Reverse-Topology Mechanism of ESCRTs DuringHIV-1 Viral Release

HIV-1病毒释放过程中ESCRT的反向拓扑机制

• 批准号: 10012755
• 负责人: Abraham King Cada
• 金额: $ 4.16万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10012755
• 关键词: Acquired Immunodeficiency Syndrome; Antiviral Agents; Binding; Biochemical; Biogenesis; Biological; Biological Assay; Biology; Biophysical Process; Biophysics; Carrier Proteins; Cell division; Cell membrane; Cell physiology; Cells; Collaborations; Complex; Computer Models; Development; Dimensions; Encapsulated; Ensure; Environment; Event; Failure; Generations; Goals; HIV; HIV Genome; HIV-1; Human; In Vitro; Individual; Investigation; Laboratories; Lead; Life Cycle Stages; Light; Lipids; Liposomes; Measures; Mediating; Membrane; Methods; Microscope; Molecular Biology; Molecular Virology; Nanotubes; Neck; Nucleotides; Physiological; Play; Preventive; Process; Property; Proteins; Reaction; Research; Resolution; Retroviridae; Role; Side; Sorting - Cell Movement; Speed; Stress; Structural Protein; System; Testing; Therapeutic; Vesicle; Viral; Viral Genome; Virion; Virus; Virus Replication; Virus-like particle; Visualization; Work; Yeasts; biophysical model; biophysical properties; design; exosome; experimental study; gag Gene Products; innovation; insight; instrument; lens; mechanical force; membrane reconstitution; novel; optical traps; particle; photolysis; reconstitution; recruit; repaired; success; trafficking; treadmill; unilamellar vesicle; viral RNA; virus envelope

Project Summary The human immunodeficiency virus type 1 (HIV-1) is a retrovirus that completes its viral life cycle when the newly assembled virion is released from the infected cell. The HIV-1 structural protein, Gag, recruits the host cell’s endosomal sorting complex required for transport (ESCRT) proteins towards the neck of budding virions to sever the nascent viral particle from the plasma membrane. In addition to viral-like particle (VLP) release, ESCRTs are utilized in membrane scission of other topologically equivalent cellular processes such as vesicular trafficking, cell division, exosome biogenesis, and plasma membrane repair. Recent advances in molecular biology and virology highlight the importance and timing of recruitment of ESCRTs in achieving proper viral particle release. However, a description of the biophysical mechanism of ESCRT-mediated scission during HIV release remains a major goal in the field. Newly developed methods in our laboratory have now made possible the encapsulation of functional human ESCRT and Gag proteins inside giant unilamellar vesicles (GUVs), thereby recapitulating the correct topology for ESCRT function in vitro. We have integrated a high-speed confocal microscope with optical trapping capabilities which allows the visualization and investigation, with piconewton resolution, of the force generated during scission of a single membrane neck. Control of the reaction is modulated by UV photolysis of a caged-nucleotide for this ATP-dependent process. Together, these innovations have given us a unique ability to interrogate HIV-1 release by ESCRTs under a biophysical lens. This proposed research represents a focused and innovative approach to investigate the ATP-dependent membrane scission mechanism of human ESCRT proteins in the setting of HIV-1 release; directly building and expanding on our previous success with the yeast ESCRT system. Specifically, in aim 1, I will identify the scission mechanism of human ESCRTs. Subsequently, aim 2 will provide a biophysical explanation of the interplay between Gag binding and membrane scission by the ESCRT machinery. The overarching hypothesis for this proposal is that ESCRTs apply mechanical force to membrane necks which destabilizes them and leads to their scission. Ultimately, successful completion of this work allows for a detailed biophysical understanding of HIV-1 release by ESCRTs that may lead to the design of novel antivirals.

项目摘要 人类免疫缺陷病毒1型（HIV-1）是一种逆转录病毒，当病毒感染后， 新组装的病毒体从受感染的细胞中释放出来。HIV-1结构蛋白Gag招募宿主 细胞内体分选复合物需要运输（ESCRT）蛋白质朝向出芽病毒粒子的颈部 将新生的病毒颗粒从细胞膜上分离除了病毒样颗粒（VLP）释放之外， ESCRT用于其他拓扑学上等同的细胞过程的膜断裂， 囊泡运输、细胞分裂、外来体生物发生和质膜修复。的最新进展 分子生物学和病毒学强调了ESCRT募集的重要性和时机， 适当的病毒粒子释放然而，对ESCRT介导的生物物理机制的描述 在艾滋病毒释放期间进行切割仍然是该领域的一个主要目标。 我们实验室新开发的方法现在已经使功能性微胶囊的封装成为可能。 人ESCRT和Gag蛋白在巨大的单层囊泡（GUV）内，从而重现了正确的 体外ESCRT功能的拓扑结构。我们已经集成了一个高速共焦显微镜与光学 捕获能力，使可视化和调查，与皮牛顿的分辨率，部队 在单个膜颈的断裂期间产生。反应的控制是由紫外光分解的 一种用于ATP依赖过程的笼状核苷酸。这些创新共同赋予了我们独特的能力 在生物物理学透镜下通过ESCRT询问HIV-1的释放。 这项研究提出了一个重点和创新的方法来调查ATP依赖的 人ESCRT蛋白在HIV-1释放环境中的膜断裂机制;直接构建和 扩大了我们之前在酵母ESCRT系统上取得的成功。具体而言，在目标1中，我将确定 人ESCRT的断裂机制。随后，aim 2将提供一个生物物理解释， 通过ESCRT机制的Gag结合和膜断裂之间的相互作用。总体假设 因为ESCRT向膜颈施加机械力，使其不稳定， 导致了它们的分裂最终，这项工作的成功完成允许进行详细的生物物理 了解ESCRT释放HIV-1，这可能会导致新的抗病毒药物的设计。