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: 10268187
• 负责人: Kevin Larsen
• 金额: $ 6.56万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10268187
• 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病毒颗粒的过程对于完成病毒出口和本质上是必不可少的 与宿主的细胞机械相关联，但出现的结构机制仍然很差 理解齿。而HIV-1 GAG多蛋白能够使宿主细胞膜变形并生成芽 结构，HIV-1必须劫持宿主ESCRT蛋白，最终推动膜科学事件 病毒萌芽所需。该过程始于募集早期ESCRT因素（ESCRT-I，ESCRT- II，Alix）通过HIV-1 GAG到病毒芽位。然后，这些早期的escrt负责招聘 晚期蛋白质的蛋白质，形成丝状结构，据信可以通过 主动重塑过程。尽管有越来越多的结构性，生化和生物物理数据 蛋白质，ESCRTS驱动膜科学的结构机制尚不清楚，留下了 我们对HIV-1释放的理解的显着差距。在AIM 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 膜，使用单粒子冷冻电子显微镜。赫利实验室的最近结构性工作 表明ESCRT-I可以通过其核心结构域内的相互作用来自我关联，以形成螺旋聚合物， 暗示ESCRT-I与ESCRT-II或ALIX的相互作用可能是组织的早期模板 完整的ESCRT科学机械。这个目标的结果将揭示本机中这些复合物的结构 喜欢上下文，提供有价值的信息，以了解早期ESCRT组件如何在膜上组装 并促进ESCRT-III招聘。在AIM 2中，具有科学能力的，受到控制的体外病毒出芽系统 将开发并使用这些病毒芽颈的完整ESCRT机械结构 冷冻电子层析成像。在此目标中确定的结构将代表On-的第一个快照 ESCRT机械的途径膜科学。这样的结构不仅会揭示分子 组织完整的大会，但也允许开发，测试和实现 ESCRT-膜科学背后的确切结构机制。详细的冷冻EM和Cryo-Et 对这些超分子络合物的研究将揭示HIV-1劫持和组织机械 完成病毒生命周期的重要方面。这项基础研究建议对未来很重要 抗逆转录病毒发现，因为我们当前对HIV-1释放的机械理解有限 研究其作为抗逆转录病毒疗法的靶标的潜力。这项工作将导致详细的“电影” 以及对生物物理的见解，这些步骤导致病毒率萌芽和释放。这个贡献将是 意义重大，因为它会揭示抗病毒药可能成功地靶向HIV-1的程度，包括 通过确定抗病毒的潜在靶标（例如，蛋白质 - 蛋白质相互作用） 治疗。