Role of Targeted Mutations in ViF on SHIV Pathogenesis

ViF 靶向突变在 SHIV 发病机制中的作用

• 批准号: 6947546
• 负责人: Edward Brice Stephens
• 金额: $ 22.05万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6947546
• 关键词: Macaca nemestrina; amination; aminohydrolases; biotechnology; cell line; cytidine; gene expression; gene mutation; gene targeting; helper T lymphocyte; human immunodeficiency virus; leukocyte count; ligase; protein sequence; recombinant virus; regulatory gene; simian AIDSs; simian immunodeficiency virus; site directed mutagenesis; ubiquitin; virus genetics; virus infection mechanism; virus protein

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.

描述（由申请方提供）：所有已知的灵长类慢病毒编码病毒感染因子或Vif，其与胞苷脱氨酶APOBEC 3G相互作用以防止其掺入病毒体。研究表明，将APOBEC 3G掺入病毒体导致将突变引入新合成的DNA中，研究表明这可能是在病毒基因组中引入遗传变异的机制。现已在Vif蛋白上鉴定出与APOBEC 3G相互作用和与Cul 5/延伸蛋白B/延伸蛋白C/Rbxl泛素连接酶相互作用的结构域。被称为SLQXLA结构域的高度保守结构域在灵长类慢病毒中高度保守，并且涉及与Cul 5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查该基序中的靶向突变在病毒发病机制中的作用。在SIVmac 239的遗传背景中，已经衍生出含有HIV-1的达特rev、vpu和env基因的猿猴人类免疫缺陷病毒（SHIV）的致病性分子克隆，其复制到高水平，在接种后1个月内引起CD 4 +T细胞的严重损失。使用这些克隆之一SHIVKu-tbMC 33>，我们建议研究Vif蛋白SLQXLA结构域中靶向氨基酸取代对病毒发病机制的作用。我们已经产生了SHIVKU-1bmc 33的衍生物，其中SLQXLA基序内的一个（SHIV/vifalq）或三个（SHIV/vifAAA）氨基酸被改变。在第一个具体目标中，我们建议用两种突变病毒中的每一种感染四只猕猴，并在六个月的时间内评估病毒载量、循环CD 4 + T细胞计数和组织发病机制。在第二个具体目标中，我们建议确定Vif蛋白中的靶向氨基酸取代是否稳定和/或在感染过程中是否选择补偿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中的靶向氨基酸取代是否加速感染期间病毒基因组内突变的积累。为了解决这一假设，我们提出检查病毒基因组的we/基因的增加的突变，并将其与从接种亲本SHIVKu-ibMC 33的猕猴中分离的nef基因进行比较。来自该突变体的信息将提供关于体内病毒基因组中可能的APOBEC 3G诱导的突变的有用的体内信息。拟议的研究将提供新的信息，这一Vif基序的发病机制中的作用，使用相关的猕猴模型。