AUTHOR SUMMARY The conserved CoV macrodomain (Mac1) counters the activity of host ADP-ribosyltransferases by removing ADP-ribose from target proteins and is critical for CoV replication and pathogenesis. Mac1 is a potential therapeutic target for CoV disease and several groups are actively developing Mac1 inhibitors. However, we lack a basic knowledge of how many of the key residues in the Mac1 ADP-ribose binding pocket contribute to its biochemical and virological functions. In this study, we engineered alanine mutations into two highly conserved residues in the ADP-ribose binding pocket of Mac1, both as recombinant proteins and recombinant viruses for both MERS-CoV and SARS-CoV-2 to determine their importance in both Mac1 biochemical functions and CoV infection. Interestingly, an isoleucine-to-alanine mutation in loop 2 of both MERS-CoV and SARS-CoV-2 Mac1 proteins enhanced ADP-ribose binding. But surprisingly, that proved to be detrimental to virus infection, indicating that this isoleucine functions to control Mac1 ADP-ribose binding and is beneficial for virus replication and pathogenesis. These results provide unique insight into how macrodomains control ADP-ribose binding to promote infection and will be critical for the development of novel inhibitors targeting Mac1 that could be used to treat CoV-induced disease.
【저자키워드】 SARS-CoV-2, coronavirus, interferon, MERS-CoV, MHV, non-structural protein 3, macrodomain, ADP-ribosylation, ADP-ribosylhydrolase,