SARS-CoV-2 Nucleocapsid Protein Forms Condensates with Viral Genomic RNA

Overview

Journal PLoS Biol

Specialty Biology

Date 2021 Oct 11

PMID 34634033

Citations 81

Authors

Amanda Jack

Luke S Ferro

Michael J Trnka

Eddie Wehri

Amrut Nadgir

Xammy Nguyenla

Douglas Fox

Katelyn Costa

Sarah Stanley

Julia Schaletzky

Ahmet Yildiz

Affiliations

Soon will be listed here.

Abstract

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (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. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that drives interactions between N proteins in condensates, and deletion of this region disrupts phase separation. We also identified small molecules that alter the size and shape of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.

Citing Articles

Rational design of phytovirucide inhibiting nucleocapsid protein aggregation in tomato spotted wilt virus.

Zan N, Li J, Yao J, Wu S, Li J, Chen F Nat Commun. 2025; 16(1):2034.

PMID: 40016246 PMC: 11868578. DOI: 10.1038/s41467-025-57281-z.

Swine Acute Diarrhea Syndrome Coronavirus: An Overview of Virus Structure and Virus-Host Interactions.

Baek S, Park J Animals (Basel). 2025; 15(2).

PMID: 39858149 PMC: 11758606. DOI: 10.3390/ani15020149.

The SARS-CoV-2 nucleocapsid protein interferes with the full enzymatic activation of UPF1 and its interaction with UPF2.

Nuccetelli V, Mghezzi-Habellah M, Deymier S, Roisin A, Gerard-Baraggia F, Rocchi C Nucleic Acids Res. 2025; 53(2).

PMID: 39831305 PMC: 11744187. DOI: 10.1093/nar/gkaf010.

RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as viral replication factories.

Kaundal S, Anish R, Ayyar B, Shanker S, Kaur G, Crawford S Sci Adv. 2024; 10(51):eadp9333.

PMID: 39705355 PMC: 11661447. DOI: 10.1126/sciadv.adp9333.

The Nucleocapsid (N) Proteins of Different Human Coronaviruses Demonstrate a Variable Capacity to Induce the Formation of Cytoplasmic Condensates.

Tikhomirova M, Kuzmenko O, Arifulin E, Shirokova O, Musinova Y, Sheval E Int J Mol Sci. 2024; 25(23).

PMID: 39684875 PMC: 11642284. DOI: 10.3390/ijms252313162.

References

Lorenz R, Bernhart S, Honer Zu Siederdissen C, Tafer H, Flamm C, Stadler P . ViennaRNA Package 2.0. Algorithms Mol Biol. 2011; 6:26. PMC: 3319429. DOI: 10.1186/1748-7188-6-26. View

Sahin U, Muik A, Derhovanessian E, Vogler I, Kranz L, Vormehr M . COVID-19 vaccine BNT162b1 elicits human antibody and T1 T cell responses. Nature. 2020; 586(7830):594-599. DOI: 10.1038/s41586-020-2814-7. View

Trnka M, Baker P, Robinson P, Burlingame A, Chalkley R . Matching cross-linked peptide spectra: only as good as the worse identification. Mol Cell Proteomics. 2013; 13(2):420-34. PMC: 3916644. DOI: 10.1074/mcp.M113.034009. View

Hurst K, Kuo L, Koetzner C, Ye R, Hsue B, Masters P . A major determinant for membrane protein interaction localizes to the carboxy-terminal domain of the mouse coronavirus nucleocapsid protein. J Virol. 2005; 79(21):13285-97. PMC: 1262602. DOI: 10.1128/JVI.79.21.13285-13297.2005. View

Zhao H, Wu D, Nguyen A, Li Y, Adao R, Valkov E . Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids. iScience. 2021; 24(6):102523. PMC: 8103780. DOI: 10.1016/j.isci.2021.102523. View

Chen Z, Rappsilber J . Protein Dynamics in Solution by Quantitative Crosslinking/Mass Spectrometry. Trends Biochem Sci. 2018; 43(11):908-920. PMC: 6240160. DOI: 10.1016/j.tibs.2018.09.003. View

Conforti R, Ma Y, Morel Y, Paturel C, Terme M, Viaud S . Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands. Cancer Res. 2010; 70(2):490-500. DOI: 10.1158/0008-5472.CAN-09-1890. View

Hoffmann M, Kleine-Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S . SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020; 181(2):271-280.e8. PMC: 7102627. DOI: 10.1016/j.cell.2020.02.052. View

Heinrich B, Maliga Z, Stein D, Hyman A, Whelan S . Phase Transitions Drive the Formation of Vesicular Stomatitis Virus Replication Compartments. mBio. 2018; 9(5). PMC: 6123442. DOI: 10.1128/mBio.02290-17. View

10.

Dyall J, Coleman C, Hart B, Venkataraman T, Holbrook M, Kindrachuk J . Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection. Antimicrob Agents Chemother. 2014; 58(8):4885-93. PMC: 4136000. DOI: 10.1128/AAC.03036-14. View

11.

Van Treeck B, Protter D, Matheny T, Khong A, Link C, Parker R . RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome. Proc Natl Acad Sci U S A. 2018; 115(11):2734-2739. PMC: 5856561. DOI: 10.1073/pnas.1800038115. View

12.

Patel A, Lee H, Jawerth L, Maharana S, Jahnel M, Hein M . A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell. 2015; 162(5):1066-77. DOI: 10.1016/j.cell.2015.07.047. View

13.

Chan K, Dorosky D, Sharma P, Abbasi S, Dye J, Kranz D . Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2. Science. 2020; 369(6508):1261-1265. PMC: 7574912. DOI: 10.1126/science.abc0870. View

14.

Riback J, Zhu L, Ferrolino M, Tolbert M, Mitrea D, Sanders D . Composition-dependent thermodynamics of intracellular phase separation. Nature. 2020; 581(7807):209-214. PMC: 7733533. DOI: 10.1038/s41586-020-2256-2. View

15.

Jain A, Vale R . RNA phase transitions in repeat expansion disorders. Nature. 2017; 546(7657):243-247. PMC: 5555642. DOI: 10.1038/nature22386. View

16.

Lu S, Ye Q, Singh D, Cao Y, Diedrich J, Yates 3rd J . The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein. Nat Commun. 2021; 12(1):502. PMC: 7820290. DOI: 10.1038/s41467-020-20768-y. View

17.

Kuo L, Hurst-Hess K, Koetzner C, Masters P . Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras. J Virol. 2016; 90(9):4357-4368. PMC: 4836358. DOI: 10.1128/JVI.03212-15. View

18.

King O, Gitler A, Shorter J . The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease. Brain Res. 2012; 1462:61-80. PMC: 3372647. DOI: 10.1016/j.brainres.2012.01.016. View

19.

Iserman C, Roden C, Boerneke M, Sealfon R, McLaughlin G, Jungreis I . Genomic RNA Elements Drive Phase Separation of the SARS-CoV-2 Nucleocapsid. Mol Cell. 2020; 80(6):1078-1091.e6. PMC: 7691212. DOI: 10.1016/j.molcel.2020.11.041. View

20.

Chu F, Thornton D, Nguyen H . Chemical cross-linking in the structural analysis of protein assemblies. Methods. 2018; 144:53-63. PMC: 6051896. DOI: 10.1016/j.ymeth.2018.05.023. View