Derivation and biological characterization of a molecular clone of SHIV(KU-2) that causes AIDS, neurological disease, and renal disease in rhesus macaques.

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.

Liu ZQ ，Muhkerjee S ，Sahni M ，McCormick-Davis C ，Leung K ，Li Z ，Gattone VH 2nd ，Tian C ，Doms RW ，Hoffman TL ，Raghavan R ，Narayan O ，Stephens EB ... -  《VIROLOGY》

A simian human immunodeficiency virus with a nonfunctional Vpu (deltavpuSHIV(KU-1bMC33)) isolated from a macaque with neuroAIDS has selected for mutations in env and nef that contributed to its pathogenic phenotype.

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.

Singh DK ，McCormick C ，Pacyniak E ，Lawrence K ，Dalton SB ，Pinson DM ，Sun F ，Berman NE ，Calvert M ，Gunderson RS ，Wong SW ，Stephens EB ... -  《VIROLOGY》

A molecular clone of simian-human immunodeficiency virus (DeltavpuSHIV(KU-1bMC33)) with a truncated, non-membrane-bound vpu results in rapid CD4(+) T cell loss and neuro-AIDS in pig-tailed macaques.

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 (DeltavpuSHIV(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 DeltavpuSHIV(KU-1bMC33) released viral p27 into the culture medium with slightly reduced kinetics compared with cultures inoculated with SHIV(KU-1bMC33). Inoculation of DeltavpuSHIV(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.

McCormick-Davis C ，Dalton SB ，Hout DR ，Singh DK ，Berman NE ，Yong C ，Pinson DM ，Foresman L ，Stephens EB ... -  《VIROLOGY》

Chronology of genetic changes in the vpu, env, and Nef genes of chimeric simian-human immunodeficiency virus (strain HXB2) during acquisition of virulence for pig-tailed macaques.

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 SIVmac239), 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 (SHIVKU-1) causes subtotal elimination of CD4(+) T cells and fatal opportunistic infections, usually within 6 months. Genetic characterization of SHIVKU-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.

McCormick-Davis C ，Zhao LJ ，Mukherjee S ，Leung K ，Sheffer D ，Joag SV ，Narayan O ，Stephens EB ... -  《VIROLOGY》

Innate differences between simian-human immunodeficiency virus (SHIV)(KU-2)-infected rhesus and pig-tailed macaques in development of neurological disease.

Buch SJ ，Villinger F ，Pinson D ，Hou Y ，Adany I ，Li Z ，Dalal R ，Raghavan R ，Kumar A ，Narayan O ... -  《VIROLOGY》