Modifications to the SR-Rich Region of the SARS-CoV-2 Nucleocapsid Regulate Self-Association and Attenuate RNA Interactions

Abstract

The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, and maturation. N is comprised of two folded domains that are separated by a highly conserved, disordered, Ser/Arg-rich linker, and flanked by disordered tails. Using NMR spectroscopy and analytical ultracentrifugation we identify an alpha-helical region in the linker that undergoes concentration dependent self-association. NMR and gel shift assays show that the linker binds viral RNA but this binding is dampened by both phosphorylation and a naturally occurring mutation, whereas in contrast, RNA binding to the full-length protein is not affected. Interestingly, phase separation with RNA is significantly reduced upon phosphorylation but enhanced with the mutation. We attribute these differences to changes in the linker helix self-association which dissociates upon phosphorylation but forms more stable higher order oligomers in the variant. These data provide a structural mechanism for how the linker region contributes to protein-protein interactions, RNA-protein interactions, liquid-liquid phase separation and N protein regulation.