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All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in non-structural protein 3 (nsp3) which binds and hydrolyzes mono-ADP-ribose (MAR) 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 mutation of a highly conserved asparagine residue, which makes contact with the distal ribose of ADP-ribose. To determine if additional Mac1 activities contribute to CoV replication, we compared the replication of murine hepatitis virus (MHV) Mac1 mutants, D1329A and N1465A, to the previously mentioned asparagine mutant, N1347A. These residues contact the adenine and proximal ribose in ADP-ribose, respectively. N1465A had no effect on MHV replication or pathogenesis, while D1329A and N1347A both replicated poorly in bone-marrow derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. Interestingly, D1329A was also significantly more attenuated than N1347A in all cell lines tested. Conversely, D1329A retained some ability to block IFN-β transcript accumulation compared to N1347A, indicating that these mutations have different effects on Mac1 functions. Combining these two mutations resulted in a virus that was unrecoverable, suggesting that the combined activities of Mac1 may be essential for MHV replication. We conclude that Mac1 has multiple functions that promote the replication of MHV, and that these results provide further evidence that Mac1 could be a prominent target for anti-CoV therapeutics. IMPORTANCE In the wake of the COVID-19 epidemic, there has been a surge to better understand how CoVs replicate, and to identify potential therapeutic targets that could mitigate disease caused by SARS-CoV-2 and other prominent CoVs. The highly conserved macrodomain, also termed Mac1, is a small domain within non-structural protein 3. It has received significant attention as a potential drug target as previous studies demonstrated that it is essential for CoV pathogenesis in multiple animal models of infection. However, the functions of Mac1 during infection remain largely unknown. Here, using targeted mutations in different regions of Mac1, we found that Mac1 has multiple functions that promote the replication of MHV, a model CoV, and therefore is more important for MHV replication than previously appreciated. These results will help guide the discovery of these novel functions of Mac1 and the development of inhibitory compounds targeting this domain.

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Enterococcus faecalis is an opportunistic pathogen capable of causing infections including endocarditis and urinary tract infections (UTI). One of the well characterized quorum sensing pathways in E. faecalis involves coordination of the conjugal transfer of pheromone-responsive plasmids by PrgX, a member of the RRNPP protein family. Members of this protein family in various Firmicutes have also been shown to contribute to numerous cellular processes including sporulation, competence, conjugation, nutrient sensing, biofilm formation and virulence. As PrgX is a plasmid-encoded RRNPP family member, we surveyed the genome of the multi-drug resistant strain V583 for additional RRNPP homologs using computational searches and refined those identified hits for predicted structural similarities to known RRNPP family members. This led us to investigate the contribution of the chromosomally encoded RRNPP homologs to biofilm processes and pathogenesis in a catheter associated urinary tract infection (CAUTI) model. In this study, we identified five such homologs and report that 3 of the 5 homologs, EF0073, EF1599 and EF1316, affect biofilm formation as well as outcomes in the CAUTI model. Importance Enterococcus faecalis causes healthcare-associated infections and displays resistance to a variety of broad spectrum antibiotics by acquisition of resistance traits as well as the ability to form biofilms. Even though a growing number of factors related to biofilm formation have been identified, mechanisms that contribute to biofilm formation are still largely unknown. The RRNPP protein family regulate a diverse set of biological reactions in low G-C Gram-positive bacteria (Firmicutes). Here we identify three predicted structural homologs of the RRNPP family, EF0073, EF1599 and EF1316, which affect biofilm formation and CAUTI pathogenesis.