RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid.

The nucleocapsid (N) protein of SARS-CoV-2 binds viral RNA, condensing it inside the virion, and phase separating with RNA to form liquid-liquid condensates. There is little consensus on what differentiates sequence-independent N-RNA interactions in the virion or in liquid droplets from those with specific genomic RNA (gRNA) motifs necessary for viral function inside infected cells. To identify the RNA structures and the N domains responsible for specific interactions and phase separation, we use the first 1,000 nt of viral RNA and short RNA segments designed as models for single-stranded and paired RNA. Binding affinities estimated from fluorescence anisotropy of these RNAs to the two-folded domains of N (the NTD and CTD) and comparison to full-length N demonstrate that the NTD binds preferentially to single-stranded RNA, and while it is the primary RNA-binding site, it is not essential to phase separation. Nuclear magnetic resonance spectroscopy identifies two RNA-binding sites on the NTD: a previously characterized site and an additional although weaker RNA-binding face that becomes prominent when binding to the primary site is weak, such as with dsRNA or a binding-impaired mutant. Phase separation assays of nucleocapsid domains with double-stranded and single-stranded RNA structures support a model where multiple weak interactions, such as with the CTD or the NTD's secondary face promote phase separation, while strong, specific interactions do not. These studies indicate that both strong and multivalent weak N-RNA interactions underlie the multifunctional abilities of N.