The SARS-CoV-2 nucleocapsid protein preferentially binds long and structured RNAs

Abstract

The SARS-CoV-2 nucleocapsid protein (NCAP) functions in viral RNA genome packaging, virion assembly, RNA synthesis and translation, and regulation of host immune response. RNA-binding is central to these processes. Little is known how NCAP selects its binding partners in the myriad of host and viral RNAs. To address this fundamental question, we employed electrophoresis mobility shift and competition assays to compare NCAP binding to RNAs that are of SARS-CoV-2 vs. non-SARS-CoV-2, long vs. short, and structured vs. unstructured. We found that although NCAP can bind all RNAs tested, it primarily binds structured RNAs, and their association suppresses strong interaction with single-stranded RNAs. NCAP prefers long RNAs, especially those containing multiple structures separated by single-stranded linkers that presumably offer conformational flexibility. Additionally, all three major regions of NCAP bind RNA, including the low complexity domain and dimerization domain that promote formation of NCAP oligomers, amyloid fibrils and liquid-liquid phase separation. Combining these observations, we propose that NCAP-NCAP interactions that mediate higher-order structures during packaging also drive recognition of the genomic RNA and call this mechanism recognition-by-packaging. This study provides a biochemical basis for understanding the complex NCAP-RNA interactions in the viral life cycle and a broad range of similar biological processes.

Low complexity domains of the nucleocapsid protein of SARS-CoV-2 form amyloid fibrils

“…2f). In a parallel study, we show that the structured regions of S2hp are essential for strong binding to NCAP, whereas S2 and other short, single-stranded RNA segments bind to it weakly 25 . Here, we detected fibrils of NCAP with S2hp (Supplementary Fig.…”

“…This suggests that specific LCD-RNA interactions are not required for LCD-amyloid formation. Nevertheless, the LCD does bind to at least S2hp vRNA 25 , and LCD fibril maturation is influenced by the RNA sequence and length (Fig. 2b), so LCD-RNA interactions play a role.…”

“…2a and Supplementary text). The S2 vRNA sequence was previously suggested to be a strong NCAP cross-linking site 7 , and we extended it by including the adjacent hairpin regions that improve binding to NCAP 25 .…”