Biochemistry of HIV-1 Budding

描述（由申请人提供）：先前的研究发现HIV-1利用TSG101/ESCRT-I和MVB囊泡形成的细胞途径从细胞中萌发。我们的研究还提供了MVB途径的各个组成部分的总体概述，它们的配对相互作用，以及一系列MVB蛋白和复合物的三维结构，包括：TSG101 UEV/PTAP复合物，ALIX YPXL结合域，VPS4 MIT和ATPase结构域，以及与TSG101 UEV， Npl4 NZF和EAP45 GLUE结构域的泛素复合物。然而，我们还不了解最基本的途径功能的基本机制，包括调节、蛋白质分类、膜变形和膜裂变。我们现在提议对三种不同的MVB复合物在HIV-1出芽中的作用进行生化、机制和结构研究。Aim 1的研究将集中在人类ESGRT-I的生物化学方面。ESCRT-I直接结合到HIV-1 Gag的PTAP晚期结构域并促进病毒释放。我们最近开发了表达和纯化重组ESCRT-I复合物的系统，并发现了第四个以前未被表征的ESCRT-I亚基家族。现在，我们将在这些观察结果的基础上定义ESCRT-I的精确组成，测试ESCRT-I激活和调节的模型，并表征一系列ESCRT-I结合蛋白。Aim 2的研究将重点关注ALIX/AIP1蛋白的结构和功能，该蛋白直接结合YPXL晚期结构域并促进病毒出芽。我们开发了用于分析ALIX在HIV-1释放中的作用的系统，并确定了ALIX的中心YPXL结合区域的晶体结构。我们现在将绘制YPXL结合位点，验证晚期结构域诱导激活ALIX的构象变化的假设，并描述ALIX结合伙伴在病毒出芽中的作用。Aim 3的研究将集中于了解VPS4 atp酶如何与ESCRT-III晶格相互作用，并从膜上释放组装的MVB机制。VPS4A和VPS4B是MVB途径中唯一已知的酶，它们的atp酶活性是HIV-1和许多其他病毒释放所必需的。我们现在建议确定VPS4如何识别ESCRT-III底物，并测试VPS4作为一种特殊伴侣的模型，该伴侣利用ATP水解的能量展开ESCRT-III亚基，将它们从膜上释放出来，并可能也驱动囊泡/病毒释放。

DESCRIPTION (provided by applicant): Previous research led to the discovery that HIV-1 utilizes TSG101/ESCRT-I and the cellular pathway of MVB vesicle formation to bud from cells. Our studies also provided a general overview of the individual components of the MVB pathway, their pairwise interactions, and three dimensional structures of a series of MVB proteins and complexes, including: the TSG101 UEV/PTAP complex, the ALIX YPXL binding domain, VPS4 MIT and ATPase domains, and ubiquitin complexes with the TSG101 UEV, Npl4 NZF, and EAP45 GLUE domains. Nevertheless, we do not yet understand the basic mechanisms that underlie most essential pathway functions including regulation, protein sorting, membrane deformation, and membrane fission. We now propose biochemical, mechanistic, and structural studies of three different MVB complexes that function in HIV-1 budding. Studies in Aim 1 will focus on the biochemistry of human ESGRT-I. ESCRT-I binds directly to the PTAP late domain of HIV-1 Gag and facilitates virus release. We have recently developed systems for expressing and purifying recombinant ESCRT-I complexes and discovered a fourth family of previously uncharacterized ESCRT-I subunits. We will now build on these observations to define the precise composition of ESCRT-I, test models for ESCRT-I activation and regulation, and characterize a series of ESCRT-I binding proteins. Studies in Aim 2 will focus on the structure and function of the ALIX/AIP1 protein, which binds directly to YPXL late domains and faciliates virus budding. We have developed systems for analyzing the role of ALIX in HIV-1 release and determined the crystal structure of the central YPXL binding region of ALIX. We will now map the YPXL binding site, test the hypothesis that late domains induce conformational changes that activate ALIX, and characerize the roles of ALIX binding partners in virus budding. Studies in Aim 3 will focus on understanding how the VPS4 ATPases interact with the ESCRT-III lattice and release the assembled MVB machinery from the membrane. VPS4A and VPS4B are the only known enzymes in the MVB pathway, and their ATPase activity is required for release of HIV-1 and many other viruses. We now propose to determine how VPS4 recognizes ESCRT-III substrates and to test the model that VPS4 functions as a specialized chaperone that uses the energy of ATP hydrolysis to unfold ESCRT-III subunits, release them from the membrane, and possibly also drive vesicle/virus release.