HIV-1 and SIV Envelope Glycoproteins

描述(由申请人提供)：详细了解参与HIV-1和密切相关的SIV与其靶细胞的相互作用、结合和融合的分子的结构和功能方面的知识，对于我们理解艾滋病的疾病过程以及合理设计诊断试剂、疫苗和其他治疗剂是基本的。在感染过程中最重要的两个分子，即包膜糖蛋白(Env)糖蛋白，SU(表面)和TM(跨膜)，只是在结构水平上的部分特征，许多重要的特征仍然不清楚。这些不确定的领域包括可变环的方向，结合和融合过程中发生的结构变化，以及促进或抑制抗体介导的中和的特征。另一个不确定的领域涉及Env蛋白在病毒颗粒表面的表达方式。例如，关于病毒表面的Env尖峰的数量、它们的分布和它们的稳定性存在争议。这项建议描述了旨在应用新的方法来确定HIV-1和SIV env、gp120和gp41在病毒表面表达和以纯化的重组形式表达的三维形态的研究。我们的目标是确定不同形式的HIV-1和SIV病毒颗粒表面上的Env分子的数量、位置、取向和结构细节，无论是单独的还是与单抗和配体形成的复合体。此外，我们还将通过对由纯化的单体和三聚体的Env蛋白和免疫复合物组成的二维晶体进行三维结构分析，来扩展我们之前对单个分子和复合体的表位和反应位点的研究。主要的方法将是通过负染和冷冻电子显微镜(EM)断层扫描来分析完整的病毒粒子，以及通过二维晶体阵列的电子衍射来分析纯化的重组Env和免疫复合物。

DESCRIPTION (provided by applicant): Detailed knowledge of the structural and functional aspects of the molecules involved in the interaction, binding, and fusion of HIV-1 and the closely related SIV with their target cells is fundamental to our understanding of the disease processes in AIDS and to the rational design of diagnostic reagents, vaccines and other therapeutic agents. Two of the most important molecules in the infectious process, namely the envelope glycoproteins (Env) glycoproteins, SU (surface), and TM (transmembrane), are only partially characterized at the structural level with many of the important features are still poorly defined. These areas of uncertainty include the orientation of the variable loops, the structural changes that occur during the binding and fusion processes, and the features which foster or inhibit Ab-mediated neutralization. Another area of uncertainty involves the manner by which the Env proteins are expressed on the surface of virus particles. There is, for example, controversy regarding the number of Env spikes on the viral surface, their distribution and their stability. This proposal describes investigations aimed at applying new approaches for the determination of the 3-D morphology of the HIV-1 and SIV Env, gp120 and gp41, as expressed on the viral surface and in purified recombinant form. Our goals are to determine the numbers, locations, orientations, and structural details of Env molecules, both alone and in complex with monoclonal antibodies and ligands, on the surface of various forms of HIV-1 and SIV viral particles. In addition we will expand upon our previous epitope and reactive-site mapping studies of individual molecules and complexes by conducting 3-D structural analyses on 2-D crystals composed of purified monomeric and trimeric Env proteins and immune complexes. The primary methodologies will be the analysis of intact virions by negative stain and cryo-electron microscopic (EM) tomography and the analysis of purified recombinant Env and immune complexes by electron diffraction of 2-D crystalline arrays.