BIOPHYSICAL STUDIES OF HIV ASSEMBLY AND MATURATION

描述(改编自申请人的摘要)：本提案的目标 是为了表征GAG结构域之间的相互作用 参与HIV组装和成熟的多蛋白。的结构 已经确定了单个基质、衣壳和核衣壳结构域， 但事实证明，很难获得关于这些的结构性信息 在多蛋白或病毒粒子的上下文中的结构域。多形性， 被包裹的、非二十面体的病毒粒子的性质使得这很可能 信息将通过传统的结构性手段获得。 一种确定氢/氚交换保护剖面的新方法 已经被开发出来，并将与光谱方法相结合， 适用于这个问题。这些技术使人们有可能识别 亚基间的界面，并确定它们的稳定性和动力学。之后 精炼蛋白水解酶条件，并指定多肽文库 质谱学所需的碎片，申请人将确定 GAG多聚蛋白及其蛋白的交换保护谱的变化 伴随聚合的结构域。然后，申请者将描述 聚合成球形和圆柱状衣壳蛋白的稳定性 聚合物。这些物种被认为在结构上类似于 在未成熟和成熟的病毒粒子中发现的球形和圆柱形衣壳核心。 一旦申请者确定了这些体外结构的特征，他将 描述未成熟和成熟亚基之间的相互作用 颗粒从细胞中萌发。 这些研究将导致对相互关系的深入理解。 在HIV生命周期中，Gag多聚蛋白的结构域之间的相互作用。 这一精辟的理解将被证明在发现和设计 靶向集结的抗病毒药物

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