All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in non-structural protein 3 (nsp3) which binds and hydrolyzes ADP-ribose covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutant of a highly conserved asparagine-to-alanine mutation, which is known to largely eliminate Mac1 ADP-ribosylhydrolase activity. To determine if Mac1 ADP-ribose binding separately contributes to CoV replication, we compared the replication of a murine hepatitis virus (MHV) Mac1 mutant predicted to dramatically reduce ADP-ribose binding, D1329A, to the previously mentioned asparagine mutant, N1347A. D1329A and N1347A both replicated poorly in bone-marrow derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. However, D1329A was significantly more attenuated than N1347A in all cell lines tested that were susceptible to MHV infection. In addition, D1329A retained some ability to block IFN-β transcript accumulation compared to N1347A, indicating that these two mutants impacted distinct Mac1 functions. Mac1 mutants predicted to eliminate both binding and hydrolysis activities were unrecoverable, suggesting that the combined activities of Mac1 may be essential for MHV replication. We conclude that Mac1 has multiple roles in promoting the replication of MHV, and that these results provide further evidence that Mac1 could be a prominent target for anti-CoV therapeutics.