SARS-CoV-2 polyprotein substrate regulates the stepwise Mpro cleavage reaction

Processing of polyproteins pp1a and pp1ab in Coronaviruses by main protease Mpro is a crucial event in virus replication and a promising target for antiviral drug development. Mpro recognizes multiple recognition sites within polyproteins in a defined order, but its mechanism remains enigmatic due to lack of structural information of the polyprotein substrate bound Mpro complex. Here, we present the cryo-EM structures of the SARS-CoV-2 Mpro in an apo-form and in complex with the nsp7-10 region of pp1a polyprotein. The structure shows that the interaction of Mpro with polyproteins is limited to the recognition site connecting nsp9 and nsp10 proteins without any tight association with the rest of polyprotein structure or sequence. Comparison between the apo-form and the polyprotein bound structures of Mpro highlights the flexible nature of active site region allowing the Mpro to accommodate various recognition sites connecting series of nsp proteins. These observations suggest that the role of Mpro for selecting a preferred cleavage site within the polyprotein is limited and underscore the structure, conformation and/or dynamics of polyprotein determining the sequential polyprotein cleavage by Mpro.