Role of Targeted Mutations in ViF on SHIV Pathogenesis

描述（由申请人提供）：所有已知的灵长类慢病毒编码病毒感染因子或Vif，该因子与胞苷脱氨酶APOBEC3G相互作用，以防止其并入病毒粒子。研究表明，将APOBEC3G掺入病毒粒子会导致新合成的DNA中引入突变，研究表明这可能是病毒基因组中引入遗传变异的机制。现在已经在Vif蛋白上发现了与APOBEC3G和Cul5/伸长蛋白B/伸长蛋白C/Rbxl泛素连接酶相互作用的结构域。被称为SLQXLA的高度保守结构域在灵长类慢病毒中是高度保守的，并且与Cul5泛素连接酶的相互作用有关。然而，尚无研究利用猕猴模型检测该基序的靶向突变在病毒发病机制中的作用。在SIVmac239遗传背景下，含有HTV-1的rev、vpu和env基因的猿类人类免疫缺陷病毒（SHIV）的致病性分子克隆已经被衍生出来，这些克隆在接种后1个月内复制到高水平，导致CD4+T细胞严重损失。利用其中一个克隆SHIVKu-tbMC33>，我们打算研究Vif蛋白SLQXLA结构域的靶向氨基酸替换在病毒发病中的作用。我们已经生成了SHIVKU-1bmc33的衍生物，其中SLQXLA基序中的一个（SHIV/vifalq）或三个（SHIV/vifAAA）氨基酸被改变。在第一个特定的目标中，我们建议用这两种突变病毒中的每一种接种四只猕猴，并在六个月的时间内评估病毒载量、循环CD4+ T细胞计数和组织发病机制。在第二个特定目标中，我们建议确定Vif蛋白中的靶向氨基酸替换是否稳定和/或在感染过程中是否选择了补偿Vif中氨基酸变化的突变。在第三个特定目标中，我们建议确定Vif中的靶向氨基酸替换是否会在感染期间加速病毒基因组内突变的积累。为了验证这一假设，我们建议检测病毒基因组的we/基因是否增加突变，并将其与从接种了亲代SHIVKu-ibMC33的猕猴中分离的nef基因进行比较。该突变体的信息将为体内apobec3g诱导的病毒基因组突变提供有用的体内信息。本研究将利用相关的猕猴模型，为该Vif基序在发病机制中的作用提供新的信息。

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.