Author: Lin, Cheng-Wen; Tsai, Chang-Hai; Tsai, Fuu-Jen; Chen, Pei-Jer; Lai, Chien-Chen; Wan, Lei; Chiu, Hua-Hao; Lin, Kuan-Hsun
Title: Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CL(pro) protease: basis for the in vitro screening of anti-SARS drugs Cord-id: kczhig5k Document date: 2004_9_10
ID: kczhig5k
Snippet: 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 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CL(pro) in Escherichia coli and Vero cells, and then characterized the in vitro trans-cleav
Document: 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 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CL(pro) in Escherichia 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 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 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.
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