Biophysical Studies of HIV Assembly and Maturation

艾滋病病毒的装配过程分为两步，其中一个不成熟的病毒粒子由Gag多蛋白组成， 在质膜上组装，获得包膜糖蛋白，并从受感染的细胞中出芽。在 第二步，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。裂解导致了深刻的形态重排 的th结构域的标志是形成一个锥形核心CA周围的复杂的NC和 病毒RNA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， 所得病毒是非传染性的。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，还需要对未成熟和成熟的病毒粒子有详细的了解 作为推动转变的事件序列。 我们开发了一种基于质谱的氢/氘交换和交联方法 这让我们能够在分子水平上观察伴随成熟的结构重排。在这 我们建议使用该技术来： 目标1-确定CA是否存在于未成熟或成熟的域交换配置中 病毒体 目标2-在分子水平上详细了解艾滋病毒的成熟过程 目的3 -组装稳定的HIV-1 CA六聚体，并进行详细的生物物理和结构分析， 充分表征成熟后形成的N-末端结构域/C-末端结构域相互作用。

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 suggest 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.