THE ROLE OF VPU IN HIV-1 PATHOGENESIS

Vpu蛋白是一种小的膜结合蛋白，其已显示：a）通过将CD 4分流至蛋白酶体途径而下调来自细胞表面的CD 4; B）促进病毒体从感染细胞释放;和c）具有离子通道活性。研究Vpu在慢病毒发病机制中的作用的一个主要缺点是缺乏猴免疫缺陷病毒（SIV）中的Vpu基因，SIV是研究AIDS发病机制最常用的猕猴模型。作为SIV/猕猴模型的替代方案，已经开发了嵌合的猿猴-人免疫缺陷病毒（SHIV），其含有来自HIV-1的达特、rev、vpu和env基因，具有SIVmac 239的遗传背景，并且已经用于衍生致病性变体，当将其接种到猪尾猕猴或恒河猴中时，引起高病毒负荷、大量CD 4 + T细胞损失和AIDS，6个月至1年内死亡。我们实验室的研究表明，Vpu确实在这些病毒引起的CD 4 + T细胞损失中发挥作用。我们最近开发了一种Vpu/增强型绿色荧光蛋白融合蛋白报告系统（VpuEGFP），该系统模拟病毒感染细胞内Vpu蛋白的细胞内转运、细胞内稳定性和CD 4下调。在本研究中，我们将使用最近开发的VpuEGFP报告系统：a）评估不同Vpu结构域对B亚型Vpu在高尔基复合体中保留的作用; B）使用定点突变来分析组成B亚型Vpu胞质结构域的氨基酸的结构-功能关系的作用;和c）与来自HIV-1的C亚型分离物的不同Vpu蛋白的生物学特性进行比较。最后，我们将介绍Vpu蛋白改变了SHIV的分子克隆中的生物学特性，以评估不同的生物学特性的Vpu对猪尾猕猴中SHIV引起的CD 4 + T细胞丢失的作用。这些研究将为Vpu不同结构域的结构-功能关系以及Vpu不同生物学特性在HIV-1发病机制中的作用提供新的信息。

The Vpu protein is a small membrane bound protein that has been shown to: a) down-modulate CD4 from the cell surface by shunting CD4 to the proteasome pathway; b) facilitate the release of virions from the infected cell; and c) have an ion channel activity. A major drawback in studying the role of Vpu in lentiviral pathogenesis has been lack of a vpu gene in simian immunodeficiency virus (SIV), which has been the most commonly used macaque model to study AIDS pathogenesis. As an alternative to the SIV/macaque model, chimeric simian-human immunodeficiency viruses (SHIVs) have been developed that contain the tat, rev, vpu, and env genes from HIV-1 in a genetic background of SIVmac239 and have been used to derive pathogenic variants that when inoculated into pig-tailed or rhesus macaques, cause high virus burdens, massive CD4+ T cell loss and AIDS, and death within 6 months to 1 year. Studies from our laboratory have indicated that the Vpu does play a role in the CD4+ T cell loss caused by these viruses. We have recently developed a Vpu/enhanced green fluorescent protein fusion protein reporter system (VpuEGFP) that mimics the intracellular transport, intracellular stability and CD4 down-regulation of Vpu protein within virus infected cells. In this proposed studies, we will use a recently developed VpuEGFP reporter system to: a) assess the role of the different Vpu domains on the retention of subtype B Vpu in the Golgi complex; b) to use site-directed mutagenesis to analyze the role of the structure-function relationships of the amino acids comprising the cytoplasmic domain of the subtype B Vpu; and c) to compare the biological properties with the divergent Vpu proteins from subtype C isolates of HIV-1. Finally, we will introduce Vpu proteins having altered biological properties in a molecular clone of SHIV to assess the role of the different biological properties of Vpu on CD4+ T cell loss caused by SHIV in pig-tailed macaques. The proposed studies will provide new information on the structure-function relationships of the different domain s of Vpu and the role of the different biological properties of Vpu on HIV-1 pathogenesis.