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: 8409911
• 负责人: Akira Ono
• 金额: $ 37.27万
• 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 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8409911
• 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. PUBLIC HEALTH RELEVANCE: Proper localization of viral components in cells is critical for efficient production of virus particles and the spread from infected to uninfected cells. The goal of the proposed research is to elucidate the mechanisms that direct Gag, a structural protein of HIV-1 that causes AIDS, to the proper sites within virus-expressing cells. Knowledge obtained from the proposed studies will help us to develop novel antiviral strategies that may either directly suppress assembly and release of HIV-1 particles or inhibit the spread of this virus to uninfected host cells.

描述（由申请人提供）：HIV-1（艾滋病的病原体）的病毒颗粒组装发生在大多数细胞类型（包括天然宿主 T 细胞）的质膜（PM）处。 PM 的 Gag 定位由矩阵 (MA) 域驱动。 MA 通过 N 端肉豆蔻酰部分和结合酸性脂质的高碱性区域 (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 Gag 的 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 还与 RNA 相互作用，从而抑制 Gag 与非 PI(4,5)P2 酸性脂质的结合，表明 RNA 参与 MA 膜相互作用。然而，PI(4,5)P2 和 RNA 调节 PM 特异性 Gag 定位的机制仍有待阐明。一旦到达 PM，Gag 进一步与称为脂筏的膜微域以及更大的 PM 域发生特异性关联。在接触未感染细胞的产生病毒的 T 细胞中，Gag 与其他病毒和细胞蛋白一起，特异性地积聚在 PM 区域，形成称为病毒突触 (VS) 的细胞-细胞连接处。 VS 通过新形成的病毒颗粒的有效转移来促进细胞间的病毒传播。值得注意的是，在 T 细胞中，Gag 多聚体定位于称为尾足的后端突起，最终构成 VS。然而，尽管 Gag 多聚体在病毒传播中很重要，但其定位于尾足并最终定位于 VS 的机制尚不清楚。我们的长期目标是阐明决定 HIV-1 组装亚细胞位点的机制。我们在该应用中的中心假设是，酸性脂质和RNA之间对于MA结合的竞争决定了Gag在质膜上的定位，其中Gag多聚化和微结构域关联促进了Gag在尾足的积累，最终形成病毒学突触。为了检验这一假设，我们计划追求以下三个具体目标： [目标 1] 确定脂质和 RNA 调节 Gag PM 结合的机制。利用体外和基于细胞的 Gag-膜相互作用测定，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 多聚化的下游影响。 [目标 2] 阐明 Gag 和膜微域之间关联的决定因素。使用新型体外系统，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。 [目标 3] 确定核衣壳驱动的多聚化将 Gag 引导至尾足的机制。使用生化和高分辨率显微镜方法，我们将分析 Gag 多聚体与尾足定向膜蛋白的关联，这些膜蛋白可能将 Gag 与向后肌动蛋白流联系起来。从本提案中概述的实验中获得的知识可能会帮助我们制定药物干预策略，调节 Gag 定位到 PM 和 VS 的机制，从而抑制细胞外病毒释放和细胞间传播。 公共卫生相关性：病毒成分在细胞中的正确定位对于病毒颗粒的有效产生以及从感染细胞到未感染细胞的传播至关重要。拟议研究的目的是阐明将 Gag（一种导致艾滋病的 HIV-1 结构蛋白）引导至病毒表达细胞内适当位点的机制。从拟议研究中获得的知识将帮助我们开发新的抗病毒策略，这些策略可以直接抑制 HIV-1 颗粒的组装和释放，或抑制该病毒向未感染的宿主细胞传播。