SARS-CoV-2 nucleocapsid protein variants have differential RNA chaperone activity

The single-stranded RNA genome of the SARS-CoV-2 virus is characterized by a complex secondary structure formed by patches of intramolecular RNA double-strands. Here, we show that the nucleocapsid (N) protein is not only the specific viral RNA packaging protein, but also acts as an RNA chaperone, facilitating RNA folding. RNA chaperones are classified by their non-specific RNA binding and the presence of intrinsically disordered regions (IDRs). N possesses three IDRs, separated by the structured RNA-binding domain (RBD) and the C-terminal domain (CTD). Our study identifies the amino acids 46-364 (RBD-IDR2-CTD) as crucial for chaperone activity, with flanking IDRs either enhancing or repressing this function, revealing the essential role of IDRs for the chaperone mechanism. Furthermore, a comparison between the Wuhan and Omicron BA.5 variant N shows reduced chaperone activity of the Omicron N protein. However, mimicking the cellular phosphorylation state of Omicron N restored its chaperone activity to the levels of the Wuhan variant. Our results identify N-phosphorylation as a regulatory mechanism of chaperone activity, emphasizing an intricate regulatory role of post-translational modifications in the dynamics of viral RNA secondary structure establishment. The regulation of RNA chaperoning could serve as a potential therapeutic target for future treatment of RNA viruses.