PMID- 33821264 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20211124 DP - 2021 Mar 31 TI - An MHV macrodomain mutant predicted to lack ADP-ribose binding activity is severely attenuated, indicating multiple roles for the macrodomain in coronavirus replication. LID - 2021.03.30.437796 [pii] LID - 10.1101/2021.03.30.437796 [doi] AB - 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-beta 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. 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 various roles and functions of Mac1 during infection remain largely unknown. Here, utilizing recombinant Mac1 mutant viruses, we have determined that different biochemical functions of Mac1 have distinct roles in the replication of MHV, a model CoV. These results indicate that Mac1 is more important for CoV replication than previously appreciated, and could help guide the development of inhibitory compounds that target unique regions of this protein domain. FAU - Voth, Lynden S AU - Voth LS FAU - O'Connor, Joseph J AU - O'Connor JJ FAU - Kerr, Catherine M AU - Kerr CM FAU - Doerger, Ethan AU - Doerger E FAU - Schwarting, Nancy AU - Schwarting N FAU - Sperstad, Parker AU - Sperstad P FAU - Johnson, David K AU - Johnson DK FAU - Fehr, Anthony R AU - Fehr AR AUID- ORCID: 0000-0003-1560-1573 LA - eng GR - P20 GM113117/GM/NIGMS NIH HHS/United States PT - Preprint DEP - 20210331 PL - United States TA - bioRxiv JT - bioRxiv : the preprint server for biology JID - 101680187 UIN - J Virol. 2021 May 19;:. PMID: 34011547 PMC - PMC8020964 EDAT- 2021/04/07 06:00 MHDA- 2021/04/07 06:01 PMCR- 2021/04/05 CRDT- 2021/04/06 05:56 PHST- 2021/04/06 05:56 [entrez] PHST- 2021/04/07 06:00 [pubmed] PHST- 2021/04/07 06:01 [medline] PHST- 2021/04/05 00:00 [pmc-release] AID - 2021.03.30.437796 [pii] AID - 10.1101/2021.03.30.437796 [doi] PST - epublish SO - bioRxiv [Preprint]. 2021 Mar 31:2021.03.30.437796. doi: 10.1101/2021.03.30.437796.