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.

The primary purpose of the nucleocapsid (N) protein of the Sars-CoV-2 coronavirus is to interact with viral RNA and drive the formation of the viral capsid, mediated by multivalent interaction of both the N- and C-terminal domains (NTD and CTD) of N with RNA. N additionally forms liquid-liquid phase separated droplets with RNA, both in vitro and in infected cells, in a process believed to be important to viral transcription and replication. The degree to which N phase separates with RNA is dependent on the specific RNA being tested, suggesting that the secondary structure of the RNA plays a role in the propensity for phase separation. The ∼32 kb CoV-2 genomic RNA contains a heterogeneous mix of paired and unpaired bases, and while studies suggest that paired RNA is the primary driver of phase separation, little is known at the structural level about how different RNA structures interact with the nucleocapsid. Here we present investigation of binding between N and short designer oligos built to be homogenously single-stranded or double-stranded (ssRNA or dsRNA). Interestingly, we find that our 14-mer dsRNA phase separates readily with the nucleocapsid, while the ssRNA does not, indicating underlying structural differences between the two interactions. Further, we demonstrate by nuclear magnetic resonance (NMR) spectroscopy, fluorescence anisotropy and gel shift assays, that the NTD binds preferentially to ssRNA, and the CTD to dsRNA. We show that the NTD interacts nonspecifically with dsRNA, and that mutations in the NTD that disrupt RNA binding promote this nonspecific interaction. Lastly, we demonstrate that the tendency of N domains to phase separate correlates with weak, nonspecific binding interactions with RNA, suggesting that this weak, nonspecific binding is the primary driver of phase separation.