AN ACTIVE-SITE MUTATION IN THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEINASE (PR) CAUSES REDUCED PR ACTIVITY AND LOSS OF PR-MEDIATED CYTOTOXICITY WITHOUT APPARENT EFFECT ON VIRUS MATURATION AND INFECTIVITY
AN ACTIVE-SITE MUTATION IN THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEINASE (PR) CAUSES REDUCED PR ACTIVITY AND LOSS OF PR-MEDIATED CYTOTOXICITY WITHOUT APPARENT EFFECT ON VIRUS MATURATION AND INFECTIVITY
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DOI：
10.1128/jvi.69.11.7180-7186.1995
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发表时间：
1995-11-01
影响因子：
5.4
通讯作者：
KRAUSSLICH, HG
中科院分区：
文献类型：
--
作者：
KONVALINKA, J;LITTERST, MA;KRAUSSLICH, HG
感染性逆转录病毒颗粒来源于在病毒体形态发生期间被病毒蛋白酶（PR）切割的结构多聚蛋白。除了切割病毒多聚蛋白（其对于感染性是必需的）之外，人类免疫缺陷病毒（HIV）的PR还切割细胞蛋白，并且PR表达引起显著的细胞毒性作用。逆转录病毒PR是天冬氨酸蛋白酶并且在活性中心含有两个拷贝的三联体Asp-Thr-Gly，其中邻近催化天冬氨酸的苏氨酸被认为具有重要的结构作用，我们将HIV 1型PR中的苏氨酸改变为丝氨酸，纯化的突变酶对HIV 1型多聚蛋白和肽底物的活性比野生型酶低约5- 10倍。它不会诱导对细菌表达的毒性，并在体外产生显著降低的细胞骨架蛋白裂解。在突变体感染的T细胞系中波形蛋白的切割也显著减少，然而，突变体病毒确实引起了几种T细胞系和原代人淋巴细胞的生产性感染，与野生型病毒相比，多蛋白切割没有显著差异，并且具有相似的感染动力学和滴度，减少的体外加工和正常的病毒粒子成熟之间的差异可以通过观察到减少的活性是由于增加的蛋白质来解释。K-m，其可能与病毒颗粒中的高底物浓度无关。因此，该突变使我们能够将PR在病毒成熟中的基本功能与其细胞毒性作用分离。
Infectious retrovirus particles are derived from structural polyproteins which are cleaved by the viral proteinase (PR) during virion morphogenesis. Besides cleaving viral polyproteins, which is essential for infectivity, PR of human immunodeficiency virus (HIV) also cleaves cellular proteins and PR expression causes a pronounced cytotoxic effect, Retroviral PRs are aspartic proteases and contain two copies of the triplet Asp-Thr-GLy in the active center with the threonine adjacent to the catalytic aspartic acid presumed to have an important structural role, We have changed this threonine in HIV type 1 PR to a serine, The purified mutant enzyme had an approximately 5- to l0-fold Lower activity against HIV type 1 polyprotein and peptide substrates compared with the wild-type enzyme. It did not induce toxicity on bacterial expression and yielded significantly reduced cleavage of cytoskeletal proteins in vitro. Cleavage of vimentin in mutant-infected T-cell lines was also markedly reduced, Mutant virus did, however, elicit productive infection of several T-cell lines and of primary human lymphocytes with no significant difference in polyprotein cleavage and with similar infection kinetics and titer compared with wild-type virus, The discrepancy between reduced processing in vitro and normal virion maturation can be explained by the observation that reduced activity was due to an increase in K-m which may not be relevant at the high substrate concentration in the virus particle, This mutation enables us therefore to dissociate the essential function of PR in viral maturation from its cytotoxic effect.