Role of Targeted Mutations in ViF on SHIV Pathogenesis

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DESCRIPTION (provided by applicant): All known primate lentiviruses encode for a viral infectivity factor or Vif, which interacts with the cytidine deaminase APOBEC3G to prevent its incorporation into virions. Studies have shown that incorporation of APOBEC3G into the virion results in the introduction of mutations into newly synthesized DNA and studies suggest that this could be a mechanism for the introduction of genetic variation in the viral genome. Domains have now been identified on the Vif protein that interact with the APOBEC3G and the interact with the Cul5/Elongin B/Elongin C/Rbxl ubiquitin ligase. The highly conserved domain known as the SLQXLA domain is highly conserved among the primate lentiviruses and has been implicated in the interaction with the Cul5 ubiquitin ligase. However, no studies have examined the role of targeted mutations in this motif in viral pathogenesis using a macaque model. Pathogenic molecular clones of simian human immunodeficiency viruses (SHIV) containing the tat rev, vpu and env genes of HTV-1 in a genetic background of SIVmac239 have been derived that replicate to high levels, cause severe loss of CD4+T cells within 1 month after inoculation. Using one of these clones, SHIVKu-tbMC33> we propose to investigate the role of targeted amino acid substitutions in the SLQXLA domain of Vif protein on viral pathogenesis. We have generated derivatives of SHIVKU-1bmc33 in which one (SHIV/vifalq) or three (SHIV/vifAAA) amino acids within the SLQXLA motif have been altered. In the first specific Aim, we propose to inoculate four macaques with each of the two mutant viruses and to assess viral loads, circulating CD4+ T cell counts and tissue pathogenesis over the course of a six month period. In the second specific aim, we propose to determine if targeted amino acid substitutions in the Vif protein are stable and/or whether mutations compensating amino acid changes in Vif are selected for during the course of infection. In the third specific Aim, we propose to determine if targeted amino acid substitutions in Vif accelerate the accumulation of mutations within the viral genome during infection. In order to address this hypothesis, we propose to examine the we/gene of the viral genome for increased mutations and the compare it with the nef genes isolated from macaques inoculated with the parental SHIVKu-ibMC33- The information from this mutant will provide useful in vivo information on the possible APOBEC3G-induced mutations in the viral genome in vivo. The proposed studies will provide new information on the role of this Vif motif in pathogenesis using a relevant macaque model.