Early Phase HIV-1 Host Cell Interactions: the proteasome

早期 HIV-1 宿主细胞相互作用：蛋白酶体

• 批准号: 7338622
• 负责人: Nikunj V Somia
• 金额: $ 23.43万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7338622
• 关键词: 26S proteasome; ATP phosphohydrolase; Acquired Immunodeficiency Syndrome; Address; Antiviral Agents; Biochemical; Biochemical Genetics; Biological Assay; Cell Communication; Cell Cycle Kinetics; Cell Extracts; Cell Fractionation; Cell Line; Cells; Complement; Cytoplasm; Data; Development; Disease; Dominant-Negative Mutation; Equilibrium; Event; Fluorescence Microscopy; Fractionation; Gagging; HIV; HIV-1; Human; Immunoblot Analysis; In Vitro; Incubated; Infection; Kinetics; Laboratories; Lead; Life Cycle Stages; Literature; Location; Mediating; Microscopy; Modeling; Monitor; Movement; Natural regeneration; Nucleosome Core Particle; Phase; Proteasome Inhibitor; Proteins; Relative (related person); Resistance; Retroviridae Infections; Role; Signal Transduction; Signaling Protein; Somatic Cell; Staging; Stress; Sucrose; System; Testing; Time; Translational Research; Viral; Viral Proteins; Virus; Virus Diseases; Work; cell type; density; drug discovery; in vivo; innovation; insight; killings; multicatalytic endopeptidase complex; mutant; novel; particle; prevent; virus host interaction

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.

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质。我们已经分离出逆转录病毒感染的细胞系突变体。它通过蛋白酶体介导的降解传入病毒来限制感染。这项工作和其他工作使我们假设蛋白酶体直接参与逆转录病毒核心的脱壳。关于HIV- 1的未包被阶段知之甚少，这一假设是探索性的和新颖的。具体地说，我们假设一个或多个蛋白酶体AAA ATP酶与解折叠活动的外壳HIV-1。为了验证这一假设，我们提出了两个具体目标。在具体目标1中，我们将研究病毒和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识别病毒蛋白，我们将讨论这是否是HIV-1生命周期中的正常事件，或者是由病毒在突变细胞中的错误定位引起的。为了区分这些替代模型，我们将通过感染后细胞提取物的分级分离和追踪病毒组分来精确确定进入的病毒在野生型（V79-4）和突变型（67-1）细胞中的位置和命运。我们将进一步跟踪病毒感染后，通过使用去卷积荧光显微镜。我们将通过在人U373细胞中的蛋白酶体的19 S调节颗粒中引入6种关键ATP酶的显性负突变体并检查对HIV-1感染的后果，来遗传地测试蛋白酶体的AAA ATP酶的参与。我们假设的一个预测是这些突变体之一将减少感染。在具体目标2中，我们将开发创新和新颖的检测方法来检测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，通过开发HIV-1核心的降解试验，测试纯化病毒核心和蛋白酶体的相互作用。我们还将开拓新的检测方法，用于检测核心颗粒与纯化的19 S调节、20 S催化和26 S蛋白酶体（19 S和20 S）亚基在体外孵育的命运。从这些遗传和生化分析的结果将测试我们的假设，即逆转录病毒的脱壳是由蛋白酶体介导的，从而有助于更好地了解一个阶段的病毒生命周期，我们目前知道的很少。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抗HIV-1感染药物发现的转化研究。