NANOSCALE ARCHITECTURE OF ESCRT MACHINERY IN HIV RELEASE

 描述(由申请人提供)：艾滋病毒和艾滋病仍然是美国和世界各地的一个长期存在的问题。缺乏更好的药物来预防艾滋病毒感染的一个原因是对病毒如何从受感染的宿主细胞释放的了解不足。艾滋病毒和其他逆转录病毒依赖于宿主细胞因子，即运输所需的内体分选复合体(ESCRT)机制，以完成萌发和释放所需的膜断裂的关键步骤。病毒编码的Gag蛋白组装在质膜上，并产生必要的曲率来包装病毒基因组。GAG还具有与ESCRT-I和/或Alix结合的特定基序，可以招募ESCRT-III蛋白。ESCRT-III蛋白在膜上组装成细丝，促进病毒的断裂和释放。然而，ESCRT-III招募和激活以释放组装的病毒粒子的分子机制仍不清楚。在这个方案中，我们将使用深蚀刻电子显微镜(EM)结合相关的光学显微镜来研究HIV Gag与细胞ESCRT机制的关系。深度蚀刻EM是一种显示细胞膜和膜表面蛋白的强大技术，可以提供病毒颗粒组装所涉及的蛋白质机制的纳米分辨率视图。我们将应用这一点来(I)定义分子结构 HIV Gag和ESCRT之间的联系因缺乏Vps4而稳定，以及(Ii)结合相关的光学显微镜，将这些研究扩展到检查正常病毒颗粒组装过程中存在的瞬时ESCRT结构。这些实验的结果将推动对艾滋病毒和其他逆转录病毒萌芽的理解，为制定干预艾滋病毒感染的新战略铺平道路。

 DESCRIPTION (provided by applicant): HIV and AIDS remains a persistent problem in the US and around the world. One reason for the lack of better drugs to prevent HIV infection is insufficient understanding of how the virus is released from infected host cells. HIV and other retroviruses depend on host cell factors known as endosomal sorting complex required for trafficking (ESCRT) machinery for the critical step of membrane scission necessary for budding and release. The virally encoded Gag protein assembles on plasma membranes and produces the necessary curvature to package the viral genome. Gag also has specific motifs to bind to ESCRT-I and/or Alix, which can recruit ESCRT-III proteins. ESCRT-III proteins assemble into filaments on membranes to facilitate scission and release virus. However, the molecular mechanisms responsible for ESCRT-III recruitment and activation to release assembled virions remain unclear. In this proposal, we will use deep-etch electron microscopy (EM) in combination with correlative light microscopy to study the relationship between HIV Gag and the cellular ESCRT machinery. Deep-etch EM is a powerful technique for visualizing cellular membranes and membrane surface proteins and can provide nm resolution views of the protein machinery involved in viral particle assembly. We will apply this to (i) define the molecular architecture of connections between HIV Gag and ESCRTs stabilized by the absence of Vps4 and (ii) together with correlative light microscopy extend these studies to examine transient ESCRT structures present during normal viral particle assembly. Results from these experiments will move understanding of HIV and other retrovirus budding forward, paving the way for developing new strategies to intervene in HIV infection.