Sharlin, N.; Mulloy, R. P.; Day, M.; Corcoran, J.

During infection, coronaviruses produce abundant double-stranded RNA (dsRNA) which can induce antiviral innate immune responses such as the interferon, 2'-5'-oligoadenylate synthetase (OAS)/RNase L, and protein kinase R (PKR) pathways. Coronaviruses must antagonize these dsRNA responses for successful replication. The SARS-CoV-2 nucleocapsid (N) protein plays a central role in evasion of dsRNA responses, interacting with dsRNA to block interferon-{beta} production and the activation of OAS/RNase L and PKR. Despite intensive study of SARS-CoV-2 N, our understanding of the innate immune evasion abilities of N proteins produced by other human coronaviruses (HCoVs) remains incomplete. Here, we provide a comprehensive comparison of HCoV N proteins expressed in a human lung cell line and show that their abilities to block dsRNA-induced innate immune responses differ. Highly pathogenic HCoV N proteins inhibited the production of interferon-{beta} mRNA and activation of OAS/RNase L, while common cold HCoV N proteins did not. While most HCoV N proteins inhibited PKR phosphorylation, HCoV-OC43 N did not, an observation that correlated with high levels of PKR activation observed during HCoV-OC43 infection. The ability of HCoV N proteins to antagonize PKR required colocalization with dsRNA, yet the overall decrease in PKR phosphorylation mediated by N was not due to sequestration of dsRNA away from PKR. Rather, N colocalized with dsRNA and PKR at dsRNA-induced foci (dRIFs) and inhibited PKR phosphorylation within dRIFs. Sarbecovirus N proteins also relocalized PKR to G3BP1 foci, suggesting that these N proteins can inhibit PKR using two distinct mechanisms. Collectively, our work reveals an unexpected level of functional and mechanistic diversity among the innate immune evasion abilities of human coronavirus N proteins. These findings challenge existing presumptions that observations made in one coronavirus can be extrapolated to others, because even conserved essential proteins such as N can exhibit considerable functional heterogeneity.