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， 病毒特异性蛋白酶的生化特性。 这些 研究是由从比较中获得的信息指导的。 劳斯肉瘤病毒和HIV-1蛋白酶（PR）的晶体结构。 在目前的研究中，HIV-1和RSV PR的特性将被进一步研究。 通过表征它们对各种合成的 肽和/或截短的前体多肽底物 类型和系统突变的切割位点。 这些实验将 测试酶亚位点和探针决定簇的结构预测， 具体底物选择和催化效率。 与酶促研究平行，将进行定点诱变。 用于在RSV和HIV-1 PR的氨基酸 残基，1）是不同的两种酶，但发现在 结构上保守的区域，2）在逆转录病毒PR中是保守的，和 3)在相关的细胞内乙酰基蛋白酶中是保守的。 一个主要 这一建议的优点在于它对结构差异的评估 通过分析细菌来源的HIV-1和RSV PR之间的差异， PR分子将一种氨基酸替换为另一种氨基酸。 一 在同源和非同源细胞上每种纯化的突变体PR的稳态动力学分析 异源底物将被进行。 此外，突变的PR将 被置于细菌和病毒前体表达载体中， 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.