The glycosylation pattern of the external envelope glycoprotein of human immunodeficiency virus type 2 (HIV-2) was studied in dependence on host cells and virus isolates. Strains HIV-2ALT, HIV-2ROD and HIV-2D194, differing in their biological properties and in the amino acid sequences of their env genes, were propagated in MOLT4, HUT78 and U937 cells, in human peripheral blood lymphocytes and monocytes/macrophages in the presence of [6-3H]glucosamine. Radiolabelled viral glycoproteins were isolated from the cell-free supernatants and digested with trypsin. Glycans were sequentially liberated by endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F, and fractionated according to charge and size. Comparison of the oligosaccharide profiles revealed that the envelope glycoproteins of different virus isolates, propagated in the same host cells, yielded very similar glycan patterns, whereas cultivation of an isolate in different host cells resulted in markedly divergent oligosaccharide maps. Variations concerned the proportion of high-mannose-, hybrid- and complex-type substituents, as well as the state of charge and structural parameters of the complex-type species. As a characteristic feature, complex-type glycans of macrophage-derived viral glycoprotein were almost exclusively substituted by lactosamine repeats. Hence, glycosylation of the HIV-2 external envelope glycoprotein seems to be primarily governed by host cell-specific factors rather than by the amino acid sequence of the corresponding polypeptide backbone.
Human immunodeficiency virus type 2 (HIV-2)-related viruses were isolated from a Gambian dying of exclusively neurological disease (HIV-2D194) and from an asymptomatic Ghanian (HIV-2D205). Both strains exhibited properties of HIV-1 biological subtype c: they grew slowly and induced few or no syncytia but eventually produced high levels of particle-associated reverse transcriptase in cultures of fresh peripheral blood lymphocytes, and they established stable infection of T-lymphoma (HUT-78) and monocytic (U937) cell lines. Each produced even higher levels of reverse transcriptase when fresh human monocytes/macrophages were used as target cells. The viruses were molecularly cloned after a single passage in culture, in order to minimize in vitro selection of subtypes present in vivo. Restriction-site analysis showed heterogeneity within each isolate. Nucleotide sequence analysis of a portion of the HIV-2D194 genome revealed that it is a member of the prototypic HIV-2 family, displaying 13% divergence versus HIV-2ROD and HIV-2NIHZ, as compared to 9% divergence between HIV-2ROD and HIV-2NIHZ. In contrast, HIV-2D205 is the most highly divergent HIV-2 strain yet described: it is equidistant in relation between the known HIV-2 strains and the simian immunodeficiency virus isolates from rhesus macaque monkeys (23-25% divergence).
It has been suggested that the human immunodeficiency virus type 2 (HIV-2) and the simian immunodeficiency virus from rhesus macaques (SIVmac) evolved from the sooty mangabey virus SIVsm (ref. 1). We now describe an HIV-2-related isolate, HIV-2-D205, from a healthy Ghanaian woman that is genetically equidistant to the prototypic HIV-2 strains and to SIVsm and SIVmac. Supported by the observation that HIV-2D205 differs in a step of envelope glycoprotein processing, our data indicate that it could represent an alternative HIV-2 subtype and that viruses of the HIV-2/SIVsm/SIVmac group could have already infected humans before HIV-2 and SIVsm/SIVmac diverged.
The Nef gene of the human and simian immunodeficiency viruses HIV and SIV has been implicated in pathogenicity; however, the mechanism by which Nef induces disease is still unknown. An impact on signal transduction in cells has been suggested by the interaction of Nef from an HIV-1 strain and tyrosine kinases like HCK and LCK as well as serine/threonine kinases. We have confirmed the binding of HCK to HIV-1 subtype B Nef and demonstrated an equally strong interaction with a subtype E Nef protein but weaker binding to Nef of HIV-2 subtype A (HIV-2D194). No binding, however, was observed to HIV-2 subtype B Nef (HIV-2D205). Instead, this protein bound to a novel cellular protein, Nefin 1, with characteristics of an adaptor protein and strong expression in all human hematopoietic tissues. Nefin 1 binds through an amino-terminal domain, which is related to SH3 domains. For interaction of Nef with Nefin 1, the PxxP motif and the three-dimensional conformation of the molecule appear necessary. In conclusion, this study demonstrates that Nef proteins of divergent strains of HIV-1 and HIV-2 may use different elements of signal transduction pathways for the induction of pathogenicity in vivo.
The polysulfated polyxylan HOE/BAY946, which has been tested in two pilot studies in ARC/AIDS patients and in asymptomatic HIV carries in Germany, was believed to act by inhibiting virus attachment to the cell. However, the drug was also found to reduce the amount of HIV particles released from infected peripheral blood mononuclear cells (PBMC) in vitro. Furthermore, preincubation of PBMC with the drug led to a partial inhibition of a following HIV infection, suggesting that the drug also affects virus entry. Electron Paramagnetic Resonance (EPR) measurements on uninfected human lymphocytes using 5-proxyl-nonane as spin label demonstrated smaller hyperfine coupling constant (aN) values in the presence of HOE/BAY946 or dextran sulfate 5000. Accordingly, h-1p/h-1H ratios were decreased, indicating increased plasma membrane hydrophobicity and a membrane-stabilizing effect of the drugs. Culture of the chronically HIV-infected monocytic cell line U937/HIV-2D194 in the presence of HOE/BAY946 specifically and drastically reduced the release of virions and the intracellular synthesis of viral proteins as determined by radioimmunoprecipitation and reverse transcriptase assays. In conclusion, although the EPR studies showed a physico-chemical effect on membrane polarity, HOE/BAY946 and dextran sulfate clearly affect processes beyond the cell membrane. Thus, in contrast to previous reports suggesting that polysulfated sugars affect HIV only by inhibiting virus binding to uninfected cells, they clearly inhibit HIV in infected cells as well and appear to have a pleiotropic mode of action. Such drugs may be less likely to result in viral resistance after prolonged application than substances acting only on one step in the life cycle of the virus.
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