Cellular Protein Involved in Trafficking of HIV-1

参与 HIV-1 贩运的细胞蛋白

• 批准号: 8924052
• 负责人: Carol A Carter
• 金额: $ 2.5万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-15 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8924052
• 关键词: Acquired Immunodeficiency Syndrome; Avian Sarcoma Viruses; Binding; Biochemical; Biological; Biological Assay; Biotinylation; Calcium; Calcium Signaling; Calmodulin; Cell Line; Cell Surface Proteins; Cell Surface Receptors; Cell membrane; Cell model; Cells; Cessation of life; Complement; Complex; Confocal Microscopy; Data; Development; Drug Formulations; Drug resistance; Endoplasmic Reticulum; Event; Fluorescence; Funding; Gagging; Gene Targeting; Genes; HIV-1; Hela Cells; Housing; ITPR1 gene; Immunoelectron Microscopy; Immunoprecipitation; Infection; Inositol; Intervention; Link; Lysosomes; Maps; Mediating; Membrane; Mono-S; Myristates; Pharmacologic Substance; Phospholipids; Play; Process; Production; Proteins; Proteomics; Recruitment Activity; Resistance; Role; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Sorting - Cell Movement; T-Lymphocyte; Testing; Time; Vaccines; Variant; Viral; Viral Genes; Viral Proteins; Virus; Virus-like particle; base; cellular targeting; defined contribution; drug development; gag Gene Products; knock-down; macrophage; multicatalytic endopeptidase complex; mutant; novel; pandemic disease; prevent; protein protein interaction; receptor; trafficking; tripolyphosphate

DESCRIPTION (provided by applicant): The human immunodeficiency virus type 1 (HIV) is the causative agent of the AIDS pandemic. In the absence of a vaccine, identification of targets for development of agents directed against the rapidly emerging resistant variants is critical. The virus must be released from infected cells to spread infection and for this process it exploits cellular proteins. The objective of this projet is to identify key proteins and determine how they facilitate assembly and release. Previous studies established the role of Tsg101 and ESCRT (endosomal sorting complexes required for transport) machinery in trafficking of the viral proteins to budding sites on the plasma membrane. Since this machinery functions to sort and deliver cell surface receptors whose signaling is no longer required to lysosomal or proteasomal compartments in the cell interior for degradation, understanding how the virus circumvents this fate will provide information for formulation of novel anti-viral strategies. Preliminary data derived through proteomic, biochemical and cell biological analyses conducted in the previous funding period indicates that the structural precursor polyprotein, Gag, which is the only viral gene product required for formation and release of virus-like particles, directs the recruitment of calcium signaling machinery for this purpose. We propose to identify the determinants in Gag that are required for recruitment (Specific Aim 1), define the contributions of the recruited cellular proteins (Specific Aim 2) and characterize their physical association to the viral protein and each other (Specific Aim 3). Chimeric Gag proteins with domains derived from HIV- 1 Gag, whose budding is dependent on the calcium machinery and avian sarcoma virus Gag, whose release is not, will be used to identify the key determinants. This approach will be complemented by mutational analysis of specific residues in the identified domains. Functional contributions of the recruited proteins in model cells (e.g., HeLa), T cells and macrophages will be investigated through siRNA-mediated depletion and replacement studies. Physical associations or proximity will be assessed using bimolecular fluorescence complementation, immunoprecipitation, immuno-electron microscopy and biotinylation assays. Direct interactions will be checked using protein- protein interaction assays. These studies will define the mechanism underlying viral exploitation of the ESCRT cellular machinery and provide new strategies for anti-viral drug development by identifying required cellular proteins and events that are potential targets for pharmaceutical interventions.

描述(由申请人提供)： 人类免疫缺陷病毒1型(HIV)是艾滋病大流行的病原体。在缺乏疫苗的情况下，确定针对快速出现的耐药变种的药物开发目标至关重要。病毒必须从受感染的细胞中释放出来 以传播感染，并在此过程中利用细胞蛋白质。该项目的目标是识别关键蛋白质，并确定它们如何促进组装和释放。以前的研究证实了Tsg101和ESCRT(运输所需的内体分选复合体)机制在将病毒蛋白运输到质膜上萌芽位置的过程中所起的作用。由于这种机制的作用是分类和传递细胞表面受体，这些受体的信号不再需要细胞内部的溶酶体或蛋白酶体隔间进行降解，因此了解病毒如何规避这一命运将为制定新的抗病毒策略提供信息。在前一个供资期间进行的蛋白质组、生化和细胞生物学分析得出的初步数据表明，结构前体多蛋白GAG是形成和释放病毒样颗粒所需的唯一病毒基因产物，指导为此目的招募钙信号机制。我们建议确定GAG中招募所需的决定因素(特定目标1)，定义招募的细胞蛋白的贡献(特定 目的2)，并表征它们与病毒蛋白的物理联系以及它们之间的相互作用(特定目的3)。具有HIV-1 Gag结构域的嵌合Gag蛋白(其出芽依赖于钙机制)和禽肉瘤病毒Gag(其释放不依赖于钙机制)将被用于识别关键决定因素。这一方法将得到对已确定区域中特定残基的突变分析的补充。招募的蛋白质在模型细胞(如HeLa)、T细胞和巨噬细胞中的功能贡献将通过siRNA介导的耗尽和替换研究来研究。将使用双分子荧光互补、免疫沉淀、免疫电子显微镜和生物素化分析来评估物理关联或接近程度。直接相互作用将使用蛋白质-蛋白质相互作用分析来检查。这些研究将确定ESCRT细胞机制的潜在病毒利用机制，并通过确定可能成为药物干预目标的所需细胞蛋白质和事件，为抗病毒药物开发提供新的战略。