HUMAN IMMUNODEFICIENCY VIRUS PROTEINASE

拟议研究计划的总体目标是审查 生物化学的后果广泛的遗传多样性的序列 蛋白水解酶，HIV PR。主要方法是克隆， 突变形式的表达、纯化和动力学表征 HIV感染者体内病毒种群遗传漂变产生的酶 个体为了探索这个问题，将研究HIV-1 PR变异体 关于它们被充分表征的蛋白酶抑制 抑制剂的展开活动站点的详细分析 在逆转录病毒蛋白酶类的相互作用，以下具体 目标将是： a. HIV-1PR基因变异及其功能分析 后果 1. HIV-1PR等位基因的遗传变异研究 将通过以下方式识别来自个体患者的定期样本： DNA扩增和核苷酸序列分析 血清阳性母亲的外周血单核细胞及其 被感染的孩子 2.变体蛋白酶分子的功能活性的分析将是 通过表达和使用动力学测定的仔细研究进行， 底物和抑制剂。一个特别的目标是研究 潜在的抗艾滋病药物，针对蛋白酶，以确定是否 突变蛋白酶表现出对抑制的抗性。 3.寡核苷酸定向诱变、表达和纯化 基于动力学和结构分析选择突变。具体 与结构-功能问题相关的假设将由 嵌合构建体的形成。 B。卵裂顺序变异的影响分析 交叉点 1.含蛋白酶和切割的蛋白质片段的加工 将通过分析来自克隆片段的位点， 通过SDS-PAGE/Western分析对表达的蛋白质进行处理a） 其中变体蛋白酶序列与pol基因融合的构建体 表达系统; B）表达系统，其中变体蛋白酶 与上游gag序列同框表达; 其中蛋白酶末端的切割位点突变为 不可裂解的形式，以允许研究其他裂解的加工 在"延伸的"蛋白酶的范围内的位点。 2.以前对裂解连接A和B的研究将扩展到 通过合成代表性的 寡肽和研究切割率的艾滋病毒PR和一些 上面发展的变化形式。连接序列的变化 将通过以下方法探索在患者样本克隆中观察到的 结合这些变化的寡肽组的合成，以及 比较正常和变异PR的切割速率。此外 为分析突变体、嵌合体和其他 形式的PR，寡肽研究将有助于理解， 通过对前驱体的进一步探索， 逆转录病毒蛋白酶特异性的问题。

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.