Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain

Wu, Chao; Qavi, Abraham J.; Hachim, Asmaa; Kavian, Niloufar; Cole, Aidan R.; Moyle, Austin B.; Wagner, Nicole D.; Sweeney-Gibbons, Joyce; Rohrs, Henry W.; Gross, Michael L.; Peiris, J. S. Malik; Basler, Christopher F.; Farnsworth, Christopher W.; Valkenburg, Sophie A.; Amarasinghe, Gaya K.; Leung, Daisy W.

Nucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.