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Previous studies have shown that the gene coding for the Vpu protein of the human immunodeficiency virus type 1 (HIV-1) is 5′ to the env gene, is in a different reading frame, and overlaps the env by 90 nucleotides. In this study, we examined the processing of the Env protein as well as the maturation and infectivity of a virus (SHIV Vpenv ) in which a single nucleotide was removed at the vpu–env junction, fusing the first 162 bases of vpu to the env ORF. Pulse-chase analysis revealed that SHIV Vpenv -infected cells gave rise to two precursor glycoprotein species (gp160 and gp175). Immune precipitation results also revealed that an anti-Vpu serum could immune precipitate the gp175 precursor, suggesting that the amino-terminal Vpu sequence was fused to the Env protein. Growth curves revealed that the SHIV Vpenv -inoculated cultures released approximately three times more p27 into the culture medium than parental SHIV KU-1bMC33 . Electron microscopy revealed that while both viruses matured at the cell plasma membrane, significantly higher quantities of virus particles were cell associated on SHIV Vpenv -infected cells compared to cultures inoculated with parental SHIV KU-1bMC33 . Furthermore, virus was observed maturing into intracellular vesicles of SHIV Vpenv -infected cells. To assess the pathogenicity of SHIV Vpenv , three pig-tailed macaques were inoculated with the SHIV Vpenv and monitored for 6 months for CD4 + T cell levels, viral loads, and the stability of the deletion at the vpu–env junction. Our results indicated that SHIV Vpenv caused a severe CD4 + T cell loss in all three macaques within weeks of inoculation. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that this mutation was stable during the period of rapid CD4 + T cell loss. Sequence analysis showed that with increasing time of infection, the one base pair deletion was repaired in all three macaques inoculated with SHIV Vpenv with the reversion occurring at 10 weeks in macaque CT1G and at 12 weeks in macaque CP3R and CT1R. These results indicate that fusion of the first 54 amino acids of Vpu to Env results in intracellular maturation of virus, and accumulation of virus within intracellular vesicles as well as on the cell plasma membrane. Our results indicate that while fusion of the vpu gene to env results in a virus that is still pathogenic for pig-tailed macaques, there is a selective pressure to maintain the vpu and env genes in separate reading frames.

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.

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 Δ vpu SHIV 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 Δ vpu SHIV 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.

We compared the Vpu sequences from 101 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. In addition to the highly conserved casein kinase II (CKII) phosphorylation sites, our analysis identified additional invariant residues in the transmembrane domain and in the first and second alpha-helical domains. Our analysis revealed that all subtype C sequences had a conserved LRLL motif at the C terminus that was also found in A/C intersubtype recombinants. While our analysis demonstrated the conservation of CKII domains in HIV-1 group M and O isolates, the number of potential CKII phosphorylation sites was variable in SIVcpz sequences. The results of this study will provide a basis for future mutagenesis studies to examine the role of certain amino acid residues in the structure and function of Vpu.

We report on the role of vpu in the pathogenesis of a molecularly cloned simian-human immunodeficiency virus (SHIV KU-1bMC33 ), in which the tat , rev , vpu , env , and nef genes derived from the uncloned SHIV KU-1b virus were inserted into the genetic background of parental nonpathogenic SHIV-4. A mutant was constructed (Δ vpu SHIV KU-1bMC33 ) in which 42 of 82 amino acids of Vpu were deleted. Phase partitioning studies revealed that the truncated Vpu was not an integral membrane protein, and pulse-chase culture studies revealed that cells inoculated with Δ vpu SHIV KU-1bMC33 released viral p27 into the culture medium with slightly reduced kinetics compared with cultures inoculated with SHIV KU-1bMC33 . Inoculation of Δ vpu SHIV KU-1bMC33 into two pig-tailed macaques resulted in a severe decline of CD4 + T cells and neurological disease in one macaque and a more moderate decline of CD4 + T cells in the other macaque. These results indicate that a membrane-bound Vpu is not required for the CD4 + T cell loss and neurological disease in SHIV-inoculated pig-tailed macaques. Furthermore, because the amino acid substitutions in the Tat and Rev were identical to those previously reported for the nonpathogenic SHIV PPc , our results indicate that amino acid substitutions in the Env and/or Nef were responsible for the observed CD4 + T cell loss and neurological disease after inoculation with this molecular clone.

Recently, we developed a highly pathogenic variant of simian–human immunodeficiency virus, SHIV-4 (containing the tat, rev, vpu, and env of the HXB2 strain of HIV-1 in a genetic background of SIV mac 239), through a series of four bone marrow-bone marrow passages—first in rhesus monkeys and then in pig-tailed macaques [Joag et al. (1996) J. Virol. 70, 3189–3197]. Inoculation of pig-tailed macaques with this pathogenic virus (SHIV KU-1 ) causes subtotal elimination of CD4 + T cells and fatal opportunistic infections, usually within 6 months. Genetic characterization of SHIV KU-1 showed that it has a functional vpu gene (the first codon is ATG vs ACG for the vpu of SHIV-4) and several amino acid substitutions in Env and nef [Stephens et al. (1997) Virology 231, 313–321]. Two pig-tailed macaques, PPc and PQc, were the first to develop a severe loss of CD4 + T cells and the acquired immune deficiency syndrome and were euthanized at 26 and 105 weeks, respectively. In this report, we analyzed the changes that occurred in the vpu, nef, and env (gp120) genes of the virus used to inoculate macaques PPc and PQc and established the chronology of changes that occurred in these viral genes as these two animals lost their CD4 + T cells and progressed to develop acquired immune deficiency syndrome. Compared with SHIV-4, the virus used to inoculate macaques PPc and PQc had 0, 3, and 0 consensus amino acid changes in the Vpu, gp120, and Nef, respectively. An analysis of the viral sequences amplified from peripheral blood mononuclear cells samples taken at various times after inoculation of PPc revealed that the vpu had not reverted to an open reading frame (closed vpu, ACG) at 4 weeks after inoculation, but by 16 weeks vpu had reverted to an open reading frame (open vpu, ATG). Macaque PQc, which had a longer course of disease, had a closed vpu at 4 and 16 weeks, but by 28 weeks, both closed and open vpu were detected. From 39 to 105 weeks, only an open vpu was detected. In both macaques, the reversion to an open vpu correlated well with the second phase (major) of CD4 + T cell loss. An analysis of the nef and env sequences isolated from the same times after inoculation revealed an association between the reversion of vpu to an open reading frame and the accumulation of increased numbers of consensus changes in these two viral proteins. These data suggest that the concomitant reversion of vpu to an open reading frame along with increased substitutions in Nef and gp120 were important genetic changes in the viral genome that were responsible for the increased and highly efficient rate of replication of the virus in CD4 + T cells and macrophages, which in turn led to elimination of the CD4 + T cells and profound loss of immunocompetence in the infected animals.

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.

A chimeric simian-human immunodeficiency virus (SHIV-4) containing the tat, rev, vpu, and env genes of HIV type 1 (HIV-1) in a genetic background of SIVmac239 was used to develop an animal model in which a primate lentivirus expressing the HIV-1 envelope glycoprotein caused acquired immune deficiency syndrome (AIDS) in macaques. An SHIV-infected pig-tailed macaque that died from AIDS at 24 weeks postinoculation experienced two waves of viremia: one extending from weeks 2-8 and the second extending from week 18 until death. Virus (SHIVKU-1) isolated during the first wave was neutralized by antibodies appearing at the end of the first viremic phase, but the virus (SHIVKU-1b) isolated during the second viremic phase was not neutralized by these antibodies. Inoculation of SHIVKU-1b into 4 pig-tailed macaques resulted in severe CD4(+) T cell loss by 2 weeks postinoculation, and all 4 macaques died from AIDS at 23-34 weeks postinoculation. Because this virus had a neutralization-resistant phenotype, we sequenced the env gene and compared these sequences with those of the env gene of SHIVKU-1 and parental SHIV-4. With reference to SHIV-4, SHIVKU-1b had 18 and 6 consensus amino acid substitutions in the gp120 and gp41 regions of Env, respectively. These compared with 10 and 3 amino acid substitutions in the gp120 and gp41 regions of SHIVKU-1. Our data suggested that SHIVKU-1 and SHIVKU-1b probably evolved from a common ancestor but that SHIVKU-1b did not evolve from SHIVKU-1. A chimeric virus, SHIVKU-1bMC17, constructed with the consensus env from the SHIVKU-1b on a background of SHIV-4, confirmed that amino acid substitutions in Env were responsible for the neutralization-resistant phenotype. These results are consistent with the hypothesis that neutralizing antibodies induced by SHIVKU-1 in pig-tailed macaque resulted in the selection of a neutralization-resistant virus that was responsible for the second wave of viremia.

Previously, we described the derivation of a pathogenic strain of simian–human immunodeficiency virus (SHIV KU-2 ) consisting of the tat, rev, vpu, and env genes of HIV-1 (strain HXB2) in a genetic background of SIV mac 239 that causes AIDS and productive infection of the CNS in rhesus macaques ( Macca mulatta ) (Raghavan et al., 1997, Brain Pathol. 7, 851–861). We report here on the characterization of a molecular clone of SHIV KU-2 , designated SHIV KU-2MC4 , that caused CD4 + T cell loss as well as neurological and renal disease in macaques. DNA sequence analysis of selected SIV regions of SHIV KU-2MC4 revealed 10 nucleotide changes in the LTR, whereas Gag, Vif, Vpr, Vpx, and Nef had 1, 1, 1, 2, and 13 predicted amino acid substitutions, respectively, compared to SIV mac 239. DNA sequence analysis of HIV-1 derived regions of SHIV KU-2MC4 revealed 2, 1, 2, and 18 predicted amino acid substitutions in the Tat, Rev, Vpu, and Env proteins, respectively, when compared to SHIV-4. Unlike the parental SHIV-4, which is not tropic for macrophages, SHIV KU-2MC4 replicated efficiently in macrophage cultures as determined by p27 assays. However, despite the numerous changes in the Env protein and newly acquired tropism for macrophages, SHIV KU-2MC4 , like the parental SHIV-4, used CXCR4 exclusively as its coreceptor for entry into susceptible cells. Inoculation of SHIV KU-2MC4 into two rhesus macaques resulted in severe infection in which the numbers of circulating CD4 + T cells in the blood declined rapidly by 2 weeks postinoculation and virus producing cells in the peripheral blood mononuclear cells were identified throughout the course of infection. At the time of euthanasia (20 and 22 weeks), both macaques had lost a significant amount of weight and had no circulating CD4 + T cells. In addition, one macaque developed intension tremors and uncoordinated movements. Virological examination of tissues at necropsy revealed active virus replication in both lymphoid and nonlymphoid tissues such as the lung and brain. Histological examination revealed that the induced immunodeficiency was associated with lymphoid depletion of the lymph nodes and spleen, opportunistic infections, lentiviral encephalitis, and severe glomerulosclerosis of the kidney. This molecular clone will serve as the basis for analyzing the molecular determinants through which SHIV KU-2 causes severe CD4 + T cell loss, neurological disease, and SHIV nephropathy in rhesus macaques.

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 (no vpu SHIV KU-1bMC33 ). While both SHIV KU-1bMC33 and no vpu SHIV KU-1bMC33 synthesized comparable amounts of env mRNA in infected cells, the no vpu SHIV KU-1bMC33 -infected cells synthesized more Env precursor when standardized against the p57 Gag precursor protein. While more Env was synthesized than Gag in no vpu SHIV 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 no vpu SHIV 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.