BIOPHYSICAL STUDIES OF HIV ASSEMBLY AND MATURATION

描述（改编自申请人摘要）：本提案的目的 是表征Gag结构域之间的相互作用， 参与HIV装配和成熟的多聚蛋白。的结构 已经确定了各个基质、衣壳和核衣壳结构域， 但事实证明，很难获得这些结构的信息， 在多蛋白或病毒体的情况下，多形， 病毒粒子的有包膜、非二十面体的性质使其可能是 信息将通过传统的结构化手段获得。 一种测定氢/氘交换保护分布的新方法 已经开发，并结合光谱方法，将 适用于这个问题。这些技术可以识别 亚基间的接口，并确定其稳定性和动力学。后 改进蛋白水解消化条件，并分配肽文库， 质谱分析所需的碎片，申请人将确定 Gag多聚蛋白交换保护谱的改变及其 域伴随聚合。然后，申请人将描述 衣壳蛋白聚合成球形和圆柱形稳定性 聚合物这些物种被认为在结构上类似于 在未成熟和成熟病毒体中发现的球形和圆柱形衣壳核心。 一旦申请人表征了这些体外结构，他将 描述未成熟和成熟的亚基间相互作用 从细胞中出芽的颗粒。 这些研究将使人们对相互关系有更深入的了解 在HIV生命周期中Gag多聚蛋白的结构域之间。 这种精细的理解将证明是无价的发现和设计， 靶向组装的抗病毒药

Description (Adapted from Applicant's abstract): The objective of this proposal is to characterize the interaction between the structural domains of the Gag polyprotein involved in the assembly and maturation of HIV. The structure of the individual matrix, capsid, and nucleocapsid domains have been determined, but it has proven difficult to obtain structural information about these domains in the context of the polyprotein or virion. The pleomorphic, enveloped, non-icosahedral nature of the virion makes it likely that this information will be obtained by traditional structural means. A novel approach to determining hydrogen/deuterium exchange protection profiles has been developed and, in combination with spectroscopic approaches, will be applied to this problem. These techniques make it possible to identify inter-subunit interfaces and determine their stability and dynamics. After refining proteolytic digestion conditions, and assigning a library of peptide fragments as required for the mass spectrometry, the applicant will determine the alterations in exchange protection profiles of the Gag polyprotein and its domain which accompany polymerization. The applicant will then characterize the stability of capsid protein polymerized into spherical and cylindrical polymers. These species are thought to be structurally analogous to the spherical and cylindrical capsid core found in the immature and mature virion. Once the applicant has characterized these in vitro structures, he will characterize the inter-subunit interactions within immature and mature particles budded from cells. These studies will lead to a refined understanding of the inter-relationship between the structural domains of the Gag polyprotein in the HIV lifecycle. This refined understanding will prove invaluable in the discovery and design of antivirals targeted at assembly