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

Infectious retrovirus particles are derived from structural polyprotei ns which are cleaved by the viral proteinase (PR) during virion morpho genesis. Besides cleaving viral polyproteins, which is essential for i nfectivity, PR of human immunodeficiency virus (HIV) also cleaves cell ular proteins and PR expression causes a pronounced cytotoxic effect, Retroviral PRs are aspartic proteases and contain two copies of the tr iplet Asp-Thr-GLy in the active center with the threonine adjacent to the catalytic aspartic acid presumed to have an important structural r ole, 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 activi ty against HIV type 1 polyprotein and peptide substrates compared with the wild-type enzyme. It did not induce toxicity on bacterial express ion and yielded significantly reduced cleavage of cytoskeletal protein s in vitro. Cleavage of vimentin in mutant-infected T-cell lines was a lso markedly reduced, Mutant virus did, however, elicit productive inf ection of several T-cell lines and of primary human lymphocytes with n o significant difference in polyprotein cleavage and with similar infe ction kinetics and titer compared with wild-type virus, The discrepanc y between reduced processing in vitro and normal virion maturation can be explained by the observation that reduced activity was due to an i ncrease in K-m which may not be relevant at the high substrate concent ration in the virus particle, This mutation enables us therefore to di ssociate the essential function of PR in viral maturation from its cyt otoxic effect.