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

ABSTRACT: 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 characterized by single-stranded RNA flanked by structured elements 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 therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.

SUBMITTER: Iserman C

PROVIDER: S-EPMC7691212 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description: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 characterized by single-stranded RNA flanked by structured elements 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.

2020-12-17 | GSE162569 | GEO

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

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

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RNA-induced liquid phase separation of SARS-CoV-2 nucleocapsid protein facilitates NF-κB hyper-activation and inflammation.

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Project description:The current work identified the protein interactors of SARS-CoV-2 Nucleocapsid RNA binding protein (NCAP or N protein) in lung cancer A-549 cells, using a co-immunoprecipitation strategy, which is coupled with SILAC-based mass spec. A significant proportion of the identified NCAP interacting proteins included stress granule (SG) and immunoregulators. SG nucleator G3BP1 showed specific interaction wit NCAP in unstressed and oxidative- or heat-stressed cells. Following stress treatment, NCAP showed enhanced association with SGs. Recombinant NCAP exhibited in vitro liquid-liquid phase separation (LLPS)to form liquid droplets. RNA stimulated NCAP to develop droplets in vitro and SG assembly in cells, and RNase A treatment completely blocked both of these functions. An RNA intercalating mitoxantrone disrupted NCAP assembly in SGs invitro and in cells. This study provides insight into the biological processes and biophysical properties of the SARS-CoV-2 NCAP and identifies a candidate way to target this protein.

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SARS CoV-2 nucleocapsid protein forms condensates with viral genomic RNA.

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