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: 10188411
• 负责人: Abraham King Cada
• 金额: $ 4.27万
• 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/10188411
• 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 新组装的病毒体从受感染的细胞释放。 HIV-1结构蛋白，插科打g，招募了宿主 细胞的内体分选复合物需要运输（ESCRT）蛋白向萌芽病毒的颈部 从质膜上的几种新生病毒颗粒。除了病毒样粒子（VLP）释放之外， ESCRT用于其他拓扑等效的细胞过程的膜科学，例如 囊泡运输，细胞分裂，外泌体生物发生和质膜修复。最近的进步 分子生物学和病毒学强调了招募ESCRT在实现方面的重要性和时机 适当的病毒颗粒释放。但是，描述了ESCRT介导的生物物理机制 艾滋病毒发行期间的分裂仍然是该领域的主要目标。 我们实验室中新开发的方法现在使功能的封装成为可能 巨型Unilamelar蔬菜（GUVS）内的人类ESCRT和GAG蛋白，从而概括了正确的 体外ESCRT功能的拓扑。我们已经将高速共聚焦显微镜与光学整合在一起 捕获功能，可以通过piconnewton分辨率进行可视化和调查。 在单个膜颈部分裂过程中产生。反应的控制通过紫外线光解调节 该ATP依赖性过程的笼核苷酸。这些创新共同使我们具有独特的能力 在生物物理透镜下询问ESCRT的HIV-1释放。 这项拟议的研究代表了一种集中和创新的方法来研究ATP HIV-1释放环境中人类ESCRT蛋白的膜科学机制；直接建造和 扩大了我们以前在酵母菌系统中的成功。具体来说，在AIM 1中，我将确定 人类章程的刻度机制。随后，AIM 2将提供生物物理的解释 ESCRT机械的插科打结合与膜科学之间的相互作用。总体假设 对于该建议是，ESCRT将机械力施加到膜颈部，这使它们不稳定，并且 导致他们的科学。最终，这项工作的成功完成允许详细的生物物理 通过ESCRT对HIV-1释放的理解，可能导致新型抗病毒药的设计。