The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein that plays a variety of roles in the viral life cycle including replication, transcription, and genome packaging. Despite its critical and multifunctional nature, the molecular details that underlie how N protein mediates these functions are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to the function of SARS-CoV-2 N protein. N protein contains three intrinsically disordered regions and two folded domains. All three disordered regions are highly dynamic and contain regions of transient helicity that appear to act as local binding interfaces for protein-protein or protein-RNA interactions. The two folded domains do not significantly interact with one another, such that full-length N protein is a flexible and multivalent RNA binding protein. As observed for other proteins with similar molecular features, we found that N protein undergoes liquid-liquid phase separation when mixed with RNA. Polymer models predict that the same multivalent interactions that drive phase separation also engender RNA compaction. We propose a simple model in which symmetry breaking through specific binding sites promotes the formation of metastable single-RNA condensate, as opposed to large multi-RNA phase separated droplets. We speculate that RNA compaction to form dynamic single-genome condensates may underlie the early stages of genome packaging. As such, assays that measure how compounds modulate phase separation could provide a convenient tool for identifying drugs that disrupt viral packaging.

The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA Binding Domain (RBD) and its flanking disordered N-Terminal Domain (NTD) tail, using single-molecule Förster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs.