Genomic RNA Elements Drive Phase Separation of the SARS-CoV-2 Nucleocapsid

We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5'-End) under physiological conditions and condensation is enhanced at human body temperatures. Other regions of gRNA promote dissolution, counteracting aggregation of the large genome. This combination of elements ensures condensates of both specific molecular and physical identity, leading to exclusion of non-packaged RNA sequences. N-protein binds single-stranded RNA flanked by stable structured elements and these features specify the number and location 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 sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2.