Conserved CXCR4 usage and enhanced replicative capacity of HIV-2/287, an isolate highly pathogenic in Macaca nemestrina.

OBJECTIVE: To investigate viral properties that contribute to the pathogenic potential of HIV-2 in macaques. DESIGN: We compared HIV-2/287, a virus highly pathogenic in Macaca nemestrina, with its non-pathogenic progenitor HIV-2 EHO, for coreceptor usage and ability to infect human and macaque peripheral blood mononuclear cells (PBMC). METHODS: Coreceptor usage was determined in GHOST cells expressing known coreceptors, and in PBMC with coreceptor-specific inhibitors. Infectivity in PBMC was determined by virus titration and p27 antigen production. Early and late products of reverse transcription were measured by PCR with primers specific for the long terminal repeat (LTR), or the gag region, respectively. RESULTS: Both viruses preferentially infect HOS-CD4 cells expressing CXCR4. Inhibition by CXCR4-specific peptide TW70 and monoclonal antibody 12G5 indicated that both viruses use predominantly CXCR4 to infect macaque and human PBMC. HIV-2/287 showed greater infectivity than HIV-2 EHO in macaque cells, but the situation was reversed in human cells. Kinetic analysis of reverse transcription products revealed no restriction in reverse transcription following HIV-2 EHO infection of macaque PBMC. However, comparison of the level of newly initiated HIV-2 EHO DNA in macaque and human PBMC indicated that there is an early restriction, prior to the initiation of reverse transcription. CONCLUSIONS: Results indicate that the adaptation of HIV-2 EHO in M. nemestrina to a highly pathogenic virus HIV-2/287 is not correlated with a shift in or an expansion of coreceptor usage, but with the acquisition of an ability to overcome restrictions for growth in macaque PBMC.

N-linked glycosylation in the V3 region of HIV type 1 surface antigen modulates coreceptor usage in viral infection.

The V3 hypervariable region of HIV-1 surface protein has been identified as a major determinant for viral tropism and coreceptor usage. However, the role of the highly conserved N-linked glycan at the V3 loop remains controversial. To further examine its role in viral infection, we introduced a conservative amino acid substitution (asparagine to glutamine) in the V3-proximal glycosylation motif (Asn-X-Ser/Thr) in the surface glycoprotein of a CXCR4-using virus (BRU), a CCR5-using virus (SF162), and a dual-tropic virus (89.6). The effect of the mutation was determined by complementation assays, and by infectivity on CEMx174 and U373-MAGI cells expressing either CXCR4 or CCR5. The mutation resulted in decreased CXCR4 usage by SHIV89.6, but increased usage by BRU. Similarly, it abrogated CCR5 usage by SHIV89.6, but had no effect on SF162. This effect was not dependent on the specific amino acid substitution used, because a threonine-toalanine mutation in the same motif in 89.6 Env yielded identical results as the asparagine-to-glutamine mutation. These findings support the notion that multiple factors, including glycosylation at V3, contribute to coreceptor usage and that the particular effects exerted by the N-linked glycan itself appear to be isolate dependent.

Derivation and characterization of a highly pathogenic isolate of human immunodeficiency virus type 2 that causes rapid CD4+ cell depletion in Macaca nemestrina.

With few exceptions, humans are the only species known to develop acquired immunodeficiency syndrome (AIDS) after human immunodeficiency virus (HIV) infection. We report here that an isolate of HIV type 2, EHO, readily established persistent infection in 100% of Macaca nemestrina in three consecutive transmission studies. Of the eight infected animals, five showed persistently high virus load and six developed AIDS-like diseases or CD4+ cell depletion within 4 years of infection. The pathology and clinical signs closely parallel those of HIV-1 infection of humans, including lymphadenopathy, anemia, CD4+ cell depletion, and opportunistic infections. A cell-free virus stock was established from the lymph nodes of an animal that developed AIDS-like diseases. This virus, HIV-2/287, was highly pathogenic in M. nemestrina, causing CD4+ cell depletion within 2-8 weeks postinfection. While both HIV-2 EHO and HIV-2/287 use predominantly CXCR4, the latter shows greatly enhanced replicative capacity in macaque peripheral blood mononuclear cells (PBMCs). The establishment of a human immunodeficiency virus that causes rapid and reproducible CD4 cell depletion in macaques could facilitate the study of HIV pathogenesis and the development of effective vaccines and therapy against AIDS.

Simian immunodeficiency virus replicates to high levels in sooty mangabeys without inducing disease.

A serologic survey of primates living in a French zoo allowed identification of three cases of infection with simian immunodeficiency virus in sooty mangabeys (Cercocebus atys) (SIVsm). Viral isolates, which were designated SIVsmFr66, SIVsmFr74, and SIVsmFr85, were obtained after short-term culture of mangabey lymphoid cells. Phylogenetic analysis of gag and env sequences amplified directly from mangabey tissues showed that the three SIVsmFr were genetically close and that they constituted a new subtype within the diverse SIVsm-SIVmac-human immunodeficiency virus type 2 (HIV-2) group. We could reconstruct the transmission events that likely occurred in 1986 between the three animals and evaluate the divergence of SIVsmFr sequences since transmission. The estimated rate of mutation fixation was 6 x 10(-3) substitutions per site per year, which was as high as the rate found for SIVmac infection in macaques. These data indicated that SIVsmFr replicated at a high rate in mangabeys, despite the nonpathogenic character of infection in this host. The viral load evaluated by competitive PCR reached 20,000 viral DNA copies per 10(6) lymph node cells. In addition, productively infected cells were readily detected in mangabey lymphoid tissues by in situ hybridization. The amounts of viral RNA in plasma ranged from 10(5) to 10(7) copies per ml. The cell-associated and plasma viral loads were as high as those seen in susceptible hosts (humans or macaques) during the asymptomatic stage of HIV or SIVmac infections. Thus, the lack of pathogenicity of SIVsm for its natural host cannot be explained by limited viral replication or by tight containment of viral production.

Nucleotide sequence of the HIV-2 EHO genome, a divergent HIV-2 isolate.

PIP: The HIV-2 EHO isolate from Cote d'Ivoire has been characterized as a highly cytopathic HIV-2 strain, which can be differentiated from other isolates by the smaller size of its external envelope glycoprotein. The entire nucleotide sequence (10,352 bp) of the HIV-2 EHO genome is filed in the EMBL/Genbank Data Libraries under Accession No. U27200. Despite its high degree of variability, the genetic organization of HIV-2 EHO was found to be similar to other HIV-2 and simian immunodeficiency virus (SIV) isolates: it contains the long terminal repeats (LTR) and the 5' and 3' ends, the structural genes, and the genes encoding proteins which transactivate the HIV genome and those for accessory proteins. McClure et al. have demonstrated that HIV-2 EHO can infect Macaca nemestrina, resulting in progression to an AIDS-like disease in 50% of the macaques challenged. The researchers have more recently reported that passage of HIV-2 EHO in such macaques increases its virulence, resulting in a persistent infection and progression to AIDS in all inoculated animals. Given these observations, its unique biological and genetic properties, and the fact that it was isolated from a full-blown AIDS patient, it is plausible to suggest that HIV-2 EHO represents a distinct subtype of HIV-2 with an increased potential to trigger a rapid disease progression toward AIDS. The nucleotide sequence reported in this paper should be helpful in determining the prevalence of HIV-2 EHO in HIV-2 seropositive patients, and in evaluating its role in the evolution of AIDS.^ieng

Characterization of monoclonal antibodies to human immunodeficiency virus type 2 envelope glycoproteins.

Twelve murine monoclonal antibodies (MAbs) to human immunodeficiency virus type 2 (isolate ROD) envelope glycoproteins have been generated and characterized. Nine MAbs were specific to the external gp125 and three reacted with the transmembrane gp36. A large majority of MAbs displayed a significant affinity for the native gp140 precursor and were shown to bind to viral antigens on the surface of fixed HIV-2-infected cells. In Western blot analysis, the 12 MAbs showed varying profiles of cross-reactivity, but none of the MAbs cross-reacted with the HIV-1LAI envelope. Six MAbs reacted exclusively with the homologous HIV-2ROD isolate whereas only two MAbs displayed cross-reactivity with HIV-2ROD, HIV-2EHO, and SIVmac251. The four other MAbs cross-reacted with either HIV-2EHO or SIVmac251. Results of competitive binding assays indicated that the three anti-gp36 MAbs shared the same competition group, whereas at least eight competition groups were defined with the nine anti-gp125 MAbs. The epitopes of the three anti-gp36 and four anti-gp125 MAbs have been delineated using synthetic peptides or by immunological screening of an SIVmac251 peptide library expressed in yeast. The anti-gp36 MAbs are directed against the same domain of the transmembrane gp36 corresponding to the major antigenic determinant of HIV-2 and HIV-1. The four anti-gp125 MAbs recognize four distinct epitopes localized in the V2, V3, and C1 domains. None of the 12 MAbs displayed neutralizing activity against HIV-2ROD, including the 2 MAbs directed against the V2 and V3 domains.(ABSTRACT TRUNCATED AT 250 WORDS)

HIV-2 EHO isolate has a divergent envelope gene and induces single cell killing by apoptosis.

The human immunodeficiency virus type 2 (HIV-2)-related isolate, referred to as HIV-2 EHO, has been isolated from an Ivory Coast patient with acquired immunodeficiency syndrome (AIDS). Infection of CD4 expressing cells with this highly infectious virus mediates a cytopathic effect characterized by single-cell killing as a consequence of apoptosis. Nucleotide sequence analysis of the HIV-2 EHO genome revealed a significant degree of divergence of its envelope gene from that of other known HIV-2 strains. This divergence for the deduced amino acid sequence corresponding to the extracellular envelope glycoprotein was 26 to 30%. These unique genetic and biological properties suggest that the HIV-2 EHO isolate is a distinct prototype in the HIV-2 family.

The cytopathic effect of HIV is associated with apoptosis.

Large amounts of histones, H1, H2A, H2B, H3, and H4, were observed in total extracts of T4 lymphocytes and derived cell lines infected with the human immunodeficiency virus (HIV) type 1 or type 2. These histones were simply detectable by analysis of crude cellular extracts by polyacrylamide gel electrophoresis in SDS and staining the proteins with Coomassie blue or by immunoblot assays using specific polyclonal antibodies. The histones were found to be localized in the nucleoplasm, bound to low molecular weight (LMW) DNA in the form of nucleosomes. The mechanism responsible for the accumulation of nucleosomes during HIV infection was found to be due to fragmentation of cellular DNA, a mechanism referred to as apoptosis or programmed cell death in which a nuclear endonuclease becomes activated and cleaves DNA at internucleosomal regions. Accordingly, the LMW DNA accumulated in the course of infection was found to have a characteristic pattern of nucleosomal ladder and its accumulation was reduced in the presence of zinc, a known inhibitor of the endonuclease. Routinely in acute HIV infections, the accumulation of nucleosomes was observed at least 24 hr before lysis of infected cells. In a particular HIV-1 infection, in which the first signals of the cytopathic effect (vacuolization of cells and appearance of syncytia) was observed at Days 6-7 whereas maximal virus production occurred at Days 10-17, the accumulation of nucleosomes was at its maximal level already on Day 6 postinfection. In the nucleoplasm of chronically infected cells producing virus but not manifesting a cytopathic effect, no LMW DNA or histones were detectable. These observations indicate that the cytopathic effect of HIV infection is associated with apoptosis. The detection of histones and oligonucleosomal DNA fragments in the nucleoplasm can be used as a convenient marker for chromatin fragmentation during this process.