Mechanisms that determine subcellular sites of HIV-1 assembly

决定 HIV-1 组装亚细胞位点的机制

• 批准号: 8822791
• 负责人: Akira Ono
• 金额: $ 37.53万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8822791
• 关键词: Accounting; Acquired Immunodeficiency Syndrome; Actins; Anti-Retroviral Agents; Antiviral Agents; Area; Binding; Biochemical; Biological Assay; Cell membrane; Cells; Cytoskeleton; Data; Defect; Development; Dimerization; Ensure; Future; Gagging; Goals; HIV-1; In Vitro; Intercellular Junctions; Intervention; Knowledge; Leucine Zippers; Life Cycle Stages; Link; Lipids; Mediating; Membrane; Membrane Microdomains; Membrane Proteins; Methods; Microscopy; Molecular; Movement; Mutation; N-terminal; Nucleocapsid; Pathogenesis; Phase; Phospholipids; Play; Process; Production; Proteins; RNA; Research; Resolution; Role; Signal Transduction; Site; Structural Protein; System; T-Lymphocyte; Testing; Viral; Viral Structural Proteins; Virion; Virus; Work; base; cell type; extracellular; inhibitor/antagonist; novel; particle; prevent; research study; transmission process; virological synapse

DESCRIPTION (provided by applicant): Virus particle assembly of HIV-1, the causative agent of AIDS, takes place at the plasma membrane (PM) in most cell types including natural host T cells. Gag localization to the PM is driven by the matrix (MA) domain. MA mediates membrane binding of Gag via N-terminal myristoyl moiety and a highly basic region (HBR) that binds acidic lipids. Binding of HBR to a PM-specific acidic phospholipid PI(4,5)P2 is critical for PM localization of Gag and efficient virus release. Notably, in vitro studies showed that MA HBR also interacts with RNA, which suppresses binding of Gag to non-PI(4,5)P2 acidic lipids, suggesting that RNA is involved in MA-membrane interactions. However, mechanisms by which PI(4,5)P2 and RNA regulate PM-specific Gag localization remain to be elucidated. Once at the PM, Gag further associates specifically with membrane microdomains known as lipid rafts as well as larger PM domains. In virus-producing T cells contacting uninfected cells, Gag, along with other viral and cellular proteins, specifically accumulates at the area of the PM forming a cell-cell junction known as the virological synapse (VS). The VS facilitates cell-to-cell virus transmission via efficient transfer of newly formed virus particles. Notably, in T cells, Gag multimers localize to a rear-end protrusion termed the uropod that eventually constitutes the VS. Despite the importance in virus spread, however, the mechanism by which Gag multimers localize to uropods and eventually to the VS is not well understood. Our long-term goal is to elucidate mechanisms that determine subcellular sites of HIV-1 assembly. Our central hypothesis in this application is that competition between acidic lipids and RNA for MA binding determines Gag localization to the plasma membrane, where Gag multimerization and microdomain association facilitate Gag accumulation at the uropod that eventually forms virological synapses. To test this hypothesis, we plan to pursue the following three specific aims: [Aim 1] Determine the mechanisms by which lipids and RNA regulate PM binding of Gag. Using in vitro and cell-based assays for Gag-membrane interactions, we will elucidate molecular determinants for the competition between RNA and acidic lipids and its downstream effect on Gag multimerization. [Aim 2] Elucidate the determinants for association between Gag and membrane microdomains. Using a novel in vitro system, we will determine contributions of a unique mode of MA-PI(4,5)P2 interaction and Gag multimerization to Gag-raft association. [Aim 3] Identify the mechanism by which nucleocapsid-driven multimerization directs Gag to the uropod. Using biochemical and high-resolution microscopy methods, we will analyze association of Gag multimers with uropod-directed membrane proteins that might link Gag to rearward actin flow. The knowledge gained from experiments outlined in this proposal will likely help us develop strategies for pharmacological intervention of mechanisms regulating Gag localization to the PM and the VS, thereby inhibiting extracellular virus release and cell-to-cell transmission.

描述（由申请人提供）：艾滋病的病毒-1病毒颗粒组装（艾滋病的病毒剂）在包括天然宿主T细胞在内的大多数细胞类型中发生在质膜（PM）。插入PM的插科打位是由矩阵（MA）域驱动的。 MA通过N末端肉豆蔻酰基部分和结合酸性脂质的高度碱性区域（HBR）介导GAG的膜结合。 HBR与PM特异性酸性磷脂PI（4,5）P2的结合对于PM定位和有效的病毒释放至关重要。值得注意的是，体外研究表明，MA HBR也与RNA相互作用，RNA抑制了GAG与非PI（4,5）P2 P2酸性脂质的结合，这表明RNA参与了Ma-Membrane相互作用。然而，PI（4,5）P2和RNA调节PM特异性插入定位的机制仍有待阐明。到达PM后，GAG进一步与称为脂质筏以及较大的PM结构域的膜微区域专门关联。在产生病毒的T细胞中，与未感染细胞的T细胞以及其他病毒和细胞蛋白，特异性地积累在PM的区域，形成了称为病毒学突触（VS）的细胞 - 细胞连接。 VS通过有效地转移新形成的病毒颗粒来促进细胞到细胞病毒的传播。值得注意的是，在T细胞中，堵嘴多组局部位于后端突出中，称为uropod，最终构成Vs。但是，尽管病毒扩散至关重要，但是尚不清楚GAG多组的插头多组的机制，最终尚未得到充分了解VS。我们的长期目标是阐明确定HIV-1组装亚细胞位点的机制。在此应用中，我们的中心假设是，酸性脂质与RNA之间的竞争决定了与质膜的堵塞定位，在该质膜中，GAG多聚合和微域关联促进了在urropod上促进GAG的积累，最终形成了病毒学突触。为了检验这一假设，我们计划追求以下三个特定目的：[AIM 1]确定脂质和RNA调节GAG的PM结合的机制。我们将使用基于细胞和细胞的测定进行GAG膜相互作用，我们将阐明RNA和酸性脂质之间竞争的分子决定因素及其对GAG多次化的下游影响。 [AIM 2]阐明了堵嘴和膜微区域之间关联的决定因素。使用新型的体外系统，我们将确定MA-PI（4,5）P2相互作用的独特模式和GAG多聚化对GAG-RAFT关联的贡献。 [AIM 3]确定核素驱动的多聚化将GAG引导到uropod的机制。使用生化和高分辨率显微镜方法，我们将分析可能将GAG与后向肌动蛋白流动联系起来的GAG多聚体与乌拉托德指导的膜蛋白的关联。从本提案中概述的实验中获得的知识可能会有助于我们制定调节对PM和VS的机制的药理干预策略，从而抑制细胞外病毒释放和细胞间传播。