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.

项目概要 人类免疫缺陷病毒 1 型 (HIV-1) 是一种逆转录病毒，当 新组装的病毒颗粒从受感染的细胞中释放出来。 HIV-1 结构蛋白 Gag 招募宿主 将 (ESCRT) 蛋白转运至出芽病毒体颈部所需的细胞内体分选复合体 从质膜上切断新生病毒颗粒。除了病毒样颗粒（VLP）释放外， ESCRT 用于其他拓扑等效细胞过程的膜分裂，例如 囊泡运输、细胞分裂、外泌体生物发生和质膜修复。最近的进展 分子生物学和病毒学强调了招募 ESRT 的重要性和时机，以实现 适当的病毒颗粒释放。然而，ESCRT 介导的生物物理机制的描述 HIV释放过程中的分裂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已使功能性封装成为可能 人类 ESCRT 和 Gag 蛋白位于巨型单层囊泡 (GUV) 内，从而概括了正确的 体外 ESCRT 功能的拓扑。我们将高速共焦显微镜与光学系统集成在一起 捕获能力，允许以皮牛顿分辨率对力进行可视化和研究 在单膜颈断裂过程中产生。反应的控制是通过紫外光解来调节的 用于此 ATP 依赖性过程的笼状核苷酸。这些创新共同赋予了我们独特的能力 从生物物理角度审视 ESRT 释放的 HIV-1。 这项拟议的研究代表了一种重点和创新的方法来研究 ATP 依赖性 HIV-1释放背景下人类ESCRT蛋白的膜断裂机制；直接构建和 扩展了我们之前在酵母 SCRT 系统方面取得的成功。具体来说，在目标 1 中，我将确定 人类ESCRTs的分裂机制。随后，目标 2 将提供生物物理学解释 Gag 结合和 ESCRT 机制造成的膜分裂之间的相互作用。总体假设 该提议的理由是，ESCRT 对膜颈施加机械力，从而破坏膜颈的稳定性， 导致他们的分裂。最终，成功完成这项工作可以提供详细的生物物理学 对 ESRT 释放 HIV-1 的了解可能会导致新型抗病毒药物的设计。