HUMAN IMMUNODEFICIENCY VIRUS PROTEINASE

描述：(改编自申请者摘要)这份提案有四个 明确的目标。目标1将考察遗传背景对 通过替换病毒的一个区域，病毒的复制和传染性 从HIV-1感染者中克隆具有不同序列片段的Gag-Poll基因 病人。病毒在培养物中的生长将在没有和 相关药物的存在。目标2将研究变体的自我加工 从患者样本中提取的GAG-POL多蛋白 Gag-Pol表达载体。在六点被嵌入的蛋白酶切割 110 kDa迷你多聚蛋白前体内的连接将通过 在大肠杆菌中表达过程中中间蛋白种类的变化 SDS-PAGE和Western blotting。前驱体也将通过以下方式制备 受控加工研究中的过度表达和复性 用于结构分析的有无缓蚀剂的条件。目标3将 扩大催化潜力和对缓蚀剂敏感性的研究 亚克隆编码的HIV-1蛋白水解酶 患者的GAG/POL片段的区域。使用成套多肽进行分析 哪些含有两个或两个以上的裂解位点被提出。抑制物结合 将被量化，并将计算生命值。目标4将 将序列分析扩展到来自儿童临床试验的样本 抗蛋白酶药物的疗效。人类免疫缺陷病毒蛋白水解酶的序列和切割 患者的地点将在开始治疗之前确定，并在 协议中选定的几个点。新序列的鉴定 变种，尤其是那些因毒品挑战而产生的变种 将提供样本，作为具体目标1、2和3的一部分进行研究。

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.