SARS-CoV-2 polyprotein substrate regulates the stepwise M(pro) cleavage reaction

The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease M(pro) to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. M(pro) cleaves polyproteins in a defined order, but how M(pro) and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound M(pro). Here, we present the cryo-EM structures of SARS-CoV-2 M(pro) in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that M(pro) interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of M(pro) highlights the flexible nature of the active site region of M(pro), which allows it to accommodate ten recognition sites found in the polyprotein. These observations suggest that the role of M(pro) in selecting a preferred cleavage site is limited and underscores the roles of the structure, conformation, and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by M(pro).