Determining the structural basis for ESCRT-mediated membrane scission in HIV-1 virion budding

确定 HIV-1 病毒粒子出芽中 ESCRT 介导的膜分裂的结构基础

• 批准号: 10468241
• 负责人: Kevin Larsen
• 金额: $ 6.4万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10468241
• 关键词: 3-Dimensional; Anti-Retroviral Agents; Antiviral Agents; Antiviral Therapy; Architecture; Basic Science; Biochemical; Biophysics; Capsid; Cell membrane; Cells; Complex; Consensus; Cryo-electron tomography; Cryoelectron Microscopy; Data; Development; Disease; Event; Filament; Future; Goals; HIV-1; Image; In Vitro; Individual; Intelligence; Investigation; Knowledge; Life Cycle Stages; Link; Mediating; Membrane; Membrane Lipids; Methods; Modeling; Molecular; Neck; Pathway interactions; Plant Roots; Play; Polymers; Process; Proteins; Research; Research Proposals; Resistance; Role; Site; Structure; System; Testing; Viral; Virion; Virus Replication; Work; antiretroviral therapy; driving force; electron tomography; gag Gene Products; insight; movie; particle; protein protein interaction; recruit; virus host interaction

Project Summary The process of HIV-1 virion budding is essential for the completion of viral egress and intrinsically linked to host cellular machinery, yet the structural mechanisms by which budding occurs remain poorly understood. While the HIV-1 Gag polyprotein is capable of deforming host cells membranes and generate bud structures, HIV-1 must hijack the host ESCRT proteins, which ultimately drive the membrane scission event required for viral budding. This process begins with the recruitment of early-ESCRT factors (ESCRT-I, ESCRT- II, and ALIX) to the viral bud site by HIV-1 Gag. These early ESCRTs are then responsible for the recruitment of the late-ESCRT proteins, which form filamentous structures that are believed to drive scission through an active remodeling process. Despite growing amounts of structural, biochemical, and biophysical data on these proteins, the structural mechanism by which ESCRTs drive membrane scission is unknown, leaving a significant gap in our understanding of how HIV-1 release is accomplished. In Aim 1, I will determine the structures of 1:1 early-ESCRT complexes (ESCRT-I/ESCRT-II and ESCRT-I/ALIX) assembled on lipid membranes, using single-particle cryo-electron microscopy. Recent structural work from the Hurley lab has shown that ESCRT-I can self-associate to form spiral polymers through interactions within its core domain, implying that ESCRT-I's interactions with ESCRT-II or ALIX may serve as an early template for organizing the full ESCRT scission machinery. Results from this Aim will reveal the structures of these complexes in a native- like context, providing valuable information to how the early-ESCRT components assemble at the membrane and promote ESCRT-III recruitment. In Aim 2, a scission-competent, controllable in vitro viral budding system will be developed and structures of the full ESCRT machinery at these viral bud necks will be determined using cryo-electron tomography. The structures determined in this Aim will represent the first snapshots of on- pathway membrane scission by the ESCRT machinery. Such structures will not only reveal the molecular organization of the full ESCRT assembly, but also allow for the development, testing, and realization of the exact structural mechanism behind ESCRT-mediate membrane scission. Detailed cryo-EM and cryo-ET studies of these supramolecular complexes will reveal how HIV-1 hijacks and organizes the ESCRT machinery to complete a vital aspect of the viral life cycle. This basic research proposal is significant for future antiretroviral discovery, as our current incomplete mechanistic understanding of HIV-1 release has limited investigations into its potential as a target for antiretroviral therapies. This work will result in a detailed `movie' of—and biophysical insights into—the steps leading to virion budding and release. This contribution will be significant because it will reveal the extent to which antivirals might successfully target HIV-1 release, including by identifying potential targets (eg. protein–protein interactions governing ESCRT assembly) for antiviral therapy.

项目概要 HIV-1 病毒体出芽过程对于完成病毒排出至关重要，本质上 与宿主细胞机器相关，但出芽发生的结构机制仍然很差 虽然 HIV-1 Gag 多蛋白能够使宿主细胞膜变形并产生芽。 HIV-1 必须劫持宿主 ESCRT 蛋白，最终驱动膜断裂事件 该过程始于早期 ESCRT 因子（ESCRT-I、ESCRT-）的招募。 II和ALIX）通过HIV-1 Gag到达病毒芽位点，然后这些早期的ESCRT负责招募。 晚期 ESCRT 蛋白，其形成丝状结构，据信通过 尽管有关这些的结构、生化和生物物理数据不断增加。 蛋白质，ESCRT 驱动膜分裂的结构机制尚不清楚，因此 我们对 HIV-1 释放如何完成的理解存在重大差距。在目标 1 中，我将确定 在脂质上组装的 1:1 早期 ESCRT 复合物（ESCRT-I/ESCRT-II 和 ESCRT-I/ALIX）的结构 赫尔利实验室最近使用单粒子冷冻电子显微镜进行了结构研究。 表明 ESCRT-I 可以通过其核心域内的相互作用自缔合形成螺旋聚合物， 这意味着 ESCRT-I 与 ESCRT-II 或 ALIX 的交互可以作为组织 完整的 ESCRT 断裂机制。该目标的结果将揭示这些复合物的天然结构。 像上下文一样，为早期 ESRT 组件如何在膜上组装提供有价值的信息 并促进 ESCRT-III 募集，目标 2 是一种具有分裂能力、可控的体外病毒出芽系统。 将开发并确定这些病毒芽颈处的完整 ESCRT 机械结构 本目标中确定的结构将代表冷冻电子断层扫描的第一张快照。 ESCRT 机制的途径膜分裂不仅揭示了分子结构。 组织完整的 ESCRT 组装，还允许开发、测试和实现 ESCRT 介导膜分裂背后的确切结构机制。 对这些超分子复合物的研究将揭示 HIV-1 如何劫持和组织 ESCRT 机制 完成病毒生命周期的一个重要方面，这项基础研究提案对未来具有重要意义。 抗逆转录病毒的发现，因为我们目前对 HIV-1 释放的不完整机制了解有限 对其作为抗逆转录病毒治疗靶点的潜力的研究这项工作将产生一部详细的“电影”。 以及对导致病毒粒子萌芽和释放的步骤的生物物理学见解。 意义重大，因为它将揭示抗病毒药物可能成功靶向 HIV-1 释放的程度，包括 通过识别抗病毒的潜在靶标（例如控制 ESCRT 组装的蛋白质-蛋白质相互作用） 治疗。