Role of Targeted Mutations in ViF on SHIV Pathogenesis

ViF 靶向突变在 SHIV 发病机制中的作用

基本信息

批准号：
7026384
负责人：
Edward Brice Stephens
金额：
$ 21.53万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
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

项目摘要

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（由申请人提供）：所有已知的灵长类动病毒编码病毒感染因子或VIF，它们与胞苷脱氨酶Apobec3g相互作用，以防止其掺入病毒体中。研究表明，将APOBEC3G掺入病毒体导致将突变引入新合成的DNA中，研究表明，这可能是引入病毒基因组遗传变异的一种机制。现在已经在与apobec3g相互作用的VIF蛋白上鉴定了域，并与CUL5/ELONGIN B/ELONGIN C/RBXL泛素连接酶相互作用。高度保守的结构域称为SLQXLA结构域在灵长类动病毒中高度保守，并且与CUL5泛素连接酶的相互作用有关。但是，尚未研究使用猕猴模型在病毒发病机理中靶向突变在该基序中的作用。在SIVMAC239的遗传背景中，含有HTV-1的TAT REV，VPU和ENV基因的Simian人类免疫缺陷病毒（SHIV）的致病分子克隆已得出，复制到高水平，在接种后1个月内导致CD4+T细胞的严重丢失。使用这些克隆之一，Shivku-TBMC33>我们建议研究靶向氨基酸取代在VIF蛋白SLQXLA结构域中在病毒发病机理中的作用。我们已经生成了Shivku-1BMC33的衍生物，其中一个（SHIV/VIFALQ）或三个（Shiv/Vifaaa）氨基酸已经改变了。在第一个特定目的中，我们建议在两个突变病毒中的每一个中接种四个猕猴，并评估病毒载量，在六个月的时间内循环CD4+ T细胞计数和组织发病机理。在第二个特定目的中，我们建议确定VIF蛋白中的靶向氨基酸取代是稳定的，并且/或在感染过程中选择了补偿VIF中氨基酸变化的突变。在第三个特定目的中，我们建议确定VIF中的靶向氨基酸取代是否会在感染过程中加速病毒基因组中突变的积累。为了解决这一假设，我们建议检查病毒基因组的WE/基因，以增加突变，并将其与接种与亲戚Shivku-ibmc33接种猕猴分离的NEF基因相比 - 该突变体的信息将在VIV诱导的Vivo中的可能的APOBEC3G诱导的突变中有用的信息在Vivo中提供了有用的信息。拟议的研究将使用相关的猕猴模型提供有关该VIF基序在发病机理中的作用的新信息。