Biophysical Studies of HIV Assembly and Maturation

HIV在两个步骤中组装，其中一个未成熟的病毒粒子由Gag多聚蛋白组成 聚集在质膜上，获得包膜糖蛋白，并从受感染的细胞中发芽。在……里面 第二步，病毒编码的蛋白酶将Gag多蛋白裂解成其组成基质(MA)--衣壳 (CA)和核衣壳(NC)结构域。卵裂导致深刻的形态重排 结构域的特征是形成围绕NC的复合体和 病毒核糖核酸。如果核心没有形成良好，无论是通过受阻的切割或引入突变， 合成的病毒是非传染性的。这表明，阻止结构重组是一种潜在的 治疗方法。要做到这一点，还需要详细了解未成熟和成熟的病毒粒子。 作为推动这一转变的事件序列。 我们开发了一种基于质谱学的氢/氢交换和交联方法。 这使我们能够在分子水平上进行伴随成熟的结构重排。在这 我们建议使用该技术的应用程序： 目标1-确定域交换配置中是否存在未成熟或成熟的CA 病毒粒子 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，并对其进行详细的生物物理和结构分析 充分表征成熟时形成的N-末端结构域/C-末端结构域的相互作用。

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.