HUMAN IMMUNODEFICIENCY VIRUS PROTEINASE

描述：（改编自申请人的摘要）本提案有四个 明确的目标。 目标1将研究遗传背景对 病毒的复制和传染性，通过替换一个区域的 从HIV-1感染者中克隆的具有不同序列片段的gag-pol基因 患者 将在不存在和 相关药物的存在。 目标2将研究变体的自加工 Gag-Pol多聚蛋白，其在插入到细胞中之后来源于患者样品。 Gag-Pol表达构建体。 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E.大肠杆菌使用 SDS-PAGE和Western印迹。 前体也将通过以下步骤制备： 用于受控条件下加工研究的过表达和重折叠 在有和没有抑制剂的条件下进行结构分析。 目标3将 扩大对催化潜力和对抑制剂敏感性的研究 通过亚克隆蛋白酶编码 患者的gag/pol片段的区域。 使用肽组的测定 其具有两个或多个切割位点。 抑制剂结合 将进行定量并计算活力值。 目标4将 将序列分析扩展到来自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 将在开始治疗前确定患者的部位， 协议中的选定点。 新序列鉴定 变异，特别是那些由于药物挑战而产生的变异 将提供作为具体目标1、2和3的一部分进行研究的样本。

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.