HUMAN IMMUNODEFICIENCY VIRUS PROTEINASE

拟议研究计划的总体目标是研究 序列中广泛的遗传多样性的生化后果 蛋白水解酶，HIV PR。主要的方法将是克隆， 突变体的表达、纯化及动力学特性研究 HIV感染者病毒种群遗传漂变产生的酶 个人。为了探索这个问题，我们将研究HIV-1PR变种 关于它们被具有良好特性的蛋白酶的抑制 抑制剂。要扩展对活动站点的详细分析 逆转录病毒蛋白水解酶类中的相互作用，下列特异性 我们将致力于以下目标： A.HIV-1PR的遗传变异分析及其功能 后果。 1.HIV-1临床分离株PR等位基因的遗传变异 个别患者的定期样本将通过以下方式进行识别 猪瘟病毒DNA的扩增及核苷酸序列分析 血清阳性母亲外周血单个核细胞及其临床意义 受感染的儿童。 2.对不同的蛋白水解酶分子的功能活性进行分析 通过表达和仔细研究使用动力学分析来追求 底物和抑制剂。一个特定的目标将是研究 潜在的抗艾滋病药物，针对蛋白酶，以确定是否 突变的蛋白水解酶表现出对抑制的抗性。 3.寡核苷酸导向的诱变、表达和纯化 根据动力学和结构分析选择突变。特定的 与结构功能问题相关的假设将由 嵌合结构的形成。 B.卵裂序列变异的影响分析 交汇点。 1.含有蛋白酶和裂解的蛋白质片段的加工 从克隆片段派生的站点将通过分析 A)表达蛋白的SDS-PAGE/Western分析处理 其中变异的蛋白酶序列与pol1基因融合的构建体 表达系统；b)一种表达系统，在该表达系统中，变异的蛋白酶 与上游GAG序列一起在帧中表达；c)构建在 其中，蛋白酶末端的裂解位点突变为 不能切割的形式，允许研究其他切割的加工 在“延伸”的蛋白水解酶的背景下。 2.先前对卵裂连接A和B的研究将扩展到 通过合成其他已知的具有代表性的切割位点 低聚肽与HIV-PR及其部分分子裂解率的研究 改变的形式出现在上面。交汇序列中的变异 在来自患者样本的克隆中观察到的将通过 合成包含这些变化的多组寡肽，以及 比较正常和变异PR的卵裂率。此外 为分析突变体、嵌合体和其他 PR的形式，寡肽的研究将有助于理解， 通过进一步探讨前驱体的加工机理 逆转录病毒蛋白水解酶的特异性问题。

The overall objective of the proposed research program is to examine the biochemical consequences of the wide genetic diversity in the sequence of the proteolytic enzyme, HIV PR. The primary approach will be the cloning, expression, purification, and kinetic characterization of mutant forms of the enzyme arising from genetic drift in virus populations in HIV-infected individuals. To explore this question, HIV-1 PR variants will be studied with respect to their inhibition by well-characterized proteinase inhibitors. To expand the analysis of the details of active site interactions in the retroviral proteinase class, the following specific aims will be pursued: A. Analysis of genetic variation in HIV-1 PR and the functional consequences. 1. The genetic variability of HIV-1 PR alleles in clinical isolates from periodic samples from individual patients will be identified by amplification and nucleotide sequence analysis of DNA isolated from peripheral blood mononuclear cells of seropositive mothers and their infected children. 2. Analysis of functional activity of variant proteinase molecules will be pursued through expression and careful study with kinetic assays using substrates and inhibitors. A particular objective will be to study potential anti-AIDS drugs, targeted to the proteinase, to determine if the mutant proteinases exhibit resistance to inhibition. 3. Oligonucleotide directed mutagenesis, expression, and purification of selected mutations based on kinetics and structural analysis. Specific hypotheses related to structure-function questions will be studied by the formation of chimeric constructs. B. Analysis of the effect of variation in the sequence of cleavage junctions. 1. The processing of protein fragments containing proteinase and cleavage sites derived from cloned segments will be examined by analyzing processing of expressed protein by SDS-PAGE/Western analysis of a) constructs in which the variant proteinase sequence is fused to a pol gene expression system; b) an expression system in which the variant proteinase is expressed in frame with the upstream gag sequences; c) constructs in which the cleavage sites at the ends of the proteinase are mutated to a non-cleavable form to permit the study of the processing of other cleavage sites within the context of an "extended" proteinase. 2. Previous studies of cleavage junctions A and B will be expanded to the other known cleavage sites through the synthesis of representative oligopeptides and the study of the rates of cleavage by HIV PR and some of the altered forms developed above. The variations in junction sequences observed in the clones from patient samples will be explored through the synthesis of sets of oligopeptides incorporating these changes, and the rates of cleavage by normal and variant PR will be compared. In addition to providing the foundation for analysis of mutant, chimeric, and other forms of PR, the oligopeptide studies will contribute to understanding, of the mechanisms of precursor processing by further exploration of the question of retroviral proteinase specificity.