Mutations differentially affecting the coronavirus Mac1 ADP-ribose binding and hydrolysis activities indicate that it promotes multiple stages of the viral replication cycle.

All coronaviruses (CoVs) encode a conserved macrodomain, termed Mac1, in non-structural protein 3 (nsp3) that binds and hydrolyzes ADP-ribose covalently attached to proteins. Mac1 is a key virulence factor that counters antiviral ADP-ribosyltransferase (PARP) activity. Previously, we found that MHV strain JHM (JHMV) with a mutation in the adenine binding site, JHMV-D1329A, was extremely attenuated in all tested cell types as opposed to JHMV-N1347A, which only has a replication defect in bone marrow-derived macrophages (BMDMs). Interestingly, an N1347A/D1329A double mutant was unrecoverable, indicating an essential role for Mac1 in JHMV infection. We hypothesized that these mutations may impact different stages of the MHV life cycle. First, to clarify how these mutations affected the biochemical activities of Mac1, we generated Mac1 proteins encoding the same mutations. As expected, the D-A mutation was extremely defective in ADP-ribose binding but maintained enzyme activity. In contrast, we previously found that the N-A mutation had WT levels of ADP-ribose binding but low enzyme activity, confirming that these mutations differentially affect the biochemical functions of Mac1. Following infection, D1329A displayed a large defect in the accumulation of viral RNA compared to WT or N1347A in all cells tested. Alternatively, N1347A infection produced normal levels of viral RNA but produced reduced levels of viral protein in interferon-competent bone marrow-derived macrophages (BMDMs). These results suggest that Mac1 ADP-ribose binding and enzymatic activities promote different stages of the viral life cycle, demonstrating the critical importance of Mac1 for JHMV replication. IMPORTANCE: Over the last three decades, coronaviruses have repeatedly demonstrated their potential to become significant veterinary and public health threats. Zoonotic transmission of the myriad known coronavirus strains will remain a concern, regardless of the advances in vaccines and treatment. One difficulty in anticipating the next coronavirus outbreak is its diverse lineage and high propensity for mutation and recombination. The coronavirus macrodomain, Mac1, is conserved among all known coronaviruses and is also conserved in the Togaviridae and Hepeviridae families. Mac1 is a key factor in viral replication and pathogenesis, but its role in the replication cycle remains unclear. A deeper investigation of Mac1 function will identify conserved antiviral mechanisms and aid in the development of Mac1 inhibitors that represent a novel strategy for antiviral therapeutics.