Evolutionary origin of human and simian immunodeficiency viruses.

From what viruses the human immunodeficiency viruses (HIVs) originated is an extremely controversial question. To address this question, we have analyzed nucleotide sequences of simian immunodeficiency viruses (SIVs) and HIVs by using the techniques for understanding molecular evolution. In particular, we compared the nucleotide sequences of whole genomes, gene region by gene region, between a given pair of viruses, including four types of SIVs--isolated from mandrills (Papio sphinx), African green monkeys (Cercopithecus aethiops), sooty mangabeys (Cercocebus atys), and rhesus macaques (Macaca mulatta)--as well as HIVs. Phylogenetic trees for all gene regions examined showed that the present HIVs may have emerged as different variants of SIVs of Old World monkeys, possibly from recombination between viruses related to SIVs.     

Infection of a simian B cell line by human and simian immunodeficiency viruses

It is well known that HIV-1 does not establish infection in nonhuman primates, nor in cell lines derived from them, due to the existence of saturable resistance factors. In this study, we show that an in vitro established Macaca fascicularis-derived CD4(-) B cell line (F6) can be productively infected by the laboratory-adapted T-tropic HXBc2/HIV-1 strain at low multiplicity of infection, apparently because it does not express the restriction factor that has been detected in other simian cell lines. Moreover, efficient entry into F6 cells was obtained with pseudotyped recombinant HIV-1 viruses containing the laboratory-adapted T-tropic (HXBc2) or the dual-tropic (89.6) envelope glycoproteins, whereas entry of virus containing the envelope glycoproteins of the M-tropic Ba-L strain was less efficient. Virus containing primary T-tropic (Eli) envelope glycoproteins did not infect F6 cells. Furthermore, although CCR5 was not present on the cell surface and gpr15 and strl33 mRNAs were not expressed in the cells, a high level of infection of F6 cells by the M-tropic simian immunodeficiency virus SIVmac316 was observed. In contrast, F6 cells were poorly infected by T-tropic SIVmac239. Given the unique properties of the F6 cell line, i.e., that it is of simian origin yet is able to be infected by HIV-1 in a CD4-independent manner, F6 cells represent a useful model for studying cellular factors mediating resistance or permissivity to HIV-1 infection and may help to evaluate HIV-1 and SIV cell tropism.     

The emergence of simian/human immunodeficiency viruses.

Molecular evolutionary analyses strongly support the hypothesis that human immunodeficiency viruses have recently arisen from a diverse pool of nonhuman primate immunodeficiency viruses. Our understanding of the molecular phylogenetic relationships between primate and nonprimate lentiviruses is less certain, partly because key intermediate forms are still to be discovered. DNA and protein sequence comparisons reveal uncanny dissimilarities, as well as similarities, among the genetic sequences of these complex retroviruses, thereby giving rise to the notion that primate lentiviruses are participants in "fast-forward" evolution.     

Adaptation of human and simian immunodeficiency viruses for resistance to tetherin/BST-2

Tetherin (BST-2 or CD317) is an interferon-inducible cellular factor that prevents the detachment of enveloped viruses from infected cells. The primate lentiviruses have evolved different countermeasures to tetherin. The majority of SIVs use Nef to antagonize the tetherin proteins of their nonhuman primate hosts. However, due to the absence of sequences in human tetherin required for antagonism by Nef, HIV-1 Vpu and HIV-2 Env evolved to serve this function in humans. We recently identified compensatory changes in the Env cytoplasmic domain of a pathogenic nef-deleted SIV that confers resistance to rhesus macaque tetherin. These observations highlight the extraordinary plasticity of the primate lentiviruses in adapting to the tetherin proteins of their respective hosts, and reveal a prominent role for tetherin in shaping the evolution of the primate lentiviruses.     

Genetic variation of simian immunodeficiency viruses in nonhuman primates.

The generation of biologically active proviral DNA clones of simian immunodeficiency virus (SIV) that give rise to infectious virions has allowed the detailed examination of genetic variation in experimentally inoculated monkeys. Studies of nucleotide sequences derived directly from circulating leukocytes of infected monkeys show that the SIV genome undergoes rapid and dramatic variation during the course of infection. The env gene is a major site for variation, and within the Env protein, hypervariable regions analogous to those previously defined for the human immunodeficiency virus type 1 (HIV-1) env gene are apparent. A major exception is the region corresponding to the V3 domain in HIV-1, which has been highly conserved in all SIV studies to date. These data notwithstanding, the role of SIV genetic variation in the pathogenesis of AIDS in monkeys remains unclear. Genetic variation within the env gene does not appear to be sufficient for the development of AIDS since significant variation is observed in both pathogenic and nonpathogenic SIV infections. Furthermore, although it generally is believed that env gene variation might allow HIV and SIV to avoid recognition and elimination by host immune responses, this premise has not been rigorously proven. The use of molecularly cloned SIV in monkey models has provided important quantitative and qualitative information on in vivo sequence variation, and these data, in turn, have laid the groundwork for addressing the undoubtedly complex functional significance of this variation.     

An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection.

The activity of the human immunodeficiency virus (HIV) protease is essential for processing of the gag-pol precursor proteins and maturation of infectious virions. We have prepared a peptidomimetic inhibitor, U-75875, that inhibited HIV-1 gag-pol protein processing in an essentially irreversible manner. Noninfectious virus particles produced in the presence of the drug contained gag precursors and were morphologically immature. In human peripheral blood mononuclear cells and in a continuous cell line, U-75875 completely blocked HIV replication; in the latter case, no spread occurred over a period of 4 weeks. U-75875, on a molar basis, was as potent as 3'-azido-3'-deoxythymidine in blocking HIV-1 replication in human lymphocytes and also inhibited HIV-2 and simian immunodeficiency virus proteases, demonstrating that it has broad activity. These results provide further evidence for the therapeutic potential of protease inhibitors in HIV infection.     

The principal neutralization determinant of simian immunodeficiency virus differs from that of human immunodeficiency virus type 1.

To identify the principal neutralization determinant (PND) of simian immunodeficiency virus (SIV), antisera were generated using recombinant gp110 [the SIV analog of the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein, gp120], gp140, several large recombinant and proteolytic envelope fragments, and synthetic peptides of the SIVmac251 isolate. When purified under conditions that retain its native structure, gp110 bound CD4 and elicited antisera that neutralized SIVmac251 with high titer. Native gp110 also completely inhibited neutralizing antibody in sera from SIVmac251-infected macaques. In contrast, denatured gp110 and gp140, large envelope fragments, and synthetic peptides (including peptides analogous to the HIV-1 PND) elicited very low or undetectable neutralizing antibody titers and did not inhibit neutralizing antibody in infected macaque sera. Enzymatically deglycosylated gp110 efficiently absorbed neutralizing antibodies from macaque sera, showing that neutralizing antibodies primarily bind the protein backbone. A 45-kDa protease digest product, mapping to the carboxyl-terminal third of gp110, also completely absorbed neutralizing antibodies from infected macaque sera. These results show that the PND(s) of this SIV isolate depends on the native conformation and that linear peptides corresponding to the V3 loop of SIV envelope, in contrast to that of HIV-1, do not elicit neutralizing antibody. This may affect the usefulness of SIVmac for evaluating HIV-1 envelope vaccine approaches that rely on eliciting neutralizing antibody.     

Quantification of simian immunodeficiency virus cytotoxic T lymphocyte escape mutant viruses

Escape from cytotoxic T-lymphocyte (CTL) pressure is common in HIV-1 infection of humans and simian immunodeficiency virus (SIV) infections of macaques. CTL escape typically incurs a fitness cost as reversion back to wild-type can occur upon transmission. We utilized sequence-specific primers and DNA probes with real-time polymerase chain reaction (PCR) to sensitively and specifically track wild-type and escape mutant viremia at the Mane-A*17-restricted SIV Gag(371379) epitope AF9 in pigtail macaques. The generation of minor escape mutant populations is detected by the real-time PCR 2 weeks earlier than observed using standard sequencing techniques. We passaged the AF9 CTL escape mutant virus into two na?ve Mane-A*17-negative pigtail macaques and showed that reversion to wild-type was rapid during acute infection and then slowed considerably at later stages of the infection. These data help refine our understanding of how CTL escape mutant viruses evolve.     

Conserved immunogenic region of a major core protein (p24) of human and simian immunodeficiency viruses

A murine monoclonal antibody (MoAb), VAK 4, has been known to specifically react with a major core protein (p24) as well as with its precursor (p55-57) and intermediate precursor (p40) of human immunodeficiency virus strain IIIB (HTLV-IIIB). Radioimmunoprecipitation assays revealed that VAK 4 MoAb precipitated a major core protein and its precursors from a variety of strains of HIV and also from simian immunodeficiency virus (SIV), although the molecular weights of the precursor proteins in each viral strain were slightly different. A protein synthesized by transfected Escherichia coli containing amino acid sequences corresponding to residues 121-436 of the HTLV-IIIB gag gene was reactive with VAK 4 MoAb, but the protein carrying only residues 121-309 was not reactive, suggesting that the epitope recognized by VAK 4 MoAb resides at the carboxyl terminus of p24 protein. A competitive enzyme-linked immunosorbent assay showed that patient sera containing anticore protein antibody inhibited the binding of VAK 4 to HTLV-IIIB. These findings suggested that VAK 4 MoAb recognized an immunogenic and conserved epitope belonging to a major core protein of HIV-related viruses.     

Persistent infection of rhesus macaques with T-cell-line-tropic and macrophage-tropic clones of simian/human immunodeficiency viruses (SHIV).

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.     

Nef proteins encoded by human and simian immunodeficiency viruses induce the accumulation of endosomes and lysosomes in human T cells

The nef gene of human and simian immunodeficiency viruses encodes a 27–32-kDa myristoylated protein that is expressed at high levels early after infection. Many functions have been ascribed to the Nef protein, including the down-regulation of cell surface CD4 and a role in viral infectivity. This report describes a novel effect of the Nef protein on human T cells. Electron microscopy was used to examine human T cell lines stably expressing functionally active simian or human immunodeficiency virus type 1 Nef proteins. These studies revealed that the subcellular morphology of Nef-expressing cells was dramatically altered as compared with control cells. The Nef-expressing cells contained numerous membrane-bound vesicles prominently displayed throughout the cytoplasm. The vesicles were analyzed by immunoelectron microscopy (IEM) and by the accumulation of internalized nonspecific membrane tracer, and thus identified as late endosomes and lysosomes. The accumulation of endosomes and lysosomes in response to the expression of Nef was a consistent finding, observed with several different viral isolates and human T cell lines.     

Isolation and characterization of simian immunodeficiency viruses from two subspecies of African green monkeys

Cercopithecus aethiops (African Green monkey), a nonhuman primate species distributed throughout subsaharan Africa, has been shown to have high seroprevalence rates of antibodies to simian immunodeficiency virus (SIV), and therefore, has been proposed as a natural reservoir for immunodeficiency viruses. Our laboratories have isolated SIV-like viruses from two East African subspecies of C. aethiops designated grivet and vervet monkeys. Analysis of the structural proteins based on the molecular weights and immunologic cross-reactivity to the prototypic SIV(MAC), HIV-1, and HIV-2 isolates suggests that these viruses are distinctly different. Heterogeneity was observed in the molecular weights of the gag, pol, and env gene products between SIV isolates from vervets [SIV(AGM(VER))] and grivets [SIV(AGM(GRI))]. Phenotypically, SIV(AGM(VER)) isolates were distinguishable from SIV(AGM(GRI)) isolates by the apparent size difference of the major core antigen p24. All SIV(AGM(GRI)) and SIV(AGM(VER)) isolates were found to encode a transmembrane protein of approximately 40 kD (gp40) in contrast to gp32 of SIV(MAC). Furthermore, the transmembrane protein was shown to be encoded by the entire env open reading frame, unlike gp32 of SIC(MAC) or gp36 of SIV(AGM(TYO-1)). These data indicate that viruses from C. aethiops share common features with SIV(MAC) and HIV-1, but represent diverse SIV-like viruses which may vary according to subspecies and geographic location.     

Construction of an SIV/HIV type 1 chimeric virus with the human interleukin 6 gene and its production of interleukin 6 in monkey and human cells

The switch from a Th1- to a Th2-type cytokine response is reported to be involved in human immunodeficiency virus (HIV) disease progression. To study the effect of IL-6, one of the Th2-type cytokines, on AIDS pathogenesis, we constructed an SIV/HIV-1 chimeric virus (SHIV) having the human IL-6 gene (SHIV-IL6) SHIV-IL6 could replicate in M8166, a human T cell line, as well as in monkey and human peripheral blood mononuclear cells (PBMCs). Along with the SHIV-IL6 replication, IL-6 was detected in the culture supernatant by ELISA. The maximum level of IL-6 was 35, 15, and 8 ng/ml in M8166, human PBMCs, and monkey PBMCs, respectively. The expressed IL-6 was biologically active as shown by the proliferation of IL-6-dependent murine hybridoma (MH-60) cells. The inserted IL-6 gene was stable for at least four passages (45 days after the initial infection) in M8166 cells, suggesting the ability to achieve stable expression of IL-6 in long-term experiments. Therefore, we successfully established an SHIV system expressing IL-6, and this is the first report of an SHIV expressing a Th2-type cytokine. With this system, IL-6 should be expressed in the regions where the virus replicates, and therefore the inoculation of macaque monkeys with SHIV-IL6 is expected to provide further information on the etiology of AIDS.     

Adaptive evolution of simian immunodeficiency viruses isolated from 2 conventional-progressor macaques with encephalitis

Simian immunodeficiency virus-infected macaques may develop encephalitis, a feature more commonly observed in macaques with rapid progressive disease than in those with conventional disease. In this report, an analysis of 2 conventional progressors with encephalitis is described. Phylogenetic analyses of viruses isolated from the cerebrospinal fluid and plasma of both macaques demonstrated compartmentalization. Furthermore, these viruses appear to have undergone adaptive evolution to preferentially replicate in their respective cell targets of monocyte-derived macrophages and peripheral blood mononuclear cells. A statistically significant loss of potential N-linked glycosylation sites in glycoprotein 160 was observed in viruses isolated from the central nervous system.     

Construction of chimeric vaccinia viruses by molecular cloning and packaging.

Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology.     

Biohazards and simian viruses.

Nonhuman primates are extensively used in laboratories as experimental animals. It is necessary, however, to realize that their employment may be dangerous to man and other species of primates, if recognition of their flora and fauna, especially viral, is not considered and if appropriate controls are not followed. Several outbreaks have occurred, resulting in high mortality and morbidity of man and simian. A number of recommendations are provided which, if followed, will minimize the waste of time, money, and life.