Mutations in the macrodomain 1 of nsp3 attenuate PEDV virulence in vivo without impairing in vitro replication

Abstract

ABSTRACT The conserved macrodomain 1 (Mac1) within coronavirus nsp3 reverses ADP-ribosylation of proteins and contributes to viral virulence. While Mac1 has been characterized in several coronaviruses, its role in porcine epidemic diarrhea virus (PEDV) remains unknown. Since D445 and N462 are functionally important residues in several betacoronaviruses, we mutated the two residues in both the PEDV Mac1 recombinant protein and the infectious virus and analyzed them in vitro and in vivo. Sequence alignment revealed that N462 is highly conserved, whereas D445 is less conserved among porcine coronaviruses. The N462A mutation significantly impaired both ADP-ribose binding and hydrolysis activity of Mac1, whereas D445A had minimal effect. Compared with wild-type PEDV, both PEDV mutants (icPC22A-nsp3-D445A and icPC22A-nsp3-N462A) replicated efficiently as wild type in Vero, porcine kidney LLC-PK1, and porcine intestinal epithelial IPEC-J2 cells. Unexpectedly, both mutated viruses induced significantly lower levels of interferons in LLC-PK1 cells and IPEC-J2 cells, in contrast to previously characterized Mac1 mutants of SARS-CoV-2 and MHV-JHMV strain. Moreover, the icPC22A-nsp3-N462A virus showed significantly reduced sensitivity to IFN-β treatment in Vero cells. These results suggest that the PEDV Mac1 functions as an interferon inducer. In gnotobiotic neonatal piglets, icPC22A-nsp3-N462A showed reduced and delayed peak viral shedding and attenuated phenotype compared with wild-type PEDV. Collectively, this work provides the first in vitro and in vivo evidence that the conserved N462 residue in the PEDV Mac1 domain is critical for both ADP-ribose binding and hydrolysis, induces IFN production, and contributes to virulence. However, how these pathways are connected deserves further investigation. IMPORTANCE Coronaviruses frequently emerge from wildlife reservoirs to infect humans and livestock, causing economically devastating epidemics/pandemics marked by severe respiratory or enteric diseases. Despite advances in vaccines and therapies, the risk of diverse coronaviruses remains a global concern. PEDV is an alphacoronavirus and causes fatal diarrhea in neonatal pigs, leading to recurring outbreaks and significant economic losses in the global swine industry. The Mac1 domain antagonizes host ADP-ribosylation and impacts pathogenesis in beta-coronaviruses, such as SARS-CoV, SARS-CoV-2, and mouse hepatitis virus (MHV). However, its role in porcine or enteric coronaviruses remains undefined. Our study revealed that PEDV Mac1 mutations decreased enzymatic function and interferon response. The latter differs from respiratory SARS-CoV and neurotropic MHV, suggesting that Mac1’s functions adapt to different host or tissue environments. Our findings identify Mac1 N462 as a rational target for live attenuated vaccine design and highlight Mac1 as a potential target for antiviral therapeutics against PEDV. Coronaviruses frequently emerge from wildlife reservoirs to infect humans and livestock, causing economically devastating epidemics/pandemics marked by severe respiratory or enteric diseases. Despite advances in vaccines and therapies, the risk of diverse coronaviruses remains a global concern. PEDV is an alphacoronavirus and causes fatal diarrhea in neonatal pigs, leading to recurring outbreaks and significant economic losses in the global swine industry. The Mac1 domain antagonizes host ADP-ribosylation and impacts pathogenesis in beta-coronaviruses, such as SARS-CoV, SARS-CoV-2, and mouse hepatitis virus (MHV). However, its role in porcine or enteric coronaviruses remains undefined. Our study revealed that PEDV Mac1 mutations decreased enzymatic function and interferon response. The latter differs from respiratory SARS-CoV and neurotropic MHV, suggesting that Mac1’s functions adapt to different host or tissue environments. Our findings identify Mac1 N462 as a rational target for live attenuated vaccine design and highlight Mac1 as a potential target for antiviral therapeutics against PEDV.