STRUCTURE/FUNCTION COMPARISONS OF THE HIV-1 AND RSV PRS

这项多学科研究的长期目标是确定 通过这项研究探讨逆转录病毒复制的生物学机制 病毒特异性蛋白水解酶的生化特性。这些 研究是根据从比较中获得的信息进行的 Rous肉瘤病毒和HIV-1蛋白(PR)的晶体结构。 在目前的研究中，HIV-1和RSV PR的特性将是 通过比较它们在各种合成化合物上的活性 含有野生多肽的多肽和/或截短前体多肽底物 类型和系统突变的裂解位点。这些实验将 酶亚基和探针决定因素的测试结构预测 特定底物的选择和催化效率。 在酶学研究的同时，定点突变将被 用来在RSV和HIV-1PR的氨基酸上放置特定的突变 1)在两种酶之间不同的残基，但在 结构保守区，2)在逆转录病毒PR中保守，以及 3)在相关的细胞天冬氨酸蛋白酶中保守。一位少校 这一建议的优势在于它对结构性差异的评估 通过对细菌来源的分析发现HIV-1和RSV PR之间的差异 PR分子将一种氨基酸替代到另一种中。一个 各纯化突变体PR对同源和异构体的稳态动力学分析 将进行异源底物培养。此外，突变的公关将 被置于细菌和病毒前体表达载体中，以 确定这些突变对体内病毒复制的影响。

The long term objective of this multidisciplinary study is to define biological mechanisms involved in retrovirus replication through the study of the biochemical properties of the viral specific protease. These studies are being guided by information obtained from comparison of the crystal structures of the Rous sarcoma virus and the HIV-1 proteases (PRs). In the present studies, the properties of the HIV-1 and RSV PRs will be compared by characterizing their activities on a variety of synthetic peptides and/or truncated precursor polypeptide substrates containing wild type and systematically mutated cleavage sites. These experiments will test structural predictions of enzyme subsites and probe determinants for specific substrate selection and catalytic efficiency. In parallel to the enzymatic studies, site-directed mutagenesis will be used to place specific mutations into the RSV and HIV-1 PRs at amino acid residues that 1) are different between the two enzymes but found in structurally conserved regions, 2) are conserved among retroviral PRs, and 3) are conserved among the related cellular aspartyl proteases. A major strength of this proposal lies in its evaluation of structural differences noted between the HIV-1 and RSV PRs through analysis of bacterially-derived PR molecules that substitute amino acids found in one into the other. A steady state kinetic analysis of each purified mutant PR on homologous and heterologous substrates will be carried out. Furthermore, mutated PR will be placed into both bacterial and viral precursor expression vectors to determine the affect of these mutations on virus replication in vivo.