In Vitro Analysis of HIV Gag Protein Interactions

描述（由申请人提供）： 我们研究的最终目标是针对人类免疫缺陷病毒（HIV）结构（Gag）蛋白的抗病毒药物的合理设计。这些蛋白质被合成为前体Gag蛋白Pr 55 Gag的结构域，其可以指导未成熟病毒颗粒的组装。然而，Pr 55 Gag的正常加工产生成熟的HIV Gag蛋白基质（MA）、衣壳（CA）、p2、核衣壳（NC）、p1和p6，其组织成熟的病毒体。对于基质、核衣壳以及衣壳的N-末端结构域（NTD）和C-末端结构域（CTD）的原子模型的可用性潜在地允许设计可以干扰Gag蛋白的功能的配体。然而，为了使这种方法成功，必须表征用于编排感染性病毒的组装和成熟的Gag蛋白的分子界面。我们的研究重点是HIV CA，它沿着p2和NC，构成了病毒颗粒组装的引擎。我们已经开发了方法用于解剖HIV衣壳相互作用，并建议在体外组装表征衣壳接口，并检查推定的衣壳目标。我们获得的结果将为组装和形态发生机制提供重要的见解，这应该作为攻击HIV病毒核心的基础。我们的具体目标如下：1.在颗粒组装体中衣壳蛋白排列的测定：将表征体外组装体中的蛋白排列，以检查衣壳蛋白如何在六聚体阵列中缔合的模型。将分析MA和NC结构域的作用，并将在体内测试所得模型。2.衣壳蛋白组装靶标的评价：将采用体外组装测定来测试结构模型，并鉴定可接近的衣壳残基。这些数据应证明有助于选择适当的衣壳抗病毒靶点。

DESCRIPTION (provided by applicant): The ultimate objective of our investigations is the rational design of antivirals targeted against human immunodeficiency virus (HIV) structural (Gag) proteins. These proteins are synthesized as domains of the precursor Gag protein, Pr55Gag, which can direct the assembly of immature virus particles. However, normal processing of Pr55Gag yields the mature HIV Gag proteins matrix (MA), capsid (CA), p2, nucleocapsid (NC), pl, and p6, which organize mature virions. The availability of atomic models for matrix, nucleocapsid, and the N-terminal domain (NTD) and C-terminal domain (CTD) of capsid potentially allows the design of ligands which could interfere with the functions of the Gag proteins. However, for this approach to be successful, it is essential to characterize molecular interfaces of the Gag proteins used to choreograph the assembly and maturation of infectious viruses. The focus of our studies is HIV CA which, along with p2 and NC, constitutes the engine of virus particle assembly. We have developed methods for the dissection of HIV capsid interactions, and propose to characterize capsid interfaces in in vitro assemblies, and examine putative capsid targets. The results we obtain will provide crucial insights into mechanisms of assembly and morphogenesis, that should serve as a foundation to attack HIV at its viral core. Our specific aims are as follows: 1. Determination of capsid protein arrangements in particle assemblies: Protein arrangements in in vitro assemblies will be characterized to examine models for how capsid proteins associate in hexamer arrays. The effects of MA and NC domains will be analyzed, and resulting models will be tested in vivo. 2. Evaluation of capsid protein assembly targets: In vitro assembly assays will be employed to test structural models, and to identify accessible capsid residues. These data should prove instrumental in choosing appropriate capsid antiviral targets.