the biological phenotype of the virus.