Madeline Day, University of Calgary

Madeline Day (University of Calgary), Noga Sharlin (University of Calgary), Rory Mulloy (University of Calgary) and Jennifer A. Corcoran (University of Calgary)

Suppressing & Conquering

The nucleocapsid (N) protein is a modular, multifunctional and essential protein produced by human coronaviruses (HCoVs). N is comprised of N- and C-terminal globular domains, flanked and linked by intrinsically disordered regions (IDRs). N performs multiple essential functions required for viral replication while also antagonizing cellular antiviral responses. We recently showed that a truncated version of SARS-CoV-2 N, N*M210, is a superior antiviral response antagonist, blocking interferon production and stress granule formation. Mutagenesis of two lysine residues in the dsRNA binding motif (dsRBM) of N*M210 eliminated both dsRNA sequestration and antiviral response antagonism. We now show that 229E N also co-localizes with dsRNA utilizing a lysine-containing dsRBM, yet a truncated version of 229E-N (229E-N*M183) does not, despite retention of the dsRBM. This suggests additional features of 229E-N beyond the dsRBM are required for dsRNA co-localization, and by extension, antiviral response antagonism. To test this, we produced a panel of successively truncated 229E-N proteins and examined their co-localization with the dsRNA mimic, poly(I:C), using immunofluorescent microscopy. These studies identified that 229E-N*M172, harbouring only 10 amino acids in the linker IDR that are absent in 229E-N*M183, restores dsRNA co-localization. Ongoing work will determine if restoration of dsRNA co-localization also restores antiviral response antagonism. This work advances our mechanistic understanding of how a modular protein like N interacts with dsRNA to block antiviral responses.