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Mutational analysis of the active centre of coronavirus 3C-like proteases

Institute of Virology and Immunology, University of Würzburg, Versbacher Straße 7, 97078 Würzburg, Germany¹
Advanced Biomedical Computing Center, 430 Miller Dr. Rm 228, SAIC/NCI-Frederick, Frederick, MD 21702-1201, USA²

Author for correspondence: John Ziebuhr. Fax +49 931 2013934. e-mail ziebuhr{at}vim.uni-wuerzburg.de

Formation of the coronavirus replication–transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL^pro, was determined. Comparative sequence analyses revealed that FIPV 3CL^pro and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL^pro domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL^pro catalytic system employs His⁴¹ and Cys¹⁴⁴ as the principal catalytic residues. Second, the amino acids Tyr¹⁶⁰ and His¹⁶², which are part of the conserved sequence signature Tyr¹⁶⁰–Met¹⁶¹–His¹⁶² and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly⁸³ and Asn⁶⁴, which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn⁶⁴ mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL^pro mutants in which the equivalent Asn residue (HCoV 3CL^pro Asn⁶⁴) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.

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