Mutational and inhibitive analysis of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer-based assays.
The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays key roles in viral replication and is an attractive target for anti-SARS drug discovery. In this report, a fluorescence resonance energy transfer (FRET)-based method was developed to assess the proteolytic activity of SARS-CoV 3CL(pro). Two internally quenched fluorogenic peptides, 1NC and 2NC, corresponding to the N-terminal and the C-terminal autocleavage sites of SARS-CoV 3CL(pro), respectively, were used as substrates. SARS-CoV 3CL(pro) seemed to work more efficiently on 1NC than on 2NC in trans-cleavage assay. Mutational analysis demonstrated that the His41 residue, the N-terminal 7 amino acids, and the domain III of SARS-CoV 3CL(pro) were important for the enzymatic activity. Antibodies recognizing domain III could significantly inhibit the enzymatic activity of SARS-CoV 3CL(pro). The effects of class-specific protease inhibitors on the trans-cleavage activity revealed that this enzyme worked more like a serine protease rather than the papain protease.
Kuang
Wan-Fen
WF
Hepatitis Research Center, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan, ROC.
Chow
Lu-Ping
LP
Wu
Mei-Hua
MH
Hwang
Lih-Hwa
LH
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases; D017931:DNA Primers; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D011480:Protease Inhibitors / Q000494:pharmacology; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D011480:Protease Inhibitors; D014764:Viral Proteins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases