Role of Targeted Mutations in ViF on SHIV Pathogenesis

描述(申请人提供)：所有已知的灵长类慢病毒编码一种病毒感染性因子或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整合到病毒粒子中会导致新合成的DNA发生突变，研究表明这可能是病毒基因组中引入遗传变异的一种机制。现在已经确定了Vif蛋白上与APOBEC3G相互作用的结构域，以及与Cul5/Elongin B/Elongin C/Rbxl泛素连接酶相互作用的结构域。SLQXLA结构域在灵长类慢病毒中高度保守，参与了与Cul5泛素连接酶的相互作用。然而，还没有研究使用猕猴模型来检验该基序中的靶向突变在病毒发病机制中的作用。以SIVmac239为遗传背景的猴免疫缺陷病毒(SIV-1)Tat rev、VPU和env基因的致病分子克隆，可在接种后1个月内高水平复制，导致CD4+T细胞的严重丧失。利用其中一个克隆，SHIVKu-tbMC33&gt；我们建议研究Vif蛋白SLQXLA结构域中有针对性的氨基酸替换在病毒致病中的作用。我们已经产生了SHIVKU-1bmc33的衍生物，其中SLQXLA基序中的一个(Shiv/vifaq)或三个(Shiv/vifAAA)氨基酸发生了改变。在第一个具体目标中，我们建议用两种突变病毒分别接种四只猕猴，并在六个月的过程中评估病毒载量、循环中的CD4+T细胞计数和组织致病作用。在第二个特定目标中，我们建议确定Vif蛋白中的目标氨基酸替换是否稳定和/或是否在感染过程中选择补偿Vif中氨基酸变化的突变。在第三个具体目标中，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。为了解决这一假设，我们建议检查病毒基因组的we/基因是否有增加的突变，并将其与从接种了亲本SHIVKu-ibMC33的猕猴中分离的nef基因进行比较-来自该突变的信息将为体内可能由APOBEC3G诱导的病毒基因组突变提供有用的体内信息。这项拟议的研究将利用相关的猕猴模型提供有关Vif基序在发病机制中作用的新信息。

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.