Genomic RNA elements drive phase separation of the SARS-CoV-2 nucleocapsid

Iserman, Christiane; Roden, Christine A.; Boerneke, Mark A.; Sealfon, Rachel S.G.; McLaughlin, Grace A.; Jungreis, Irwin; Fritch, Ethan J.; Hou, Yixuan J.; Ekena, Joanne; Weidmann, Chase A.; Theesfeld, Chandra L.; Kellis, Manolis; Troyanskaya, Olga G.; Baric, Ralph S.; Sheahan, Timothy P.; Weeks, Kevin M.; Gladfelter, Amy S.

We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33°C and 37°C) and reduced at room temperature (22°C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions with single-stranded RNA flanked by structure and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.