PMID- 34011547 OWN - NLM STAT- MEDLINE DCOM- 20210719 LR - 20220113 IS - 1098-5514 (Electronic) IS - 0022-538X (Print) IS - 0022-538X (Linking) VI - 95 IP - 15 DP - 2021 Jul 12 TI - Unique Mutations in the Murine Hepatitis Virus Macrodomain Differentially Attenuate Virus Replication, Indicating Multiple Roles for the Macrodomain in Coronavirus Replication. PG - e0076621 LID - 10.1128/JVI.00766-21 [doi] LID - e00766-21 AB - All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in nonstructural protein 3 (nsp3) that 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 beta interferon (IFN-beta) 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 are 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 is 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 nonstructural 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. FAU - Voth, Lynden S AU - Voth LS AD - Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA. FAU - O'Connor, Joseph J AU - O'Connor JJ AD - Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA. FAU - Kerr, Catherine M AU - Kerr CM AD - Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA. FAU - Doerger, Ethan AU - Doerger E AD - Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA. FAU - Schwarting, Nancy AU - Schwarting N AD - Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA. FAU - Sperstad, Parker AU - Sperstad P AD - Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA. FAU - Johnson, David K AU - Johnson DK AD - Molecular Graphics and Modeling Laboratory and the Computational Chemical Biology Core, University of Kansas, Lawrence, Kansas, USA. FAU - Fehr, Anthony R AU - Fehr AR AD - Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA. LA - eng GR - Start-up funds/University of Kansas (KU)/ GR - P20 GM113117/GM/NIGMS NIH HHS/United States GR - T32 GM132061/GM/NIGMS NIH HHS/United States GR - R35GM138029/HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ GR - T32GM132061/HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ GR - K22AI134993/HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ GR - R35 GM138029/GM/NIGMS NIH HHS/United States GR - K22 AI134993/AI/NIAID NIH HHS/United States GR - P20GM113117/HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20210712 PL - United States TA - J Virol JT - Journal of virology JID - 0113724 RN - 0 (Viral Nonstructural Proteins) SB - IM UOF - bioRxiv. 2021 Mar 31;:. PMID: 33821264 MH - Amino Acid Substitution MH - Animals MH - HeLa Cells MH - Humans MH - Macrophages/metabolism/virology MH - Mice MH - Murine hepatitis virus/*physiology MH - *Mutation, Missense MH - *Viral Nonstructural Proteins/genetics/metabolism MH - Virus Replication/*genetics PMC - PMC8274620 OTO - NOTNLM OT - ADP-ribose OT - ADP-ribosylation OT - MHV OT - NAD OT - PARP OT - SARS-CoV-2 OT - coronavirus OT - interferon OT - macrodomain OT - mouse hepatitis virus EDAT- 2021/05/21 06:00 MHDA- 2021/07/20 06:00 PMCR- 2022/01/12 CRDT- 2021/05/20 05:47 PHST- 2021/05/21 06:00 [pubmed] PHST- 2021/07/20 06:00 [medline] PHST- 2021/05/20 05:47 [entrez] PHST- 2022/01/12 00:00 [pmc-release] AID - JVI.00766-21 [pii] AID - 00766-21 [pii] AID - 10.1128/JVI.00766-21 [doi] PST - ppublish SO - J Virol. 2021 Jul 12;95(15):e0076621. doi: 10.1128/JVI.00766-21. Epub 2021 Jul 12.