Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

Iserman, Christiane; Roden, Christine; Boerneke, Mark; Sealfon, Rachel; McLaughlin, Grace; Jungreis, Irwin; Park, Chris; Boppana, Avinash; Fritch, Ethan; Hou, Yixuan J.; Theesfeld, Chandra; Troyanskaya, Olga G; Baric, Ralph S.; Sheahan, Timothy P.; Weeks, Kevin; Gladfelter, Amy S.

A mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show 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 is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5’-End and find preferences for single-stranded RNA flanked by stable structured elements. 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.