Early Phase HIV-1 Host Cell Interactions: the proteasome

描述（由申请人提供）：人类免疫缺陷病毒（HIV）是导致获得性免疫缺陷综合征（AIDS）的原因。据估计，这一毁灭性疾病迄今已造成2 000万人死亡，全世界约有3 600万人艾滋病毒呈阳性。我们的目标是了解参与HIV-1生命周期早期阶段的细胞蛋白。我们已经分离出逆转录病毒感染的突变细胞系。它通过蛋白酶体介导的传入病毒降解来限制感染。这和其他的工作使我们假设蛋白酶体直接参与了逆转录病毒核心的脱壳。我们对HIV- 1的脱膜阶段知之甚少，这一假设是探索性的和新颖的。具体来说，我们假设一个或多个具有展开活性的蛋白酶体AAA atp酶剥去了HIV-1的外壳。为了验证这一假设，我们提出了两个具体目标。在特异性目的1中，我们将研究病毒与蛋白酶体之间的体内相互作用。由于蛋白酶体介导的HIV-1降解需要信号依赖性蛋白酶体对病毒蛋白的识别，我们将讨论这是HIV-1生命周期中的正常事件，还是由于突变细胞中病毒的错定位而促进的。为了区分这些替代模型，我们将通过分离感染后的细胞提取物和跟踪病毒成分，精确地确定传入病毒在野生型（V79-4）和突变型（67-1）细胞中的位置和命运。我们将在感染后使用反卷积荧光显微镜进一步目视追踪病毒。我们将在人类U373细胞中引入蛋白酶体19S调控颗粒中6个关键atp酶的显性阴性突变体，并检查其对HIV-1感染的影响，从而对蛋白酶体AAA atp酶的参与进行基因检测。我们假设的一个预测是这些突变体中的一个会减少感染。在具体目标2中，我们将开发创新和新颖的检测方法来检查病毒和蛋白酶体之间的体外相互作用。我们将使用野生型V79-4和突变型67-1细胞的细胞提取物开发HIV-1核心降解试验，以测试纯化病毒核心与蛋白酶体的相互作用。我们还将开发新的检测方法，用于纯化的19S调节、20S催化和26S蛋白酶体（19S和20S）亚基体外培养的核心颗粒的命运。这些遗传和生化分析的结果将验证我们的假设，即逆转录病毒脱膜是由蛋白酶体介导的，从而有助于我们更好地了解病毒生命周期的一个阶段，而我们目前对这个阶段知之甚少。鉴定介导病毒核心和蛋白酶体之间相互作用的蛋白质和信号将有助于发现对抗HIV-1感染的药物的转化研究。

DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS). This devastating disease is estimated to have killed 20 million people to date, and approximately 36 million people worldwide are HIV positive. We aim to understand cellular proteins involved in the early phase of the lifecycle of HIV-1. We have isolated a cell line mutant for retrovirus infection. It restricts infection by proteasome mediated degradation of incoming virus. This and other work lead us to hypothesize that the proteasome is directly involved in the uncoating of retroviral cores. Very little is known about the uncoating stage of HIV- 1 and this hypothesis is exploratory and novel. Specifically we hypothesize that one or more of the proteasome AAA ATPases with unfolding activity uncoat HIV-1. To test this hypothesis we propose two specific aims. In Specific Aim 1 we will examine the in vivo interaction between the virus and the proteasome. Since proteasome mediated degradation of HIV-1 demands recognition of viral proteins by the signal dependent proteasome, we will addresses if this is a normal event in the lifecycle of HIV-1 or is promoted by mislocalization of the virus in mutant cells. To differentiate between these alternative models we will precisely determine the location and fate of incoming virus in wild-type (V79-4) and mutant (67-1) cells by fractionation of cell extracts after infection and tracking viral components. We will further track the virus visually after infection by using deconvoluting fluorescence microscopy. We will genetically test for the involvement of the AAA ATPases of the proteasome by introducing dominant negative mutants of the 6 key ATPases in the 19S regulatory particle of the proteasome in human U373 cells and examining the consequences on HIV-1 infection. A prediction of our hypothesis is one of these mutants will decrease infection. In Specific aim 2, we will develop innovative and novel assays to examine the in vitro interaction between the virus and the proteasome. We will test the interaction of purified viral cores and the proteasome by developing a degradation assay for HIV-1 cores using cell extracts from wild type V79-4 and mutant 67-1 cells. We will also pioneer novel assays for the fate of core particles incubated with purified 19S regulatory, 20S catalytic and 26S proteasome (19S and 20S) subunits in vitro. Results from these genetic and biochemical analyses will test our hypothesis that retroviral uncoating is mediated by the proteasome, thus contributing to a greater understanding of a stage of the viral life cycle about which we currently know very little. Identification of proteins and signals that mediate interaction between viral cores and the proteasome will aid translational research for drug discovery to combat HIV-1 infection.