Human Immunodeficiency Virus Proteinase

我们在当前资助期内对HIV-1蛋白酶结构和功能的研究取得了令人兴奋的结果，为一个新的假设奠定了基础，即Gag/Pol蛋白的结构预组织使p2/核衣壳（NC）切割位点靠近蛋白酶二聚体（PR）的活性位点。处理的效率和顺序可能受到p2到p6p¿'区域内突变的影响。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，我们将把蛋白酶结构和功能的分析扩展到b亚型以外的亚型。Specific Aim 1将重点研究Gag/Pol融合蛋白内突变对加工效率和顺序的影响。我们目前的工作已经确定了从p2蛋白开始到p6po1末端[HXB2氨基酸364-440]的区域内突变影响加工的几个点。我们将在全长gag/pol基因上使用选择性诱变方法来确定364-440氨基酸范围内的其他重要点。我们将采用体外转录/翻译系统来分析诱变的影响。Specific Aim 2将研究Gag-Pol多蛋白的加工过程，并检查各种亚型（包括A2， C， D， H和F）以及重组形式a /G， a /C和B/F的蛋白酶的性质，这些蛋白酶来自NTH艾滋病研究和参考试剂计划。我们将使用与Specific Aim 1中相同的方法来检查非b亚型衍生的多蛋白加工的比率和中间产物。这将提供Gag-Pol 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Gag/Pol介于基质（MA）和PR之间的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。全球存在多种不同形式的病毒，要求我们评估除B亚型以外的其他亚型蛋白酶的特性，B亚型在美国占主导地位，并已用于耐药性发展的研究。我们将从C， A2和h亚型开始，这些基因将被亚克隆，表达和纯化，并分析动力学特性，使用临床批准的药物结合抑制剂，以及与各种相同抑制剂复合物的三维结构。特异性目标3将探索各种扩展形式的HIV-1蛋白酶的性质和结构，随着蛋白酶上游序列的增加，我们知道加工的初始步骤发生在蛋白酶上游区域。我们假设存在Gag/Pol的结构预组织，使C切割位点[p2/NC]靠近蛋白酶的活性位点裂缝，导致该连接的初始快速切割。如果通过构建无活性蛋白酶- gag融合蛋白可以通过晶体学观察到这种结构，那么我们将确定一个新的药物发现靶点，即形成预裂解结构的相互作用表面。此外，我们将探索特异性Aim 1的野生型和突变型蛋白酶与NC蛋白之间相互作用的潜力，作为药物发现的替代靶点。

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 p6p¿'. 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 p6po1 [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.