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.

项目摘要 HIV-1病毒体出芽过程是完成病毒逃逸的关键， 与宿主的细胞机制有关，但出芽发生的结构机制仍然很差 明白而HIV-1 Gag多聚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 病毒萌芽所必需的。这个过程开始于早期ESCRT因子（ESCRT-1，ESCRT-2）的募集。 II和阿利克斯）通过HIV-1 Gag作用于病毒芽位点。这些早期的ESCRT负责招募 晚期ESCRT蛋白形成丝状结构，据信可以通过细胞分裂来驱动断裂 积极的重塑过程。尽管越来越多的结构，生物化学和生物物理数据，这些 蛋白质，ESCRT驱动膜断裂的结构机制是未知的，留下了一个 我们对HIV-1释放是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/阿利克斯）的结构 膜，使用单粒子冷冻电子显微镜。赫尔利实验室最近的结构工作 表明ESCRT-I可以通过其核心结构域内相互作用自缔合形成螺旋聚合物， 这意味着ESCRT-I与ESCRT-II或阿利克斯的相互作用可以作为组织ESCRT-I的早期模板。 完整的ESCRT切割装置从这个目的的结果将揭示这些配合物的结构，在一个自然的， 类似的背景，提供了有价值的信息，早期ESCRT组件如何组装在膜上 并促进ESCRT-III的征聘。在目的2中，一种具有切割能力的、可控的体外病毒出芽系统， 将被开发，并在这些病毒芽颈处的完整ESCRT机制的结构将被确定， 冷冻电子断层扫描在本目标中确定的结构将代表第一个快照， 通过ESCRT机制的途径膜断裂。这样的结构不仅揭示了 组织完整的ESCRT组装，但也允许开发，测试和实现 ESCRT介导的膜断裂背后的确切结构机制。详细的cryo-EM和cryo-ET 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 来完成病毒生命周期的一个重要方面。这一基础研究建议对今后的研究具有重要意义 抗逆转录病毒药物的发现，因为我们目前对HIV-1释放机制的不完全理解， 研究其作为抗逆转录病毒治疗靶点的潜力。这项工作将导致详细的“电影” 以及对病毒体出芽和释放步骤的生物物理学见解。这一贡献将是 重要的是，它将揭示抗病毒药物可能成功靶向HIV-1释放的程度，包括 通过识别潜在目标（例如，控制ESCRT组装的蛋白质-蛋白质相互作用）用于抗病毒 疗法