Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs.
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 (3CLpro) mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CLpro in Escherichia coli and Vero cells, and then characterized the in vitro trans-cleavage and the cell-based cis-cleavage by the 3CLpro. Mutational analysis of the 3CLpro demonstrated the importance of His41, Cys145, and Glu166 in the substrate-binding subsite S1 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 study not only established the quantifiable and reliable assay for the in vitro and cell-based measurement of the 3CLpro activity, but also characterized the molecular interaction of the SARS-CoV 3CLpro with the substrates. The results will be useful for the rational development of the anti-SARS drugs.
Lin
Cheng-Wen
CW
Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan, ROC. cwlin@mail.cmu.edu.tw
Tsai
Chang-Hai
CH
Tsai
Fuu-Jen
FJ
Chen
Pei-Jer
PJ
Lai
Chien-Chen
CC
Wan
Lei
L
Chiu
Hua-Hao
HH
Lin
Kuan-Hsun
KH
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D002522:Chlorocebus aethiops; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D045473:Severe acute respiratory syndrome-related coronavirus / Q000187:drug effects* / Q000201:enzymology; D014709:Vero Cells; D014764:Viral Proteins / Q000378:metabolism*
D000998:Antiviral Agents; D017931:DNA Primers; D014764:Viral Proteins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases