Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes COVID-19, a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. Phase separation is driven, in part, by hydrophobic and electrostatic interactions. While the N protein forms spherical assemblies with unstructured RNA, it forms asymmetric condensates with viral RNA strands that contain secondary structure elements. Cross-linking mass spectrometry identified a region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. We also identified small molecules that alter the formation of N protein condensates. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We enclose the substantially revised version of our manuscript, both presenting new experimental evidence and refining the presentation and interpretation of our results. 1)We performed a series of FRAP and droplet fusion experiments to demonstrate that the N protein forms liquid condensates both in vitro and in vivo. 2)We performed detailed biochemical characterization of the wild-type and mutant N proteins in vitro. 3)We also tested the compounds that alter in vitro phase separation of N protein in SARS-CoV-2 infected cells. We showed that one of these drugs (nelfinavir mesylate) significantly reduces SARS-CoV-2 mediated cell death. 4)We put our work in the context of physiological relevance by discussing the growing body of recent literature showing that many viruses incorporate phase separation for replication and packaging in infected cells. We also discussed how phase separation of the nucleocapsid protein may enable more efficient replication and packaging of SARS-CoV-2 in infected cells. We also made substantial changes in the main text, figures, and extended data.