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 (N) encoded by SARS-CoV-2 plays key roles in the replication cycle and is a critical serological marker. Here we characterize essential biochemical properties of N and describe the utility of these insights in serological studies. We define N domains important for oligomerization and RNA binding and show that N oligomerization provides a high affinity RNA binding platform. We also map the RNA binding interface, showing protection in the N-terminal domain and linker region. In addition, phosphorylation causes reduction of RNA binding and redistribution of N from liquid droplets to loose-coils, showing how N/RNA accessibility and assembly may be regulated by phosphorylation. Finally, we find that the C-terminal domain of N is the most immunogenic, based upon antibody binding to patient samples. Together, we provide a biochemical description of SARS-CoV-2 N and highlight the value of using N domains as highly specific and sensitive diagnostic markers.