The Vpu protein of human immunodeficiency virus type 1 (HIV-1) has been reported to enhance virion release from infected cells and to down-regulate the expression of CD4 on infected cells. Previous studies have shown that Vpu and the envelope glycoprotein precursor (gp160) are translated from different reading frames of the same bicistronic messenger RNA (mRNA). In order to assess the effect of the Vpu sequences 5' to the Env open reading frame on Env biosynthesis and pathogenesis, we have constructed a deletion mutant of a molecularly cloned chimeric simian--human immunodeficiency virus (SHIV(KU-1bMC33)) in which the entire coding region of vpu upstream of env had been deleted (novpuSHIV(KU-1bMC33)). While both SHIV(KU-1bMC33) and novpuSHIV(KU-1bMC33) synthesized comparable amounts of env mRNA in infected cells, the novpuSHIV(KU-1bMC33)-infected cells synthesized more Env precursor when standardized against the p57 Gag precursor protein. While more Env was synthesized than Gag in novpuSHIV(KU-1bMC33)-infected cells, pulse--chase analysis revealed that p27 Gag protein was released from infected cells with delayed kinetics, a reflection of the lack of a Vpu protein. Inoculation of novpuSHIV(KU-1bMC33) into two pig-tailed macaques resulted in no loss of circulating CD4(+) T cells. However, replicating virus could be detected in the lymphoid tissues (lymph nodes, spleen, thymus) 1 year after inoculation and the thymus of one of the macaques exhibited severe atrophy. The results of these studies indicate that the Vpu coding sequences upstream of Env may attenuate the level of Env precursor biosynthesis but significantly contribute to the pathogenesis of this SHIV in pig-tailed macaques.
Proliferating cell nuclear antigen (PCNA), also called DNA polymerase δ-associated protein, is found in the cells of the proliferative compartment of normal tissues and is essential for DNA replication. It can be recognized by many monoclonal antibodies to various epitopes on the molecule. In this investigation one of these, PC 10, has been used on formalin-fixed, paraffin-embedded, human and rodent gastro-intestinal epithelial tissues to assess numerically the labelling index of PC 10 and to compare it, in the rat liver and gastrointestinal tract, with the S-phase fraction as determined by bromodeoxyuridine (BrdUrd) labelling. The distribution of PC 10-labelled cells was recorded with respect to cell position in the intestinal crypts of man. In tissues where both modes of assessment were used, PC 10 staining in the well-established proliferative compartments was found to be more extensive than that of BrdUrd. The higher labelling index with PC 10 can be explained by its identification of PCNA outside the S phase of the cell cycle and also by the long half-life of PCNA protein in post-proliferative intestinal epithelial cells as they migrate towards the villus. Nevertheless the data suggest PC 10 immunostaining in gastro-intestinal epithelia is an operational marker of cell proliferation which is reproducible, quantifiable and can be performed on routinely processed tissues.
We compared the Vif sequences from more than 100 group M and O strains of HIV-1 isolated from diverse geographical regions and various subtypes, in order to identify regions of high variability and those amino acid residues that were highly conserved or invariant. Our analysis found that there were 10 highly conserved domains with additional invariant residues located throughout the protein. Our analysis revealed that in the highly conserved amino-terminal domain, all subtype C isolates examined had a methionine-to-leucine substitution at position 8 and most subtype C isolates had an arginine-to-lysine substitution at position 17 of the protein. Our analysis revealed that the MAP kinase phosphorylation sites, and the cysteine residues at positions 114 and 133, were conserved in Vif sequences from group M, group O, and SIV cpz isolates. Our analysis also shows that the RKKR motif at positions 90--93, proposed as a nuclear transport inhibition signal (NTIS), was conserved neither in different geographical group M and O HIV-1 isolates nor in SIVcpz.
Previous studies have shown that passage of nonpathogenic SHIV-4 through a series of macaques results in the selection of variants of the virus that are capable of causing rapid subtotal loss of CD4(+) T cells and AIDS within 6-8 months following inoculation into pig-tailed macaques. Using a pathogenic variant of SHIV-4 known as SHIV(KU-1bMC33), we reported that a mutant of this virus with the majority of the vpu deleted was still capable of causing profound CD4(+) T cell loss and neuroAIDS in pig-tailed macaques (McCormick-Davis et al., 2000, Virology 272, 112-116). In this study, we have analyzed the tissue-specific changes in the env and nef in one macaque that developed neuroAIDS (macaque 50 O) and in three macaques that developed only a moderate or no significant loss of CD4(+) T cells and no neurological disease (macaques 50 Y, 20220, 20228) following inoculation with DeltavpuSHIV(KU-1bMC33). Sequence analysis of the gp120 region of env isolated from lymphoid tissues (lymph node and spleen) of macaques 50 Y, 20220, and 20228 revealed no consensus amino acid substitutions. In contrast, analysis of the gp120 sequences isolated from lymphoid and CNS tissues (parietal cortex, basal ganglia, and pons) of macaque 50 O revealed numerous amino acid substitutions. The significance of the amino acid substitutions in gp120 was supported by neutralization assays which showed that the virus isolated from the lymph node of macaque 50 O was neutralization resistant compared to the parental SHIV(KU-1bMC33). Analysis of changes in the nef gene from macaque 50 O revealed in-frame deletions in Nef that ranged from 4 to 13 amino acids in length, whereas the nef genes isolated from the other three macaques revealed no deletions or consensus amino acid substitutions. Inoculation of the virus isolated from the lymph node of the macaque which developed neuroAIDS, SHIV(50OLNV), into four pig-tailed macaques resulted in a severe loss of the circulating CD4(+) T cells within 2 weeks postinoculation, which was maintained for up to 20 weeks postinoculation, confirming that this virus had indeed become more pathogenic in pig-tailed macaques. Taken together, these observations suggest that DeltavpuSHIV(KU-1bMC33) has a low pathogenic phenotype in macaques but that individual pig-tailed macaques can select for additional mutations within the Env and Nef which can compensate for the lack of an intact Vpu and ultimately increase its pathogenicity.
Several studies have shown that deletion of the nef gene of simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) results in attenuated viruses. However, studies have not critically examined trafficking of attenuated viruses to the central nervous system (CNS) at early stages after inoculation. In this study, we investigated the colocalization of pathogenic and vpu-negative, nef-interrupted SHIVs at early stages following inoculation. The first virus, designated SHIV(50OLNV), was isolated from the lymph node of a pig-tailed macaque which developed severe CD4+ T cell loss and neurological disease. The second virus was a molecularly cloned virus in which the vpu gene was deleted and the gene for the enhanced green fluorescent protein from the jellyfish Aequoria victora had been inserted in-frame within the nef gene of the pathogenic SHIV(KU-1bMC33) (designated SHIV(KU-1bEGFP)). Three pig-tailed macaques were inoculated intravenously with equivalent amounts of two viruses, two macaques were inoculated with SHIV(KU-1bEGFP), and two macaques were inoculated with SHIV(50OLNV). The peripheral blood mononuclear cells (PBMCs) were isolated from bleeds obtained 3, 7, 10, and 14 days postinoculation and monitored for syncytia-inducing virus and for fluorescent cells. Virus was detected in the PBMCs as early as 3 days postinoculation and was present throughout the course of this short-term study. At 14 days postinoculation, the macaques were sacrificed and examined for virus in lymphoid tissues and different regions of the CNS following necropsy. Our results revealed the presence of both viruses in lymphoid and CNS tissues, although SHIV(50OLNV) was present to a much greater extent. Histological examination revealed that one macaque displayed signs of meningitis and all three macaques developed massive cortical astrocyte activation as demonstrated by immunostaining for glial fibrillary acidic protein, but only limited microglial activation. In the two macaques inoculated with SHIV(50OLNV), astrocyte activation similar to that in the macaques inoculated with both viruses was observed while no astrocyte activation was observed in macaques inoculated with SHIV(KU-1bEGFP). Thus, this study demonstrates that SHIVs with an intact nef(SHIV(50OLNV)) as well as those lacking a vpu gene and with a nonfunctional nef gene (SHIV(KU-1bEGFP)) are capable of invading the CNS and that pathogenic SHIVs are capable of causing reactive astrocytosis early after inoculation.
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