Journal
FEBS LETTERS
Volume 574, Issue 1-3, Pages 131-137Publisher
WILEY
DOI: 10.1016/j.febslet.2004.08.017
Keywords
SARS-coronavirus; 3C-like protease; trans- and cis-cleavaae; substrate specificity
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Severe acute respiratory syndrome (SARS) has been globally reported. A novel coronavirus (CoV), SARS-CoV, was identified as the etiological agent of the disease. SARS-CoV 3C-like protease (3CL(pro)) mediates the proteolytic processing of replicase polypeptides la and lab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CL(pro) in Escheichia coli and Vero cells, and then characterized the in vitro trans-cleavage and the cell-based cis-cleavage by the 3CL(pro). Mutational analysis of the 3CL(pro) demonstrated the importance of His41, Cys145, and Glu166 in the substrate-binding subsite SI for keeping the proteolytic activity. In addition, alanine substitution of the cleavage substrates indicated that Gln-(p1) in the substrates mainly determined the cleavage efficiency. Therefore, this stud), not only established the quantifiable and reliable assay for the in vitro and cell-based measurement of the 3CL(pro) activity, but also characterized the molecular interaction of the SARS-CoV 3CL(pro) with the substrates. The results will be useful for the rational development of the anti-SARS drugs. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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