A chimeric murine coronavirus with altered deISGylase and proteolytic activities exhibits attenuated viral pathogenesis

ABSTRACT All coronaviruses encode one or two multifunctional papain-like proteases (PLPs) with protease activity that cleaves the viral polyprotein, along with deubiquitinating (DUB) and deISGylase activities that remove ubiquitin and interferon-stimulated gene 15 (ISG15) from viral and host proteins. Studying the role of coronavirus deISGylase activity in viral infection and pathogenesis has been challenging due to the inability to generate a viable deISGylase-deficient coronavirus. To address this issue, we used a recently characterized PLP domain from human coronavirus HKU1 that has intact protease and DUB activity, but minimal deISGylase activity. Switching the HKU1 PLP2 domain with the mouse hepatitis virus strain A59 (MHV-A59) PLP2 domain allowed us to generate a viable chimeric virus, MHV-HP2, that replicated to high titers in cell culture. Analysis of the PLP activities of MHV-HP2 revealed that the virus was impaired in deISGylase activity, intact in its DUB activity, and delayed in proteolytic processing at early time points. Detailed analysis of MHV-HP2 replication kinetics revealed that the delay in proteolytic processing coincided with a transient replication delay in cell culture. This replication delay was not rescued by knockdown of ISG15, suggesting proteolytic delay rather than protein ISGylation drives the delay in replication. Importantly, we found that MHV-HP2 is attenuated for replication and pathogenesis in vivo, with infected mice showing more rapid clearance of MHV-HP2 and reduced evidence of liver damage and inflammation as compared to MHV-A59-infected mice. These results document the generation of a murine coronavirus with diminished deISGylase activity and altered protease activity that highlight the importance of coronavirus PLP in coronavirus pathogenesis. IMPORTANCE Coronaviruses are adept at circumventing host antiviral defenses by encoding the multifunctional papain-like protease (PLP). PLP modulates the host cellular environment with its proteolytic, deubiquitinase, and deISGylase activities. Coronavirus deISGylase activity targets the host protein interferon-stimulated gene 15 (ISG15), which can be conjugated to host and viral proteins to promote an antiviral state. To assess the contribution of viral deISGylase activity to pathogenesis, we generated a chimeric mouse coronavirus lacking robust deISGylase activity. Characterization of the chimeric virus revealed that the virus was also inefficient in proteolytic processing. We determined that diminished deISGylase activity and altered PLP protease activity resulted in attenuated pathogenesis in a mouse model system, since mice infected with the chimeric virus exhibited rapid clearance of infection and less severe tissue damage as compared to infection with the parental virus. These results reveal that modulating coronavirus PLP deISGylase and proteolytic activities can reduce disease burden.