Chronology of Genetic Changes in thevpu, env,andnefGenes of Chimeric Simian–Human Immunodeficiency Virus (Strain HXB2) during Acquisition of Virulence for Pig-Tailed Macaques

Abstract

Recently, we developed a highly pathogenic variant of simian–human immunodeficiency virus, SHIV-4 (containing thetat, rev, vpu,andenvof 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 [Joaget 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-1showed that it has a functionalvpugene (the first codon is ATG vs ACG for thevpuof SHIV-4) and several amino acid substitutions in Env andnef[Stephenset al.(1997)Virology231, 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 thevpu, nef,andenv(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 thevpuhad not reverted to an open reading frame (closedvpu,ACG) at 4 weeks after inoculation, but by 16 weeksvpuhad reverted to an open reading frame (openvpu,ATG). Macaque PQc, which had a longer course of disease, had a closedvpuat 4 and 16 weeks, but by 28 weeks, both closed and openvpuwere detected. From 39 to 105 weeks, only an openvpuwas detected. In both macaques, the reversion to an openvpucorrelated well with the second phase (major) of CD4+T cell loss. An analysis of thenefandenvsequences isolated from the same times after inoculation revealed an association between the reversion ofvputo 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 ofvputo 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.