Biophysical Studies of HIV Assembly and Maturation

描述（由申请方提供）：HIV在两步过程中组装，其中由Gag多蛋白组成的未成熟病毒体在质膜上组装，获得包膜糖蛋白，并从感染细胞中出芽。在第二步中，病毒编码的蛋白酶将Gag多蛋白切割成其组成基质（MA）、衣壳（CA）和核衣壳（NC）结构域。切割导致结构域的深刻的形态学重排，其特征在于围绕NC和病毒RNA的复合物形成CA的圆锥形核心。如果核心没有很好地形成，无论是通过阻断切割还是引入突变，所得病毒都是非感染性的。这表明阻断结构重排是一种潜在的治疗方法。要做到这一点，需要详细了解未成熟和成熟的病毒体以及驱动转化的事件序列。我们已经开发了一种基于质谱的氢/氘交换和交联方法，使我们能够在分子水平上进行伴随成熟的结构重排。在本申请中，我们建议使用该技术来：目的1 -确定CA是否存在于未成熟或成熟病毒体中的结构域交换构型目的2-在分子水平上详细了解HIV成熟过程目的3 -组装HIV-1 CA的稳定六聚体并进行详细的生物物理和结构分析，以充分表征N-末端结构域/C-结构域。成熟后形成的末端结构域相互作用。

DESCRIPTION (provided by applicant): HIV assembles in a two step process in which an immature virion composed of the Gag polyprotein assembles at the plasma membrane, acquires the envelope glycoprotein, and buds from the infected cell. In the second step, a viral encoded protease cleaves the Gag polyprotein into its constituent matrix (MA), capsid (CA), and nucleocapsid (NC) structural domains. Cleavage results in a profound morphological rearrangement of th structural domains marked by the formation of a conical core of CA surrounding a complex of the NC and viral RNA. If the core is not well formed, either through blocked cleavage or the introduction of mutations, the resultant virus is non-infectious. This suggests that blocking the structural rearrangement is a potential therapeutic approach. To do so requires a detailed understanding of the immature and mature virions as well as the sequence of events driving the transformation. We have developed a mass spectrometry based hydrogen/deuterium exchange and crosslinking approach that allows us to the structural rearrangements that accompany maturation at the molecular level. In this application we propose to use that technology to: Aim 1 - Determine whether CA exists in a domain swapped configuration in either the immature or mature virion Aim 2- Obtain a detailed understanding of the process of HIV maturation at the molecular level Aim 3 - Assemble stable hexamers of HIV-1 CA and peform a detailed biophysical and structural analysis to fully characterize the N-terminal domain/C-terminal domain interaction that is formed upon maturation.