Reverse-Topology Mechanism of ESCRTs DuringHIV-1 Viral Release

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 新组装的病毒粒子从感染的细胞中释放出来。HIV-1结构蛋白GAG招募宿主 向萌芽病毒粒子颈部转运所需的细胞内体分选复合体(ESCRT)蛋白 把新生的病毒颗粒从质膜上割下来。除了病毒样颗粒(VLP)释放外， ESCRT被用于其他拓扑学上等价的细胞过程的膜断裂，例如 囊泡运输、细胞分裂、外切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招募ESCRT的重要性和时机，以实现 适当的病毒颗粒释放。然而，对ESCRT介导的生物物理机制的描述 艾滋病毒释放过程中的裂解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的封装成为可能 人类ESCRT和GAG蛋白在巨大的单层囊泡(GUV)内，从而概括了正确的 体外ESCRT功能的拓扑学。我们已经将高速共聚焦显微镜和光学系统集成在一起 诱捕能力，允许以皮牛顿分辨率可视化和调查原力 在单个膜颈部断裂时产生。反应的控制受紫外光分解的调节。 这一依赖于三磷酸腺苷的过程中的一种笼子核苷酸。这些创新加在一起，赋予了我们独特的能力 在生物物理透镜下审问ESCRT对HIV-1的释放。 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 HIV-1释放环境中人ESCRT蛋白的膜断裂机制；直接构建和 在我们之前利用酵母ESCRT系统取得的成功的基础上进行了扩展。具体地说，在目标1中，我将确定 人类ESCRT的断裂机制。随后，目标2将提供一个生物物理解释 ESCRT机制在Gag结合和膜断裂之间的相互作用。最重要的假设 因为这一提议是，ESCRT向膜颈部施加机械力，从而破坏它们的稳定 会导致它们的分裂。最终，这项工作的成功完成将允许详细的生物物理 了解ESCRT释放HIV-1可能导致新型抗病毒药物的设计。

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.