THE ROLE OF VPU IN HIV-1 PATHOGENESIS

VPU蛋白是一种小的膜结合蛋白，已被证明：a)通过将CD4分流到蛋白酶体途径，下调细胞表面的CD4；b)促进病毒粒子从感染细胞中释放；以及c)具有离子通道活性。研究VPU在慢病毒致病机制中的作用的一个主要缺陷是在猴免疫缺陷病毒(SIV)中缺乏VPU基因，SIV已成为研究艾滋病发病机制的最常用的猕猴模型。作为SIV/猕猴模型的替代，猴-人类免疫缺陷嵌合病毒(SHIV)已被开发出来，它包含来自HIV-1的TAT、REV、VPU和env基因，其遗传背景为SIVmac239，并已被用于衍生致病变异，当接种到猪尾或恒河猴体内时，会导致高病毒负担、大量CD4T细胞丢失和艾滋病，以及在6个月至1年内死亡。我们实验室的研究表明，VPU在这些病毒引起的CD4T细胞损失中确实发挥了作用。我们最近开发了一个VPU/增强型绿色荧光蛋白融合蛋白报告系统(VpuEGFP)，它可以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CD4下调。在这项拟议的研究中，我们将使用最近开发的VpuEGFP报告系统来：a)评估不同的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细胞质结构域的氨基酸的结构-功能关系的作用；以及c)将其生物学特性与来自HIV-1 C亚型分离株的不同Vpu蛋白进行比较。最后，我们将在SHIV的分子克隆中引入生物学特性发生改变的VPU蛋白，以评估VPU的不同生物学特性在SIV引起的猪尾猕猴CD4T细胞损失中的作用。这些研究将为VPU不同结构域的结构与功能关系以及VPU不同生物学特性在HIV-1致病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.