Human Immunodeficiency Virus Proteinase

人类免疫缺陷病毒蛋白酶

• 批准号: 7846703
• 负责人: Ben M. Dunn
• 金额: $ 8.97万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-05 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7846703
• 关键词: Acquired Immunodeficiency Syndrome; Active Sites; Affect; Amino Acid Sequence; Amino Acids; Antiviral Agents; Binding; Biochemical; Biological Assay; Chimeric Proteins; Cleaved cell; Complement; Complex; Crystallography; Funding; Gagging; Genes; Genetic Polymorphism; Genetic Transcription; Growth; HIV; HIV-1 protease; In Vitro; Kinetics; Length; Letters; Methods; Mutagenesis; Mutation; Myelin P2 Protein; N-terminal; Nature; Nucleocapsid; Nucleocapsid Proteins; Pathway interactions; Pattern; Peptide Hydrolases; Peptide Sequence Determination; Pharmaceutical Preparations; Phenotype; Polyproteins; Positioning Attribute; Process; Property; Proteins; Reagent; Recombinants; Research; Research Personnel; Resistance development; Site; Structure; Surface; System; Systems Analysis; Translations; United States National Institutes of Health; Variant; Viral; Virus; Work; base; dimer; drug discovery; gag-pol Fusion Proteins; inhibitor/antagonist; mutant; pol Gene Products; pol genes; programs; recombinant virus; three dimensional structure

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.

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已经取得了令人兴奋的结果，这些结果构成了新假设的基础，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 p6Pol 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，我们还将把蛋白酶结构和功能的分析扩展到B亚型以外的亚型。 具体目标 1 将重点关注 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 起始点到 p6Pol [HXB2 氨基酸 364-440] 末端的区域内的几个突变影响加工的点。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 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。 具体目标 2 将研究 Gag-Pol 多蛋白加工并检查各种亚型（包括 A2、C、D、H 和 F）以及重组形式 A/G、A/C 和 B/F 的蛋白酶的特性，我们从 NTH 艾滋病研究和参考试剂计划获得。我们将使用与特定目标 1 中相同的方法来检查源自非 B 亚型的变体的多蛋白加工的速率和中间体。这将提供更多 Gag-Pol 多聚蛋白序列的天然变体，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，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，要求我们评估 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 将探索各种延伸形式的 HIV-1 蛋白酶的特性和结构，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的上游区域。我们假设 Gag/Pol 存在结构预组织，使 C 裂解位点 [p2/NC] 靠近蛋白酶的活性位点裂解，导致该连接处最初快速裂解。如果这种结构可以通过晶体学、通过构建无活性的蛋白酶-Gag融合蛋白来观察，那么我们将确定药物发现的新靶点，即形成预切割结构的相互作用表面。此外，我们将探索 Specific 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。