Pathogenesis of simian immunodeficiency virus encephalitis: viral determinants of neurovirulence.

To examine the relationship between macrophage tropism and neurovirulence, macaques were inoculated with two recombinant hybrid viruses derived from the parent viruses SIVmac239, a lymphocyte-tropic, non-neurovirulent clone, and SIV/17E-Br, a macrophage-tropic, neurovirulent virus strain. The first recombinant, SIV/17E-Cl, contained the portion of the env gene that encodes the surface glycoprotein and a short segment of the transmembrane glycoprotein of SIV/17E-Br in the backbone of SIVmac239. Unlike SIVmac239, SIV/17E-Cl replicated productively in macrophages, demonstrating that sequences in the surface portion of env determine macrophage tropism. None of five macaques inoculated with SIV/17E-Cl developed simian immunodeficiency virus (SIV) encephalitis. The second recombinant, SIV/17E-Fr, which contained the entire env and nef genes and the 3' long terminal repeat of SIV/17E-Br in the SIVmac239 backbone, was also macrophage tropic. Six of nine macaques inoculated with SIV/17E-Fr developed SIV encephalitis ranging from mild to moderate in severity, indicating a significant (P = 0.031) difference in the neurovirulence of the two recombinants. In both groups of macaques, CD4+ cell counts declined gradually during infection and there was no significant difference in the rate of the decline between the two groups of macaques. This study demonstrated that macrophage tropism alone is not sufficient for the development of neurological disease. In addition, it showed that while sequences in the surface portion of the envelope gene determine macrophage tropism, additional sequences derived from the transmembrane portion of envelope and/or nef confer neurovirulence.     

Expression of simian immunodeficiency virus (SIV) nef in astrocytes during acute and terminal infection and requirement of nef for optimal replication of neurovirulent SIV in vitro

As the most numerous cells in the brain, astrocytes play a critical role in maintaining central nervous system homeostasis, and therefore, infection of astrocytes by human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) in vivo could have important consequences for the development of HIV encephalitis. In this study, we establish that astrocytes are infected in macaques during acute SIV infection (10 days postinoculation) and during terminal infection when there is evidence of SIV-induced encephalitis. Additionally, with primary adult rhesus macaque astrocytes in vitro, we demonstrate that the macrophage-tropic, neurovirulent viruses SIV/17E-Br and SIV/17E-Fr replicate efficiently in astrocytes, while the lymphocyte-tropic, nonneurovirulent virus SIV(mac)239 open-nef does not establish productive infection. Furthermore, aminoxypentane-RANTES abolishes virus replication, suggesting that these SIV strains utilize the chemokine receptor CCR5 for entry into astrocytes. Importantly, we show that SIV Nef is required for optimal replication in primary rhesus macaque astrocytes and that normalizing input virus by particle number rather than by infectivity reveals a disparity between the ability of a Nef-deficient virus and a virus encoding a nonmyristoylated form of Nef to replicate in these central nervous system cells. Since the myristoylated form of Nef has been implicated in functions such as CD4 and major histocompatibility complex I downregulation, kinase association, and enhancement of virion infectivity, these data suggest that an as yet unidentified function of Nef may exist to facilitate SIV replication in astrocytes that may have important implications for in vivo pathogenesis.     

Isolation and Characterization of a Neuropathogenic Simian Immunodeficiency Virus Derived from a Sooty Mangabey

Transfusion of blood from a simian immunodeficiency virus (SIV)- and simian T-cell lymphotropic virus-infected sooty mangabey (designated FGb) to rhesus and pig-tailed macaques resulted in the development of neurologic disease in addition to AIDS. To investigate the role of SIV in neurologic disease, virus was isolated from a lymph node of a pig-tailed macaque (designated PGm) and the cerebrospinal fluid of a rhesus macaque (designated ROn2) and passaged to additional macaques. SIV-related neuropathogenic effects were observed in 100% of the pig-tailed macaques inoculated with either virus. Lesions in these animals included extensive formation of SIV RNA-positive giant cells in the brain parenchyma and meninges. Based upon morphology, the majority of infected cells in both lymphoid and brain tissue appeared to be of macrophage lineage. The virus isolates replicated very well in pig-tailed and rhesus macaque peripheral blood mononuclear cells (PBMC) with rapid kinetics. Differential replicative abilities were observed in both PBMC and macrophage populations, with viruses growing to higher titers in pig-tailed macaque cells than in rhesus macaque cells. An infectious molecular clone of virus derived from the isolate from macaque PGm (PGm5.3) was generated and was shown to have in vitro replication characteristics similar to those of the uncloned virus stock. While molecular analyses of this virus revealed its similarity to SIV isolates from sooty mangabeys, significant amino acid differences in Env and Nef were observed. This virus should provide an excellent system for investigating the mechanism of lentivirus-induced neurologic disease.     

Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus.

The simian immunodeficiency virus (SIV) macaque model of AIDS has provided a valuable system with which to investigate vaccine approaches for protection against human immunodeficiency virus type 1 (HIV-1) infection. In particular, the ability of macaques persistently infected with attenuated infectious molecular clones of SIV to resist challenge with the pathogenic parental swarm has conclusively demonstrated that protective immunity can be achieved by immunization prior to exposure. The breadth of these protective responses and the immunological correlates of protection, however, have not been identified. In addition, vaccine studies have mainly employed lymphocyte-tropic strains of HIV-1 and SIV. Recent studies have implicated macrophage-tropic strains in the transmission of HIV-1 and have suggested that these virus strains should be examined in vaccine strategies. Macrophage-tropic viruses may confer additional advantages in the induction of protective immunity by replication in antigen-presenting cells. In this study, the immune response of rhesus macaques inoculated with an attenuated macrophage-tropic recombinant of SIVmac239 (SIV/17E-Cl) was evaluated with respect to protective immunity by heterologous challenge at various times after infection. Vigorous type-specific neutralizing-antibody responses restricted to SIV/17E-Cl were evident by 2 weeks postinfection. By 7 months, however, cross-reactive neutralizing antibodies emerged which neutralized not only SIV/17E-Cl but also the heterologous primary isolate SIV/DeltaB670. Challenge of SIV/17E-Cl-infected monkeys with SIV/DeltaB670 at various times postinfection demonstrated that protective responses were associated with the appearance of cross-reactive neutralizing antibodies. Furthermore, passive transfer of sera from SIV/17E-Cl-infected animals passively protected two of four naive recipients.     

Role of microglial cells in selective replication of simian immunodeficiency virus genotypes in the brain

An accelerated, consistent macaque simian immunodeficiency virus (SIV) model in which over 90% of pigtailed macaques (Macaca nemestrina) coinoculated with SIV/17E-Fr and SIV/DeltaB670 developed encephalitis was used to determine whether central nervous system (CNS) lesions are associated with the replication of specific genotypes in the brain and, more specifically, in the microglia. Ten of 11 inoculated macaques had severe (n = 3), moderate (n = 5), or mild (n = 2) encephalitis at 3 months postinoculation. To compare actively replicating viral genotypes in the CNS and in microglia with those in the periphery, the V1 region of the SIV envelope gene was amplified and sequenced from RNA extracted from basal ganglia, from microglial cells isolated from the brain, and from peripheral blood mononuclear cells (PBMC) isolated from blood at the time of death. To distinguish between actively replicating with latent viral genotypes in the CNS, viral genotypes in RNA and DNA from basal ganglia were compared. Two macrophage-tropic, neurovirulent viruses, SIV/17E-Fr and SIV/DeltaB670 Cl-2, predominated in the brain RNA of macaques with encephalitis, comprising 95% of the genotypes detected. The same two viral genotypes were present at the same frequencies in microglial cell RNA, suggesting that microglia are pivotal in the selective replication of neurovirulent viruses. There was a significantly greater number of viral genotypes in DNA than there were in RNA in the brain (P = 0.004), including those of both the macrophage- and lymphocyte-tropic viral strains. Furthermore, significantly fewer viral genotypes were detected in brain RNA than in PBMC RNA at the time of death (P = 0.004) and the viral strain that predominated in the brain frequently was different from that which predominated in the PBMC of the same animal. These data suggest that many viral genotypes enter the brain, but only a limited subset of macrophage-tropic, neurovirulent viruses replicate terminally in the brains of macaques with encephalitis. They further suggest that the selection of macrophage-tropic, neurovirulent viruses occurs not at the level of the blood-brain barrier but at a stage after virus entry and that microglial cells may play an important role in that selection process.     

Genotypic analysis of infant macaques infected transplacentally and orally

Abstract: The SIV-infected macaque provides an excellent model to study factors involved in maternal-fetal transmission of HIV. In our prenatal transmission studies, female macaques were inoculated intravenously during midgestation with either SIV/DeltaB670 or a combination of SIV/DeltaB670 and the macrophage-tropic molecular clone SIV/17E-Fr. The females harbored a genetically diverse virus population at parturition, whereas a single genotype from the maternal quasispecies was identified in the infants. One of two variants was transplacentally transmitted to the infants, SIV/17E-Fr or B670-Cl 12, a genotype contained within the SIV/DeltaB670 inoculum. Both of these variants have been identified in the central nervous system of macaques that have developed encephalitis and they replicate in vitro on primary rhesus macrophages. These results suggest a critical role for macrophages in fetal infection in utero. In our perinatal transmission studies we have evaluated the viral genotypes found in two newborn macaques infected orally with SIV/DeltaB670 and in one infant infected via amniotic inoculation in late gestation. More than one viral genotype was identified in each infant, moreover, each infant harbored different genotypes. These results suggest different mechanisms are responsible for viral infection via these routes.     

Genotypic selection of simian immunodeficiency virus in macaque infants infected transplacentally.

To understand viral and host factors that contribute to transplacental transmission of human immunodeficiency virus, we developed an animal model using pregnant female macaques infected with simian immunodeficiency virus (SIV). Pregnant females were inoculated intravenously during midgestation with either a well-characterized primary isolate of SIV (SIV/DeltaB670) or a combination of SIV/DeltaB670 and the macrophage-tropic molecular clone SIV/17E-Fr. The viral genetic diversity in five infected female macaques and their in utero-infected infants was analyzed. All of the mothers harbored a genetically diverse virus population at parturition, whereas a single genotype from the maternal quasispecies was identified in the infants at birth. Only one of two variants was found in the infants: SIV/17E-Fr (two cases) or a genotype contained within the SIV/DeltaB670 quasispecies (three cases). The macrophage-tropic properties of both transmitted genotypes were suggested by productive replication in primary rhesus macrophage cultures in vitro and the clonal presence in central nervous system tissue of infected monkeys with encephalitis. These observations provide compelling evidence for both genotypic and phenotypic selection in transplacental transmission of SIV and suggest a critical role for macrophages in fetal infection in utero.     

CD4-independent entry and replication of simian immunodeficiency virus in primary rhesus macaque astrocytes are regulated by the transmembrane protein

Previous studies have demonstrated that the genetic determinants of simian immunodeficiency virus (SIV) neurovirulence map to the env and nef genes. Recent studies from our laboratory demonstrated that SIV replication in primary rhesus macaque astrocyte cultures is dependent upon the nef gene. Here, we demonstrate that macrophage tropism is not sufficient for replication in astrocytes and that specific amino acids in the transmembrane (TM) portion of Env are also important for optimal SIV replication in astrocytes. Specifically, a Gly at amino acid position 751 and truncation of the cytoplasmic tail of TM are required for efficient replication in these cells. Studies using soluble CD4 demonstrated that these changes within the TM protein regulate CD4-independent, CCR5-dependent entry of virus into astrocytes. In addition, we observed that two distinct CD4-independent, neuroinvasive strains of SIV/DeltaB670 also replicated efficiently in astrocytes, further supporting the role of CD4 independence as an important determinant of SIV infection of astrocytes in vitro and in vivo.     

Role of the SH3-Ligand Domain of Simian Immunodeficiency Virus Nef in Interaction with Nef-Associated Kinase and Simian AIDS in Rhesus Macaques

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, it is essential for high virus loads and progression to simian AIDS (SAIDS) in SIV-infected adult rhesus macaques. Nef proteins from HIV type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) motif. The region of Nef with this motif is similar to the Src homology region 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/threonine kinase, designated Nef-associated kinase (NAK). In this study, analysis of viral clones containing point mutations in the nef gene of the pathogenic clone SIVmac239 revealed that several strictly conserved residues in the PxxP region were essential for Nef-NAK interaction. The results of this analysis of Nef mutations in in vitro kinase assays indicated that the PxxP region in SIV Nef was strikingly similar to the consensus sequence for SH3 ligand domains possessing the minus orientation. To test the significance of the PxxP motif of Nef for viral pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P¹⁰⁴A/P¹⁰⁷A. This clone, expressing Nef that does not associate with NAK, was inoculated into seven juvenile rhesus macaques. In vitro kinase assays were performed on virus recovered from each animal; the ability of Nef to associate with NAK was restored in five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversion in the mutated PxxP motif. These revertant genotypes, which included a second-site suppressor mutation, restored the ability of Nef to interact with NAK. Additionally, the proportion of revertant viruses increased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo selection for the ability of SIV Nef to associate with NAK was correlated with the induction of SAIDS. Accordingly, these studies implicate a role for the conserved SH3 ligand domain for Nef function in virally induced immunodeficiency.     

Assessing genetic-based therapies for AIDS using the simian immunodeficiency virus

Abstract: A plasmid encoding the full-length infectious molecular proviral clone of SIV_mac239 was generated. Virus derived from cells transfected with this clone replicated to high levels and was cytopathic for some transformed human CD4⁺ cell lines and primary rhesus macaque peripheral blood mononuclear cells. Since replication of SIV requires the functional expression of the viral encoded rev protein, transient co-transfection studies were initiated with the infectious proviral clone and a well-characterized trans-dominant negative HIV-1 rev mutant.     

Identification of viral determinants of macrophage tropism for simian immunodeficiency virus SIVmac.

Simian immunodeficiency virus (SIV), a lymphocytopathic lentivirus, induces an AIDS-like disease in rhesus macaques (Macaca mulatta). A pathogenic molecular clone of rhesus macaque SIV (SIVmac), SIVmac-239, replicates and induces cytopathology in T lymphocytes but is restricted for replication in macrophages. In contrast, a nonpathogenic molecular clone of SIVmac, SIVmac-1A11, replicates and induces syncytia (multinucleated giant cells) in cultures of both T lymphocytes and macrophages. SIVmac-1A11 does not cause disease in macaques. To map the viral determinants of macrophage tropism, reciprocal recombinant genomes were constructed between molecular clones of SIVmac-239 and SIVmac-1A11. Infectious recombinant viruses were rescued by transfection of cloned viral genomes into permissive lymphoid cells. Analysis of one pair of reciprocal recombinants revealed that an internal 6.2-kb DNA fragment of SIVmac-1A11 was necessary and sufficient for both syncytium formation and efficient replication in macrophages. This region includes the coding sequences for a portion of the gag gene, all of the pol, vif, vpr, and vpx genes, the first coding exons of tat and rev, and the external env glycoprotein gp130. Thus, the transmembrane glycoprotein of env, the nef gene, the second coding exons of tat and rev, and the long terminal repeats are not essential for in vitro macrophage tropism. Analysis of additional recombinants revealed that syncytium formation, but not virus production, was controlled by a 1.4-kb viral DNA fragment in SIVmac-1A11 encoding only the external env glycoprotein gp130. Thus, gp130 env of SIVmac-1A11 is necessary for entry of virus into macrophages but is not sufficient for a complete viral replication cycle in this cell type. We therefore conclude that gp130 env and one or more genetic elements (exclusive of the long terminal repeats, transmembrane glycoprotein of env, and second coding exons of tat and rev, and nef) are essential for a complete replication cycle of SIVmac in rhesus macaque macrophages.     

Spontaneous substitutions in the vicinity of the V3 analog affect cell tropism and pathogenicity of simian immunodeficiency virus.

Simian immunodeficiency virus (SIV) exists within tissues of infected macaques as a mixture of diverse genotypes. The goal of this study was to investigate the biologic significance of this variation in terms of cellular tropism and pathogenicity. PCR was used to amplify and clone 3'-half genomes from the spleen of an immunodeficiency SIV-infected pig-tailed macaque (Macaca nemestrina). Eight infectious clones were generated by ligation of respective 3' clones into a related SIVsm 5' clone, and virus stocks were generated by transient transfection. Four of these viruses were infectious for macaque peripheral blood mononuclear cells (PBMC) or monocyte-derived macrophages (MDM). Three viruses with distinct tropism for macaque PBMC or MDM were tested for in vivo infectivity and pathogenicity. The ability of these three viruses to infect PBMC and macrophages correlated with differences in infectivity and pathogenicity. Thus, a virus that was infectious for both PBMC and MDM was highly infectious for macaques and induced AIDS in half of the inoculated animals. In contrast, virus that was less infectious for PBMC and not infectious for MDM induced only transient viremia. Finally, a virus that was not infectious for either primary cell type did not infect macaques. Chimeric clones exchanging portions of the envelope gene of the 62A and smH4 molecular clones and a series of point mutants were used to map the determinant of tropism to a 60-amino-acid region of gp120 encompassing the V3 analog of SIV. Naturally occurring mutations within this region were critical for determining tropism and, as a result, pathogenicity of these SIVsm clones.     

Characterization of infectious molecular clones of simian immunodeficiency virus (SIVmac) and human immunodeficiency virus type 2: persistent infection of rhesus monkeys with molecularly cloned SIVmac.

Infection of macaque monkeys with simian immunodeficiency virus (SIV) is probably the best animal model currently available for studying acquired immunodeficiency syndrome. In this report, we describe three infectious molecular clones of SIVmac and one of human immunodeficiency virus type 2 (HIV-2) and their use in the study of cell and species specificity, animal infection, and the relationship of gene sequence to function. Replication of the cloned viruses in different cell lines varied dramatically. Some human CD4+ cell lines (HUT 78 and MT-4) supported the replication of SIVmac and HIV-2, while others (CEM and Jurkat-T) supported the replication of HIV-2 but not SIVmac. Growth of cloned virus in macaque lymphocytes in vitro was predictive of macaque infection in vivo. Macaque lymphocytes supported the replication of SIVmac239 and SIVmac251 but not SIVmac142 or HIV-2ROD. Using virus recovery and antibody response as criteria for infection, macaques that received cloned SIVmac251 and SIVmac239 became infected, while macaques receiving cloned SIVmac142 and HIV-2ROD did not become infected. Nucleotide sequences from the envelope region of all four cloned viruses demonstrated that there is considerable flexibility in the location of the translational termination (stop) signal. These infectious molecular clones will be very useful for future studies directed at the molecular basis for persistence, pathogenicity, tropism, and cell and species specificity.     

The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable.

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.     

In Vivo Replication Capacity Rather Than In Vitro Macrophage Tropism Predicts Efficiency of Vaginal Transmission of Simian Immunodeficiency Virus or Simian/Human Immunodeficiency Virus in Rhesus Macaques

We used the rhesus macaque model of heterosexual human immunodeficiency virus (HIV) transmission to test the hypothesis that in vitro measures of macrophage tropism predict the ability of a primate lentivirus to initiate a systemic infection after intravaginal inoculation. A single atraumatic intravaginal inoculation with a T-cell-tropic molecular clone of simian immunodeficiency virus (SIV), SIVmac239, or a dualtropic recombinant molecular clone of SIV, SIVmac239/1A11/239, or uncloned dualtropic SIVmac251 or uncloned dualtropic simian/human immunodeficiency virus (SHIV) 89.6-PD produced systemic infection in all rhesus macaques tested. However, vaginal inoculation with a dualtropic molecular clone of SIV, SIVmac1A11, resulted in transient viremia in one of two rhesus macaques. It has previously been shown that 12 intravaginal inoculations with SIVmac1A11 resulted in infection of one of five rhesus macaques (M. L. Marthas, C. J. Miller, S. Sutjipto, J. Higgins, J. Torten, B. L. Lohman, R. E. Unger, H. Kiyono, J. R. McGhee, P. A. Marx, and N. C. Pedersen, J. Med. Primatol. 21:99–107, 1992). In addition, SHIV HXBc2, which replicates in monkey macrophages, does not infect rhesus macaques following multiple vaginal inoculations, while T-cell-tropic SHIV 89.6 does (Y. Lu, P. B. Brosio, M. Lafaile, J. Li, R. G. Collman, J. Sodroski, and C. J. Miller, J. Virol. 70:3045–3050, 1996). These results demonstrate that in vitro measures of macrophage tropism do not predict if a SIV or SHIV will produce systemic infection after intravaginal inoculation of rhesus macaques. However, we did find that the level to which these viruses replicate in vivo after intravenous inoculation predicts the outcome of intravaginal inoculation with each virus.     

High viral load in the cerebrospinal fluid and brain correlates with severity of simian immunodeficiency virus encephalitis

AIDS dementia and encephalitis are complications of AIDS occurring most frequently in patients who are immunosuppressed. The simian immunodeficiency virus (SIV) model used in this study was designed to reproducibly induce AIDS in macaques in order to examine the effects of a neurovirulent virus in this context. Pigtailed macaques (Macaca nemestrina) were coinoculated with an immunosuppressive virus (SIV/DeltaB670) and a neurovirulent molecularly cloned virus (SIV/17E-Fr), and more than 90% of the animals developed moderate to severe encephalitis within 6 months of inoculation. Viral load in plasma and cerebrospinal fluid (CSF) was examined longitudinally to onset of AIDS, and viral load was measured in brain tissue at necropsy to examine the relationship of systemic and central nervous system (CNS) viral replication to the development of encephalitis. In all animals, plasma viral load peaked at 10 to 14 days postinfection and remained high throughout infection with no correlation found between plasma viremia and SIV encephalitis. In contrast, persistent high levels of CSF viral RNA after the acute phase of infection correlated with the development of encephalitis. Although high levels of viral RNA were found in the CSF of all macaques (six of six) during the acute phase, this high level was maintained only in macaques developing SIV encephalitis (five of six). Furthermore, the level of both viral RNA and antigen in the brain correlated with the severity of the CNS lesions. The single animal in this group that did not have CNS lesions had no detectable viral RNA in any of the regions of the brain. The results substantiate the use of CSF viral load measurements in the postacute phase of SIV infection as a marker for encephalitis and CNS viral replication.     

A Lymph Node-Derived Cytopathic Simian Immunodeficiency Virus Mne Variant Replicates in Nonstimulated Peripheral Blood Mononuclear Cells

Lymph nodes (LNs) are sites of active human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) replication and disease at both early and late stages of infection. Consequently, variant viruses that replicate efficiently and subsequently cause immune dysfunction may be harbored in this tissue. To determine whether LN-associated SIVs have an increased capacity to replicate and induce cytopathology, a molecular clone of SIV was isolated directly from DNA extracted from unpassaged LN tissue of a pig-tailed macaque (Macaca nemestrina) infected with SIVMne. The animal had declining CD4⁺ T-lymphocyte counts at the time of the LN biopsy. In human CD4⁺ T-cell lines, the LN-derived virus, SIVMne027, replicated with relatively slow kinetics and was minimally cytopathic and non-syncytium inducing compared to other SIVMne clones. However, in phytohemagglutinin-stimulated pig-tailed macaque peripheral blood mononuclear cells (PBMCs), SIVMne027 replicated efficiently and was highly cytopathic for the CD4⁺ T-cell population. Interestingly, unlike other SIVMne clones, SIVMne027 also replicated to a high level in nonstimulated macaque PBMCs. High-level replication depended on the presence of both the T-cell and monocyte/macrophage populations and could be enhanced by interleukin-2 (IL-2). Finally, the primary determinant governing the ability of SIVMne027 to replicate in nonstimulated and IL-2-stimulated PBMCs mapped to gag-pol-vif. Together, these data demonstrate that LNs may harbor non-syncytium-inducing, cytopathic viruses that replicate efficiently and are highly responsive to the effects of cytokines such as IL-2.     

Biological characterization of nef in long-term survivors of human immunodeficiency virus type 1 infection.

We have previously shown that there were no gross deletions or obvious sequence abnormalities within nef of human immunodeficiency virus type 1 (HIV-1) in the 10 long-term survivors studied (Y. Huang, L. Zhang, and D. D. Ho, J. Virol. 69:93-100, 1995). Here we extend our study to examine these nef alleles in a functional context. Using a new technique, termed site-directed gene replacement, we have precisely replaced the nef of an infectious molecular clone, HIV-1HXB2, with nef alleles derived from 10 long-term survivors as well as from a patient with AIDS. The replication properties of these chimeric viruses demonstrated that the nef alleles derived from long-term survivors neither significantly increased nor decreased viral replication, compared with the nef allele of Nef+ HIV-1HXB2 and that derived from a patient with AIDS. However, Nef+ viruses always replicated faster than virus lacking nef. Moreover, single-cell infection analysis by the MAGI assay showed that these chimeric viruses, as well as Nef+ HIV-1HXB2, were more infectious than Nef- HIV-1HXB2 was. Therefore, we conclude that the genotypic and phenotypic features of nef are not likely to account for the nonprogression of HIV-1 infection in the 10 cases studied, unless the function of the nef gene in vivo is not accurately reflected by the in vitro assays we used.     

A molecularly cloned, pathogenic, neutralization-resistant simian immunodeficiency virus, SIVsmE543-3.

An infectious molecular clone of simian immunodeficiency virus SIVsm was derived from a biological isolate obtained late in disease from an immunodeficient rhesus macaque (E543) with SIV-induced encephalitis. The molecularly cloned virus, SIVsmE543-3, replicated well in macaque peripheral blood mononuclear cells and monocyte-derived macrophages and resisted neutralization by heterologous sera which broadly neutralized genetically diverse SIV variants in vitro. SIVsmE543-3 was infectious and induced AIDS when inoculated intravenously into pig-tailed macaques (Macaca nemestrina). Two of four infected macaques developed no measurable SIV-specific antibody and succumbed to a wasting syndrome and SIV-induced meningoencephalitis by 14 and 33 weeks postinfection. The other two macaques developed antibodies reactive in Western blot and virus neutralization assays. One macaque was sacrificed at 1 year postinoculation, and the survivor has evidence of immunodeficiency, characterized by persistently low CD4 lymphocyte subsets in the peripheral blood. Plasma samples from these latter animals neutralized SIVsmE543-3 but with much lower efficiency than neutralization of other related SIV strains, confirming the difficulty by which this molecularly cloned virus is neutralized in vitro. SIVsmE543-3 will provide a valuable reagent for studying SIV-induced encephalitis, mapping determinants of neutralization, and determining the in vivo significance of resistance to neutralization in vitro.     

Thenef gene of SIVmac239 is necessary for efficient growth in H9 cells

AIDS viruses require an intact functionalnef gene in order to inducedisease. The nonpathogenic molecular cloned virus SIVmac239nef-deletion encodes a truncatednef gene. This attenuated reading frame is expressed both in vitro and in a virus-infected animal in vivo. Encoding the first 58 amino acids of Nef, the reading frame retained its ability to down-modulate CD4 from the surface of T cells. CD4-down-modulated stable cell lines expressing full-length and truncatednef genes were significantly less infected by SIV. SIV-mac239nef-open and SIVmacnef-deletion encoding a truncatednef clearly differed in replication kinetics in H9 cells and H9-derived cell lines. SIV-mac239nef-deletion replication was delayed in H9.