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. 1995 Nov;69(11):7180–7186. doi: 10.1128/jvi.69.11.7180-7186.1995

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.

J Konvalinka 1, M A Litterst 1, R Welker 1, H Kottler 1, F Rippmann 1, A M Heuser 1, H G Kräusslich 1
PMCID: PMC189639  PMID: 7474139

Abstract

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 10-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 Km 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.

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Selected References

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