Mutation of a highly conserved isoleucine residue in loop 2 of several ????-coronavirus macrodomains indicates that enhanced ADP-ribose binding is detrimental to infection

All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in nonstructural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the GIF (glycine-isoleucine-phenylalanine) motif. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus (MHV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that led to attenuated replication and pathogenesis in cell culture and mice. In contrast, the I-A mutations had normal enzyme activity and enhanced ADP-ribose binding. Despite increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication. AUTHOR SUMMARYThe 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.