Mouse hepatitis virus A59 (MHV-A59) is a representative member of the genus betacoronavirus within the subfamily Coronavirinae, which infects the liver, brain and respiratory tract. Through different inoculation routes, MHV-A59 can provide animal models for encephalitis, hepatitis and pneumonia to explore viral life machinery and virus-host interactions. In viral replication, non-structural protein 5 (Nsp5), also termed main protease (M-pro), plays a dominant role in processing coronavirus-encoded polyproteins and is thus recognized as an ideal target of anti-coronavirus agents. However, no structure of the MHV-A59 M-pro has been reported, and molecular exploration of the catalysis mechanism remains hindered. Here, we solved the crystal structure of the MHV-A59 M-pro complexed with a Michael acceptor-based inhibitor, N3. Structural analysis revealed that the C beta of the vinyl group of N3 covalently bound to C145 of the catalytic dyad of M-pro, which irreversibly inactivated cysteine protease activity. The lactam ring of the P1 side chain and the isobutyl group of the P2 side chain, which mimic the conserved residues at the same positions of the substrate, fit well into the S1 and S2 pockets. Through a comparative study with M-pro of other coronaviruses, we observed that the substrate-recognition pocket and enzyme inhibitory mechanism is highly conservative. Altogether, our study provided structural features of MHV-A59 M-pro and indicated that a Michael acceptor inhibitor is an ideal scaffold for antiviral drugs. (C) 2019 Elsevier Inc. All rights reserved.