Unique Mutations in the Murine Hepatitis Virus Macrodomain Differentially Attenuate Virus Replication, Indicating Multiple Roles for the Macrodomain in Coronavirus Replication

Abstract

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.

The Conserved Macrodomain Is a Potential Therapeutic Target for Coronaviruses and Alphaviruses

“…Similarly, SINV with an N10A mutation is non-viable, and recovered viruses are reverted to the wild type, mutated to D or T, or have developed an A31G compensatory mutation [53]. A similar phenomenon is observed in coronaviruses: most recovered G1439V mutant MHVs are reverted to wild type or have developed a compensatory mutation, A1438T, and the D1329A/N1347A double mutant cannot be recovered [36]. Because these residues line ADP-ribose binding sites, these data indicate strong selective pressure to maintain the macrodomain structural and amino acid configurations.…”

“…Some mutations in the ADP-ribose binding regions of coronavirus, alphavirus, and HEV macrodomains are not tolerated and viruses cannot replicate, indicating an essential function for this domain [28,[34][35][36]. Alphavirus macrodomain mutants without binding or hydrolase activity are not viable due to defects in initiation of infection and viral RNA synthesis [34,35].…”

Host ADP-ribosylation and the SARS-CoV-2 macrodomain

“…PARP12 and PARP14 were shown to be required for this increased IFN signalling in cells infected with macrodomain-mutated MHV, suggesting that the ADP-ribose modifications they catalyze may be the principal targets of this CoV macrodomain [22]. Interestingly, a recent study challenged this view via the identification of MHV macrodomain mutations that severely impaired viral replication, while only modestly affecting IFN suppression [79], likely due to differences between the effect of these mutations on ADP-ribose binding compared with ADP-ribosyl hydrolase activity [69][70][71]79].…”

Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening

“…These data suggest that Mac1’s function is to counter PARP-mediated anti-viral ADP-ribosylation (20). More recently, we have identified mutations in the MHV-JHM Mac1 domain, predicted to abolish ADP-ribose binding, that resulted in severe replication defects in cell culture, indicating that for some CoVs Mac1 may be even more important than previously appreciated (21). Mutations in the alphavirus and HEV macrodomain also have substantial phenotypic effects on virus replication and pathogenesis (22-26).…”