Human Immunodeficiency Virus Proteinase

描述(由申请人提供)：我们在当前资助期对HIV-1蛋白酶结构和功能的研究取得了令人兴奋的结果，形成了一个新的假说的基础，即存在GAG/POL蛋白的结构性预组织，使p2/核衣壳(NC)裂解位点非常接近蛋白酶二聚体(PR)的活性位点。从p2到p6Pol的区域内的突变会影响处理的效率和顺序。在新的实验计划中，我们将探索GAG和PR上游区域之间的相互作用。此外，我们将把我们对蛋白酶结构和功能的分析扩展到B亚型以外的其他亚型。 具体目标1将重点放在Gag/Pol融合蛋白内的突变对处理效率和顺序的影响。我们目前的工作已经确定了从蛋白质p2的开始到p6Pol[HXB2氨基酸364-440]结束的区域中的几个突变影响加工的点。我们将使用对全长Gag/Poll基因的选择性突变方法来确定364-440氨基酸范围内的其他重要点。我们将使用体外转录/翻译系统来分析诱变的影响。 特定目的2将研究Gag-Pol多聚蛋白的加工，并检测我们从第n次艾滋病研究和参考试剂计划获得的A2、C、D、H和F亚型以及重组形式A/G、A/C和B/F的蛋白酶的性质。我们将使用与特定目标1相同的方法来检查来自非B亚型的变体在多蛋白加工中的比率和中间体。这将提供Gag-PolvProtein序列的更多自然变体，并扩大我们对序列变异后果的理解。来自非B亚型的基质(MA)和PR之间的GAG/POL区域将被放入我们的重组病毒系统中进行生长分析，以补充生化和结构研究。全球各种不同形式病毒的存在要求我们评估B亚型以外的亚型的蛋白酶的性质，B亚型在美国占主导地位，已被用于耐药性发展的研究。我们将从C、A2和H亚型开始，对这些基因进行亚克隆、表达和纯化，并分析动力学性质、使用临床批准的药物结合抑制剂以及与各种相同抑制剂形成复合体的三维结构。 具体目标3将探索HIV-1蛋白酶的各种扩展形式的性质和结构，其中蛋白酶上游的序列越来越长，因为我们知道加工的初始步骤发生在蛋白酶上游区域。我们假设，GAG/Pol有一个结构性的预组织，使C裂解位点[p2/nc]靠近蛋白酶的活性部位裂解，导致该连接的初始快速裂解。如果这种结构可以通过结晶学观察到，通过构建失活的蛋白酶-GAG融合蛋白，那么我们将识别一个新的药物开发靶点，即形成前切割结构的相互作用面。此外，我们将探索来自特定目标1的野生型和突变形式的蛋白酶与NC蛋白之间作为药物发现替代靶点的相互作用的潜力。

DESCRIPTION (provided by applicant): Our efforts to study HIV-1 protease structure and function in the current funding period have yielded exciting results that form the basis for a new hypothesis, that there is a structural pre-organization of the Gag/Pol protein that brings the p2/nucleocapsid (NC) cleavage site in close proximity to the active site of the protease dimer (PR). The efficiency and order of processing can be influenced by mutations within the region from p2 through p6Pol. In the new Experimental Plan, we will explore the interactions between upstream regions of Gag and PR. In addition, we will extend our analyses of protease structure and function to subtypes other than subtype B. Specific Aim 1 will focus on the influence of mutations within the Gag/Pol fusion protein on the efficiency and order of processing. Our current work has identified several points within the region from the start of protein p2 through the end of the p6Pol [HXB2 amino acids 364-440] where mutations affect processing. We will use a selective mutagenesis method on the full-length gag/pol gene to identify other points of importance within the limits of amino acids 364-440. We will employ an in vitro transcription/translation system to analyze the effects of mutagenesis. Specific Aim 2 will study the Gag-Pol polyprotein processing and examine the properties of the proteases from a variety of subtypes including A2, C, D, H, and F, and recombinant forms A/G, A/C, and B/F, which we have obtained from the NTH AIDS Research & Reference Reagent Program. We will use the same approach as in Specific Aim 1 to examine the rate of and intermediates in polyprotein processing of variants derived from the non-B subtypes. This will provide more natural variants of the Gag-Pol polvprotein sequence and expand our understanding of the consequences of sequence variation. The region of Gag/Pol between matrix (MA) and PR from the non-B subtypes will be placed into our recombinant virus system for analyses of growth to complement the biochemical and structural studies. The presence of a variety of different forms of the virus globally demand that we evaluate the properties of proteases from subtypes other than subtype B, which is predominant in the US and has been used in studies of resistance development,. We will begin with subtypes C, A2, and H. The genes will be subcloned, expressed and purified, and analyzed for kinetic properties, inhibitor binding using clinically-approved drugs, and three-dimensional structure in complex with a variety of the same inhibitors. Specific Aim 3 will explore the properties and the structure of a variety of extended forms of HIV-1 protease, with increasingly longer sequences upstream of the protease, as we know that the initial steps in processing occur in the region upstream of protease. We hypothesize that there is a structural pre-organization of Gag/Pol that brings the C cleavage site [p2/NC] near the active site cleft of the protease, resulting in the initial rapid cleavage of that junction. If this structure can be observed by crystallography, through the construction of an inactive protease-Gag fusion protein, then we will identify a new target for drug discovery, i.e., the interaction surface for formation of the pre-cleavage structure. In addition, we will explore the potential of interactions between the wild-type and mutant forms of protease from Specific Aim 1 and the NC protein as alternative targets for drug discovery.