Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs

FEBS Letters
Cheng-Wen LinKuan-Hsun Lin

Abstract

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.

References

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Citations

Jul 25, 2020·Journal of Biomolecular Structure & Dynamics·Nima Razzaghi-AslDanial Gholamin
Mar 24, 2006·FEMS Immunology and Medical Microbiology·Cheng-Wen LinJeng-Yi Li
May 3, 2011·Protein & Cell·Bin Xia, Xue Kang
Sep 29, 2020·Phytochemistry Reviews : Proceedings of the Phytochemical Society of Europe·Julia Solnier, Johannes-Paul Fladerer
Jan 21, 2010·The American Journal of Gastroenterology·Alexander MontoIra M Jacobson
Feb 17, 2015·Journal of Enzyme Inhibition and Medicinal Chemistry·Ji-Young ParkYoung Bae Ryu
Dec 29, 2020·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Shaomin Yan, Guang Wu
Dec 12, 2020·Frontiers in Plant Science·Tatiana MatveevaSofia Sokornova
Apr 30, 2021·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Shaomin Yan, Guang Wu
Nov 29, 2005·Virus Research·Ian Brierley, Francisco J Dos Ramos

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Methods Mentioned

BETA
ELISA
protein assay
PCR
Assay

Software Mentioned

Compute pI / Mw

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