Human Immunodeficiency Virus Proteinase

描述（由申请人提供）：我们在当前资助期内研究HIV-1蛋白酶结构和功能的努力取得了令人兴奋的结果，这些结果构成了一个新假设的基础，即Gag/Pol蛋白存在结构预组织，使p2/核衣壳（NC）切割位点非常接近蛋白酶二聚体（PR）的活性位点。加工的效率和顺序可以受到从p2到p6 Pol区域内的突变的影响。在新的实验计划中，我们将探索Gag和PR的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亚型B以外的亚型。 具体目标1将侧重于Gag/Pol融合蛋白内的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蛋白质p2开始到p6 Pol [HXB 2氨基酸364-440]结束的区域内的几个点，其中突变影响加工。我们将对全长gag/pol基因使用选择性诱变方法，以确定氨基酸364-440限度内的其他重要点。我们将采用体外转录/翻译系统来分析诱变的影响。 具体目标2将研究Gag-Pol多聚蛋白的加工过程，并检查我们从NTH AIDS Research & Reference Reagent Program获得的各种亚型（包括A2、C、D、H和F）以及重组形式A/G、A/C和B/F的蛋白酶的特性。我们将使用与具体目标1相同的方法来检查来自非B亚型的变体的多蛋白加工的速率和中间体。这将提供Gag-Pol多蛋白序列的更多天然变体，并扩展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区域将被置于我们的重组病毒系统中用于生长分析，以补充生化和结构研究。全球范围内存在多种不同形式的病毒，这要求我们评估除B亚型以外的亚型蛋白酶的特性，B亚型在美国占主导地位，并已用于耐药性发展的研究。我们将开始从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的抑制剂结合以及与多种相同抑制剂复合的三维结构。 具体目标3将探索HIV-1蛋白酶的各种延伸形式的性质和结构，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的区域。我们假设，有一个结构的前组织的Gag/Pol，使C切割位点[p2/NC]附近的活性位点分裂的蛋白酶，导致在该连接的初始快速切割。如果这种结构可以通过晶体学观察，通过构建无活性的蛋白酶-Gag融合蛋白，那么我们将确定药物发现的新靶点，即，用于形成预解理结构的相互作用表面。此外，我们将探索特异性目的1蛋白酶的野生型和突变形式与NC蛋白之间的相互作用作为药物发现的替代靶点的潜力。

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.