Characterization of a novel simian immunodeficiency virus (SIV) from L'Hoest monkeys (Cercopithecus l'hoesti): implications for the origins of SIVmnd and other primate lentiviruses

The human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) appear to have originated by cross-species transmission of simian immunodeficiency virus (SIV) from asymptomatically infected African primates. Few of the SIVs characterized to date efficiently infect human primary lymphocytes. Interesting, two of the three identified to infect such cultures (SIVsm and SIVcpz) have appeared in human populations as genetically related HIVs. In the present study, we characterized a novel SIV isolate from an East African monkey of the Cercopithecus genus, the l'hoest monkey (C. l'hoesti), which we designated SIVlhoest. This SIV isolate efficiently infected both human and macaque lymphocytes and resulted in a persistent infection of macaques, characterized by high primary virus load and a progressive decline in circulating CD4 lymphocytes, consistent with progression to AIDS. Phylogenetic analyses showed that SIVlhoest is genetically distinct from other previously characterized primate lentiviruses but clusters in the same major lineage as SIV from mandrills (SIVmnd), a West African primate species. Given the geographic distance between the ranges of l'hoest monkeys and mandrills, this may indicate that SIVmnd arose through cross-species transmission from close relatives of l'hoest monkeys that are sympatric with mandrills. These observations lend support to the hypothesis that the primate lentiviruses originated and coevolved within monkeys of the Cercopithecus genus. Regarded in this light, lentivirus infections of primates not belonging to the Cercopithecus genus may have resulted from cross-species transmission in the not-too-distant past.     

The regulation of primate immunodeficiency virus infectivity by Vif is cell species restricted: a role for Vif in determining virus host range and cross-species transmission

The primate immunodeficiency virus Vif proteins are essential for replication in appropriate cultured cell systems and, presumably, for the establishment of productive infections in vivo. We describe experiments that define patterns of complementation between human and simian immunodeficiency virus (HIV and SIV) Vif proteins and address the determinants that underlie functional specificity. Using human cells as virus producers, it was found that the HIV-1 Vif protein could modulate the infectivity of HIV-1 itself, HIV-2 and SIV isolated from African green monkeys (SIVAGM). In contrast, the Vif proteins of SIVAGM and SIV isolated from Sykes' monkeys (SIVSYK) were inactive for all HIV and SIV substrates in human cells even though, at least for the SIVAGM protein, robust activity could be demonstrated in cognate African green monkey cells. These observations suggest that species-specific interactions between Vif and virus-producing cells, as opposed to between Vif and virus components, may govern the functional consequences of Vif expression in terms of inducing virion infectivity. The finding that the replication of murine leukemia virus could also be stimulated by HIV-1 Vif expression in human cells further supported this notion. We speculate that species restrictions to Vif function may have contributed to primate immunodeficiency virus zoonosis.     

The U3 promoter and the nef gene of simian immunodeficiency virus (SIV) smmPBj1.9 do not confer acute pathogenicity upon SIVagm

Two chimeric proviruses comprising the U3 promoter and the nef gene of simian immunodeficiency virus (SIV) smmPBj1.9 in addition to other genomic regions of SIVagm3mc from African green monkeys (Cercopithecus aethiops) were constructed. The derived chimeric viruses (SIVagm3mc/SIVsmmPBj1.9) were both able to replicate in nonstimulated peripheral blood leukocytes from pig-tailed macaques (Macaca nemestrina), a biological property often correlated with acute pathogenicity. However, only one of the chimeric viruses was acutely pathogenic, inducing a rapid depletion of the peripheral CD4+ T cells in two infected pig-tailed macaques within 10 days after infection in a manner similar to infection with SIVsmmPBj1.9 itself. The other chimeric virus actively replicated during the first 8 weeks after experimental infection of two pig-tailed macaques but induced neither acute disease nor CD4+ T-cell depletion for 113 weeks after infection. Thus, the U3 promoter and the nef gene of SIVsmmPBj1.9 alone appear to be insufficient to confer acute pathogenicity to SIVagm3mc.     

Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes

Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus-1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8+ lymphocytes during primary SIV infection. Eliminating CD8+ lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.     

Vpr of simian immunodeficiency virus of African green monkeys is required for replication in macaque macrophages and lymphocytes

The genomes of simian immunodeficiency viruses isolated from African green monkeys (SIVagm) contain a single accessory gene homolog of human immunodeficiency virus type 1 (HIV-1) vpr. This genomic organization differs from that of SIVsm-SIVmac-HIV-2 group viruses, which contain two gene homologs, designated vpr and vpx, which in combination appear to share the functions of HIV-1 vpr. The in vitro role of the SIVagm homolog was evaluated with molecularly cloned, pathogenic SIVagm9063-2. These studies revealed that this gene shares properties of HIV-1 vpr, such as nuclear and virion localization. In addition, SIVagm mutants with inactivating mutations of vpr are unable to replicate in nondividing cells, such as macaque monocyte-derived macrophages, but replicate to almost wild-type levels in a susceptible human T-cell line. The transport of virus preintegration complexes into the nucleus in primary macrophages, as measured by the production of unintegrated circular viral DNA, is less efficient for the mutant viruses than it is for the wild-type virus. SIVagm mutants also replicate inefficiently in primary macaque peripheral blood mononuclear cells, with a propensity for substitutions that remove the inserted inactivating stop codon. These data, in conjunction with recent findings that the Vpr protein is capable of inducing G2 arrest, are consistent with designation of this SIVagm accessory gene as vpr to reflect its shared functions and properties with HIV-1 vpr.     

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An apparent species-specific relatedness of SIVagm suggests a coevolution with their natural hosts. However, the exact species or subspecies classification of African green monkeys, AGM, is uncertain because current classification schemes rely on phenotype markers, while more definitive genetic data are lacking. In this study, the CD4 protein involved in tissue type recognition was genetically cloned and sequenced from PBMC RNA from all AGM species, including Barbados green monkeys (BGM). Phylogenetic trees were constructed that also included genomic CD4 nucleotide sequences from patas, sooty mangabeys, rhesus and pig-tail macaques, chimpanzees, and humans. Chimpanzees and humans consistently clustered together. Monkeys within the Cercopithecus genus formed a separate cluster which included pata monkeys, supporting its grouping as a member of Cercopithecus. Surprisingly, sooty mangabeys were genetically more closely related to Asian macaques than to other African species, which might explain why macaques are more susceptible to infection by the SIVsm group than to infection by SIVagm or HIV-1 and why patas, on the other hand, are highly susceptible to SIVagm infection. Based on CD4 genetic data, tantalus, vervets, grivets, and sabaeus formed separate subgroups with BGM grouping closely with vervets. The branching order of the AGM species was related to that of their respective SIVagm env sequences. The study suggests a strong correlation between CD4 phylogeny and the susceptibility of the host species to infection by a specific lentivirus and supports the assumption of a coevolution of SIVagm and AGM. CD4 sequencing is suggested as a relevant method for genetic determination of primate species.     

A novel simian immunodeficiency virus (SIVdrl) pol sequence from the drill monkey, Mandrillus leucophaeus

The drill monkey has been shown by serology and PCR to harbor a unique simian immunodeficiency virus (SIVdrl). A pol sequence, amplified from uncultured peripheral blood cells, is most closely related to the equivalent SIV sequences from the red-capped mangabey (SIVrcm), the sabaeus African green monkey (SIVagmSAB), and the chimpanzee (SIVcpz) and to the human immunodeficiency virus type 1 (HIV-1) sequence of humans. It is as yet unclear whether SIVdrl has a mosaic genome like SIVrcm and SIVagmSAB, is a member of the SIVcpz/HIV-1 lineage, or represents a novel primate lentivirus lineage.     

Genetic diversity of simian immunodeficiency viruses from West African green monkeys: evidence of multiple genotypes within populations from the same geographical locale

High simian immunodeficiency virus (SIV) seroprevalence rates have been reported in the different African green monkey (AGM) subspecies. Genetic diversity of these viruses far exceeds the diversity observed in the other lentivirus-infected human and nonhuman primates and is thought to reflect ancient introduction of SIV in the AGM population. We investigate here genetic diversity of SIVagm in wild-living AGM populations from the same geographical locale (i.e., sympatric population) in Senegal. For 11 new strains, we PCR amplified and sequenced two regions of the genome spanning the first tat exon and part of the transmembrane glycoprotein. Phylogenetic analysis of these sequences shows that viruses found in sympatric populations cluster into distinct lineages, with at least two distinct genotypes in each troop. These data strongly suggest an ancient introduction of these divergent viruses in the AGM population.     

"Hidden" dUTPase sequence in human immunodeficiency virus type 1 gp120

A coding region homologous to the sequence for essential eukaryotic enzyme dUTPase has been identified in different genomic regions of several viral lineages. Unlike the nonprimate lentiviruses (caprine arthritis- encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, and visna virus), where dUTPase is integrated into the pol coding region, this enzyme has never been demonstrated to be present in the primate lentivirus genomes (human immunodeficiency virus type 1 [HIV-1], HIV-2, or the related simian immunodeficiency virus). A novel approach allowed us to identify a weak but significant sequence similarity between HIV-1 gp120 and the human dUTPase. This finding was then extended to all of the primate lentivirus lineages. Together with the recently reported fragmentary structural similarity between the V3 loop region and the Escherichia coli dUTPase (P. D. Kwong, R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson, Nature 393:648-659, 1998), our results strongly suggest that an ancestral dUTPase gene has evolved into the present primate lentivirus CD4 and cytokine receptor interacting region of gp120.     

Mechanism of Nef-induced CD4 endocytosis: Nef connects CD4 with the mu chain of adaptor complexes

The Nef protein of primate lentiviruses down-regulates the cell surface expression of CD4 and probably MHC I by connecting these receptors with the endocytic machinery. Here, we reveal that Nef interacts with the mu chains of adaptor complexes, key components of clathrin-coated pits. For human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) Nef, this interaction occurs via tyrosine-based motifs reminiscent of endocytosis signals. Mutating these motifs prevents the binding of SIV Nef to the mu chain of plasma membrane adaptor complexes, abrogates its ability to induce CD4 internalization, suppresses the accelerated endocytosis of a chimeric integral membrane protein harboring Nef as its cytoplasmic domain and confers a dominant-negative phenotype to the viral protein. Taken together, these data identify mu adaptins as downstream mediators of the down-modulation of CD4, and possibly MHC I, by Nef.     

Polymyositis, arthritis, and uveitis in a macaque experimentally infected with human T lymphotropic virus type I

OBJECTIVE: To establish a simian model of human T lymphotropic virus type I (HTLV-I) infection and disease. METHODS: Irradiated HTLV-I-producing cells were used to infect two 2-year-old rhesus macaque monkeys (Macaca mulatta). One monkey was also simultaneously inoculated with a cell-free suspension of simian immunodeficiency virus (SIV). Evidence of infection was monitored by serial clinical examinations and by serologic, molecular, and virologic assays. RESULTS: Both HTLV-I-inoculated monkeys became persistently infected following inoculation. Clinical disease was observed in the singly inoculated monkey, which developed arthritis (with synovial fluid positive for HTLV-I by culture and polymerase chain reaction), anterior chamber uveitis, and steroid-responsive polymyositis confirmed by electrophysiologic studies. The dually inoculated animal remained clinically healthy, despite high levels of SIV and HTLV-I virus expression and loss of HTLV-I-specific antibodies. CONCLUSION: These results indicate the utility of a nonhuman primate model for studying HTLV-I disease pathogenesis and the dynamics of SIV-1/HTLV-I retroviral coinfection.     

Common themes of antibody maturation to simian immunodeficiency virus, simian-human immunodeficiency virus, and human immunodeficiency virus type 1 infections

Characterization of virus-specific immune responses to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) is important to understanding the early virus-host interactions that may determine the course of virus infection and disease. Using a comprehensive panel of serological assays, we have previously demonstrated a complex and lengthy maturation of virus-specific antibody responses elicited by attenuated strains of SIV that was closely associated with the development of protective immunity. In the present study, we expand these analyses to address several questions regarding the nature of the virus-specific antibody responses to pathogenic SIV, SIV/HIV-1 (SHIV), and HIV-1 infections. The results demonstrate for the first time a common theme of antibody maturation to SIV, SHIV, and HIV-1 infections that is characterized by ongoing changes in antibody titer, conformational dependence, and antibody avidity during the first 6 to 10 months following virus infection. We demonstrate that this gradual evolution of virus-specific antibody responses is independent of the levels of virus replication and the pathogenicity of the infection viral strain. While the serological assays used in these studies were useful in discriminating between protective and nonprotective antibody responses during evaluation of vaccine efficacy with attenuated SIV, these same assays do not distinguish the clinical outcome of infection in pathogenic SIV, SHIV, or HIV-1 infections. These results likely reflect differences in the immune mechanisms involved in mediating protection from virus challenge compared to those that control an established viral infection, and they suggest that additional characteristics of both humoral and cellular responses evolve during this early immune maturation.     

Nucleocapsid protein zinc-finger mutants of simian immunodeficiency virus strain mne produce virions that are replication defective in vitro and in vivo

All retroviruses (except the spumaretroviruses) contain a nucleocapsid (NC) protein that encodes one or two copies of the Zn2+-finger sequence -Cys-X2-Cys-X4-His-X4-Cys-. This region has been shown to be essential for recognition and packaging of the genomic RNA during virion particle assembly. Additionally, this region has been shown to be involved in early infection events in a wide spectrum of retroviruses, including mammalian type C [e.g., murine leukemia virus (MuLV)], human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus, and other retroviruses. Mutations in the two Zn2+-fingers of the NC protein of simian immunodeficiency virus strain Mne [SIV(Mne)] have been generated. The resulting virions contained the normal complement of processed viral proteins with densities indistinguishable from wild-type SIV(Mne). All of the mutants had electron micrograph morphologies similar to those of immature particles observed in wild-type preparations. RNA packaging was less affected by mutations in the NC protein of SIV(Mne) than has been observed for similar mutants in the MuLV and HIV-1 systems. Nevertheless, in vitro replication of SIV(Mne) NC mutants was impaired to levels comparable to those observed for MuLV and HIV-1 NC mutants; replication defective NC mutants are typically 10(5)- to 10(6)-fold less infectious than similar levels of wild-type virus. One mutant, DeltaCys33-Cys36, was also found to be noninfectious in vivo when mutant virus was administered intravenously to a pig-tailed macaque. NC mutations can therefore be used to generate replication defective virions for candidate vaccines in the SIV macaque model for primate lentiviral diseases.     

The isolation and characteristics of the green monkey lentivirus

We characterized the simian immunodeficiency virus isolated from Cercopithecus aethiops (subspecies C. a. pygerythrus) originating from Kenya. SIV was isolated and continuously produced with the MOLT4 clone 8 cell line and was designated as SIV-SU1. SIV-SU1 isolate replicated with high efficiency in MOLT4 clone 8, MT-2 with moderate efficiency in CEM x 174 and with poor efficiency in HUT-78, U937, C8166. The infection of MT-2, C8166 and HUT-78 resulted in extensive cell killing. Western blotting of purified preparations of SIV-SU1 revealed viral proteins of 130, 68, 55, 41, 24, 17 kDa. Cross-reactivity of SIV-SU1 proteins with HIV-1, HIV-2, SIVmac, SIVsm, SIVmnd was studied by radioimmunoprecipitation assay. The most extensive cross-reactivity was observed with SIVmac. Total cellular DNA from chronically infected cells was hybridized to SIVagm266 DNA probes. Detection of cross-hybridizing DNA sequences required very low stringency, and the restriction endonuclease fragmentation pattern of SIV-SU1 differed from other SIVs.     

A two-component cytoskeletal system of Xenopus laevis egg cortex: concept of its contractility

We found that most peripheral CD4 cells co-express a low density of CD8 alpha antigen in African green monkeys (AGM). Further, the cell surface expression of CD4 and the expression of CD4 mRNA underwent a decrease when purified CD4CD8low cells were cultured with mitogen and IL-2. These observations suggest that AGM CD4 cells are subject to loss of CD4 expression after lymphocyte activation. Part of the peripheral CD8 fraction exhibited a significant helper activity which suggested the phenotypic conversion in helper T cells from CD4+ to CD4- in vivo. Simian immunodeficiency virus (SIV) grew well in CD4 panning cells following SIV infection. In contrast, CD4CD8low cells were resistant to SIV infection after their conversion to CD4- cells.     

The effect of simian immunodeficiency virus infection in vitro and in vivo on the cytokine production of isolated microglia and peripheral macrophages from rhesus monkey

Microglia are the major target for human immunodeficiency virus (HIV) infection within the central nervous system. Because only a few cells are productively infected, it has been suggested that an aberrant cytokine production by this cell population may be an indirect mechanism leading to the development of neurological disorders in HIV-infected patients. Therefore we decided to study the secretion pattern of several interleukins (IL) by microglial cells and peripheral blood macrophages isolated from uninfected and simian immunodeficiency virus (SIV)-infected Rhesus monkeys. We found that uninfected, unstimulated primate microglia produce more IL-6 and less TNF alpha than peripheral blood macrophages, but generate comparable levels of IL-1 beta and IL-8. After infection with SIV in vitro, synthesis of all cytokines tested is increased compared to uninfected cultures and to peripheral blood macrophages. Microglia isolated from infected animals produce more IL-8 and TNF alpha than the uninfected cultures and display a strongly increased capacity to secrete TNF alpha upon stimulation with lipopolysaccharide. In addition, production of IL-6 by in vivo-infected microglia increases with time in culture to very high levels despite the fact that only a few cells contained replicating virus. These findings clearly show that the cytokine production of microglia is impaired after SIV infection both in vitro and in vivo and that a low level of viral replication is sufficient for these alterations to occur. In conclusion, the results of this study further support a possible role of cytokines in the pathogenesis of neuro-AIDS.     

Neutralization sensitivity of cell culture-passaged simian immunodeficiency virus

CEMx174- and C8166-45-based cell lines which contain a secreted alkaline phosphatase (SEAP) reporter gene under the control of a tat-responsive promoter derived from either SIVmac239 or HIV-1(NL4-3) were constructed. Basal levels of SEAP activity from these cell lines were low but were greatly stimulated upon transfection of tat expression plasmids. Infection of these cell lines with simian immunodeficiency virus (SIV) or human immunodeficiency virus type 1 (HIV-1) resulted in a dramatic increase in SEAP production within 48 to 72 h that directly correlated with the amount of infecting virus. When combined with chemiluminescent measurement of SEAP activity in the cell-free supernatant, these cells formed the basis of a rapid, sensitive, and quantitative assay for SIV and HIV infectivity and neutralization. Eight of eight primary isolates of HIV-1 that were tested induced readily measurable SEAP activity in this system. While serum neutralization of cloned SIVmac239 was difficult to detect with other assays, neutralization of SIVmac239 was readily detected at low titers with this new assay system. The neutralization sensitivities of two stocks of SIVmac251 with different cell culture passage histories were tested by using sera from SIV-infected monkeys. The primary stock of SIVmac251 had been passaged only twice through primary cultures of rhesus monkey peripheral blood mononuclear cells, while the laboratory-adapted stock had been extensively passaged through the MT4 immortalized T-cell line. The primary stock of SIVmac251 was much more resistant to neutralization by a battery of polyclonal sera from SIV-infected monkeys than was the laboratory-adapted virus. Thus, SIVmac appears to be similar to HIV-1 in that extensive laboratory passage through T-cell lines resulted in a virus that is much more sensitive to serum neutralization.     

Longitudinal analysis of behavioral, neurophysiological, viral and immunological effects of SIV infection in rhesus monkeys

A model is proposed in which a neurovirulent, microglial-passaged, simian immunodeficiency virus (SIV) is used to produce central nervous system (CNS) pathology and behavioral deficits in rhesus monkeys reminiscent of those seen in humans infected with human immunodeficiency virus (HIV). The time course of disease progression was characterized by using functional measures of cognition and motor skill, as well as neurophysiologic monitoring. Concomitant assessment of immunological and virological parameters illustrated correspondence between impaired behavioral performance and viral pathogenesis. Convergent results were obtained from neuropathological findings indicative of significant CNS disease. In ongoing studies, this SIV model is being used to explore the behavioral sequelae of immunodeficiency virus infection, the viral and host factors leading to neurologic dysfunction, and to begin testing potential therapeutic agents.