Cellular Protein Involved in Trafficking of HIV-1

参与 HIV-1 贩运的细胞蛋白

• 批准号: 8308833
• 负责人: Carol A Carter
• 金额: $ 35.24万
• 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/8308833
• 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; public health relevance; 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. PUBLIC HEALTH RELEVANCE: Although HIV-1 is still the cause of the world-wide AIDS pandemic and no vaccine is available, infection is no longer a death sentence thanks to interventions targeted at viral gene products, which are now being used as a "functional cure" approach. Nevertheless, major barriers to a cure persist because drug-resistant variants emerge rapidly. Discovery of new anti-viral targets is thus a continuing and critical priority. A strategy that should permit us to circumvent the lattr problem is development of anti-viral agents that target cellular proteins such as Tsg101 and endocytic machinery factors that play key roles in virus production.

描述（由申请人提供）： 人类免疫缺陷病毒1型（HIV）是艾滋病流行病的病原体。在没有疫苗的情况下，确定靶点以开发针对快速出现的耐药变异体的药剂至关重要。病毒必须从受感染的细胞中释放出来 来传播感染，在这个过程中，它利用了细胞蛋白质。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。先前的研究确定了Tsg 101和ESCRT（运输所需的内体分选复合物）机制在将病毒蛋白运输到质膜上的出芽位点中的作用。由于这种机制的功能是分选和传递细胞表面受体，其信号不再需要细胞内部的溶酶体或蛋白酶体区室进行降解，因此了解病毒如何规避这种命运将为制定新的抗病毒策略提供信息。通过在上一个资助期进行的蛋白质组学、生物化学和细胞生物学分析获得的初步数据表明，结构前体多聚蛋白Gag是形成和释放病毒样颗粒所需的唯一病毒基因产物，它指导钙信号机制的募集。我们建议确定Gag中募集所需的决定因素（特异性目的1），定义募集的细胞蛋白质的贡献（特异性目的1）， 目的2）并表征它们与病毒蛋白和彼此的物理缔合（具体目的3）。嵌合Gag蛋白与来自HIV- 1 Gag的结构域，其出芽依赖于钙机制和禽肉瘤病毒Gag，其释放不是，将用于确定关键决定因素。这种方法将通过对所鉴定的结构域中的特定残基进行突变分析来补充。模型细胞中募集蛋白质的功能贡献（例如，HeLa）、T细胞和巨噬细胞将通过siRNA介导的消耗和替代研究进行研究。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试验评估物理关联或邻近性。将使用蛋白质-蛋白质相互作用试验检查直接相互作用。这些研究将确定ESCRT细胞机制的病毒利用机制，并通过确定所需的细胞蛋白和事件（药物干预的潜在靶点），为抗病毒药物开发提供新的策略。 公共卫生相关性： 虽然HIV-1仍然是世界范围艾滋病流行病的原因，而且没有疫苗，但由于针对病毒基因产物的干预措施，感染不再是死刑，现在正被用作“功能性治愈”方法。尽管如此，治愈的主要障碍仍然存在，因为耐药变异迅速出现。因此，发现新的抗病毒靶点是一个持续和关键的优先事项。一个策略，应该允许我们绕过lattr问题是抗病毒药物的发展，靶向细胞蛋白质，如Tsg 101和内吞机制的因素，在病毒的生产中发挥关键作用。