HUMAN IMMUNODEFICIENCY VIRUS PROTEINASE

人类免疫缺陷病毒蛋白酶

• 批准号: 2671966
• 负责人: Ben M. Dunn
• 金额: $ 20.59万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2671966
• 关键词: AIDS therapy; SDS polyacrylamide gel electrophoresis; clinical research; drug resistance; endopeptidases; enzyme mechanism; enzyme structure; gene mutation; human immunodeficiency virus 1; human subject; microorganism population study; molecular cloning; nucleic acid sequence; pediatric AIDS; protease inhibitor; virulence; virus replication; western blottings

DESCRIPTION: (Adapted from Applicant's Abstract) This proposal has four specific aims. Aim 1 will examine the influence of genetic background on the replication and infectivity of virus by replacing a region of the gag-pol gene with segments of varying sequences cloned from HIV-1 infected patients. Virus growth in culture will be quantitated in the absence and presence of relevant drugs. Aim 2 will study the self-processing of variant Gag-Pol polyproteins derived from patient samples after insertion into a Gag-Pol expression construct. Cleavage by the imbedded protease at six junctions within the 110 kDa mini polyprotein precursor will be studied by changes of intermediate protein species during expression in E. coli using SDS-PAGE and Western blotting. The precursor will also be prepared by over-expression and refolding for processing studies under controlled conditions with and without inhibitors for structural analysis. Aim 3 will expand studies of the catalytic potential and susceptibility to inhibitors of variant HIV-1 protease species derived by subcloning the protease coding region of the gag/pol segments from patients. Assays using sets of peptides which harbor two or more cleavage sites are proposed. Inhibitor binding will be quantitated and Vitality values will be calculated. Aim 4 will extend sequence analysis to samples from a pediatric clinical trial of the efficacy of anti-protease drugs. The sequence of HIV protease and cleavage sites from patients will be determined before starting therapy, and at selected points within the protocol. identification of new sequence variants, especially those that develop as a consequence of drug challenge will provide samples to be studied as part of Specific Aims 1, 2 and 3.

描述：（改编自申请人的摘要）该提案有四个 具体目标。 目标 1 将检查遗传背景对 通过替换病毒的某个区域来实现病毒的复制和感染性 从 HIV-1 感染者克隆的具有不同序列片段的 gag-pol 基因 患者。 病毒在培养物中的生长将在不存在的情况下进行定量 相关药物的存在。 目标2将研究变体的自我处理 Gag-Pol 多蛋白源自患者样本插入 Gag-Pol 表达构建体。 六点处被嵌入的蛋白酶切割 110 kDa 微型多蛋白前体内的连接将由 大肠杆菌表达过程中中间蛋白种类的变化 SDS-PAGE 和蛋白质印迹。 前体也将由以下方法制备 控制下加工研究的过度表达和重折叠 有和没有抑制剂的条件进行结构分析。 目标3将 扩大催化潜力和抑制剂敏感性的研究 通过亚克隆蛋白酶编码衍生的变体 HIV-1 蛋白酶种类 患者的 gag/pol 片段区域。 使用肽组进行测定 提议其中含有两个或多个切割位点。 抑制剂结合 将被量化并计算活力值。 目标4将 将序列分析扩展到儿科临床试验的样本 抗蛋白酶药物的功效。 HIV蛋白酶和切割的序列 患者的部位将在开始治疗之前确定，并且在 协议中选定的点。 新序列的识别 变异体，尤其是那些因药物挑战而产生的变异体 将提供样本作为具体目标 1、2 和 3 的一部分进行研究。