Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

Abstract

The etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription, and is essential for viral replication. Here, we show that RNA induces cooperative liquid–liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid–liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

References 38

1. 1.
   A pneumonia outbreak associated with a new coronavirus of probable bat origin · 2020

   No notice
2. 2.

   Virological.org. Novel 2019 Coronavirus Genome. Available online: http://virological.org/t/novel-2019-coronavirus-genome/319. (2020).

   Not in our library

3. 3.
   Site-specific glycan analysis of the SARS-CoV-2 spike · 2020

   No notice
4. 4.
   The Coronavirus Nucleocapsid Is a Multifunctional Protein · 2014

   No notice
5. 5.
   Return of the Coronavirus: 2019-nCoV · 2020

   No notice
6. 6.

   Liu, W. et al. Evaluation of nucleocapsid and spike protein-based ELISAs for detecting antibodies against SARS-CoV-2. J. Clin. Microbiol.58, e00461-20 (2020).

   Not in our library10.1128/jcm.00461-20

7. 7.

   Fehr AR, Perlman S. Coronaviruses: an overview of their replication and pathogenesis. Methods Mol. Biol. 2015;1282:1-23.

   Not in our library10.1007/978-1-4939-2438-7_1

8. 8.
   US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 · 2020

   No notice
9. 9.

   Burbelo, P. D. et al. Sensitivity in detection of antibodies to nucleocapsid and spike proteins of severe acute respiratory syndrome coronavirus 2 in patients with coronavirus disease 2019. J. Infect. Dis.222, 206–213 (2020).

   Not in our library10.1093/infdis/jiaa273

10. 10.

    Alberti S, Gladfelter A, Mittag T. Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell. 2019;176:419-434.

    Not in our library10.1016/j.cell.2018.12.035

11. 11.

    Forman-Kay JD, Kriwacki RW, Seydoux G. Phase Separation in Biology and Disease. J. Mol. Biol. 2018;430:4603-4606.

    Not in our library10.1016/j.jmb.2018.09.006

12. 12.

    Novoa RR, et al. Virus factories: associations of cell organelles for viral replication and morphogenesis. Biol. Cell. 2005;97:147-172.

    Not in our library10.1042/bc20040058

13. 13.
    Phase Transitions Drive the Formation of Vesicular Stomatitis Virus Replication Compartments · 2018

    No notice
14. 14.
    Negri bodies are viral factories with properties of liquid organelles · 2017

    No notice
15. 15.

    Guseva S, et al. Measles virus nucleo- and phosphoproteins form liquid-like phase-separated compartments that promote nucleocapsid assembly. Sci. Adv. 2020;6:eaaz7095.

    Not in our library10.1126/sciadv.aaz7095

16. 16.

    Ye, Q., West, A. M. V., Silletti, S. & Corbett, K. D. Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein. bioRxiv (2020).

    Not in our library10.1002/pro.3909

17. 17.

    Maharana S, et al. RNA buffers the phase separation behavior of prion-like RNA binding proteins. Science. 2018;360:918-921.

    Not in our library10.1126/science.aar7366

18. 18.

    Zhou Y, Routh A. Mapping RNA-capsid interactions and RNA secondary structure within virus particles using next-generation sequencing. Nucleic Acids Res. 2020;48:e12.

    Not in our library10.1093/nar/gkz1124

19. 19.

    White JP, Lloyd RE. Regulation of stress granules in virus systems. Trends Microbiol. 2012;20:175-183.

    Not in our library10.1016/j.tim.2012.02.001

20. 20.
    Dance with the Devil: Stress Granules and Signaling in Antiviral Responses · 2020

    No notice
21. 21.

    Gordon, D. E. et al. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature583, 459–468. (2020).

    Not in our library10.1038/s41586-020-2286-9

22. 22.

    Hofweber M, et al. Phase separation of FUS is suppressed by its nuclear import receptor and arginine methylation. Cell. 2018;173:706-719.

    Not in our library10.1016/j.cell.2018.03.004

23. 23.

    Jain S, et al. ATPase-modulated stress granules contain a diverse proteome and substructure. Cell. 2016;164:487-498.

    Not in our library10.1016/j.cell.2015.12.038

24. 24.

    Peng TY, Lee KR, Tarn WY. Phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization. FEBS J. 2008;275:4152-4163.

    Not in our library10.1111/j.1742-4658.2008.06564.x

25. 25.

    Wegener M, Muller-McNicoll M. View from an mRNP: the roles of sr proteins in assembly, maturation and turnover. Adv. Exp. Med. Biol. 2019;1203:83-112.

    Not in our library10.1007/978-3-030-31434-7_3

26. 26.

    Pietrek LM, Stelzl LS, Hummer G. Hierarchical ensembles of intrinsically disordered proteins at atomic resolution in molecular dynamics simulations. J. Chem. Theory Comput. 2020;16:725-737.

    Not in our library10.1021/acs.jctc.9b00809

27. 27.

    Kwon I, et al. Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science. 2014;345:1139-1145.

    Not in our library10.1126/science.1254917

28. 28.

    Fukuhara T, et al. Utilization of host SR protein kinases and RNA-splicing machinery during viral replication. Proc. Natl Acad. Sci. USA. 2006;103:11329-11333.

    Not in our library10.1073/pnas.0604616103

29. 29.

    Hillen, H. S. et al. Structure of replicating SARS-CoV-2 polymerase. Nature584,154–156 (2020).

    Not in our library10.1038/s41586-020-2368-8

30. 30.

    Stertz S, et al. The intracellular sites of early replication and budding of SARS-coronavirus. Virology. 2007;361:304-315.

    Not in our library10.1016/j.virol.2006.11.027

31. 31.

    Cascarina, S. M. & Ross, E. D. A proposed role for the SARS-CoV-2 nucleocapsid protein in the formation and regulation of biomolecular condensates. FASEB J.34, 9832–9842 (2020).

    Not in our library10.1096/fj.202001351

32. 32.

    Boehning M, et al. RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat. Struct. Mol. Biol. 2018;25:833-840.

    Not in our library10.1038/s41594-018-0112-y

33. 33.

    Cramer P. Organization and regulation of gene transcription. Nature. 2019;573:45-54.

    Not in our library10.1038/s41586-019-1517-4

34. 34.

    Sola I, Almazan F, Zuniga S, Enjuanes L. Continuous and discontinuous RNA synthesis in coronaviruses. Annu Rev. Virol. 2015;2:265-288.

    Not in our library10.1146/annurev-virology-100114-055218

35. 35.

    Lee W, Tonelli M, Markley JL. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics. 2015;31:1325-1327.

    Not in our library10.1093/bioinformatics/btu830

36. 36.

    Shen Y, Delaglio F, Cornilescu G, Bax A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR. 2009;44:213-223.

    Not in our library10.1007/s10858-009-9333-z

37. 37.

    Magnus M, Boniecki MJ, Dawson W, Bujnicki JM. SimRNAweb: a web server for RNA 3D structure modeling with optional restraints. Nucleic Acids Res. 2016;44:W315-W319.

    Not in our library10.1093/nar/gkw279

38. 38.

    Craft JW, Legge GB. An AMBER/DYANA/MOLMOL phosphorylated amino acid library set and incorporation into NMR structure calculations. J. Biomol. NMR. 2005;33:15-24.

    Not in our library10.1007/s10858-005-1199-0

Cited by papers we hold 15

• SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence · 2023
  No notice
• The SARS-CoV-2 nucleocapsid protein: its role in the viral life cycle, structure and functions, and use as a potential target in the development of vaccines and diagnostics · 2023
  No notice
• Gastrointestinal Manifestations of COVID-19 Infection: Clinicopathologic Findings in Intestinal Resections Performed at Single Institution · 2022
  Not checked
• NMR Provides Unique Insight into the Functional Dynamics and Interactions of Intrinsically Disordered Proteins · 2022
  No notice
• SARS-CoV-2 nucleocapsid protein adheres to replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress · 2022
  No notice
• Conformational Dynamics of Intrinsically Disordered Proteins Regulate Biomolecular Condensate Chemistry · 2022
  No notice
• RNA-induced liquid phase separation of SARS-CoV-2 nucleocapsid protein facilitates NF-κB hyper-activation and inflammation · 2021
  No notice
• Repurposing Ivermectin for COVID-19: Molecular Aspects and Therapeutic Possibilities · 2021
  Not checked
• The Integral Role of RNA in Stress Granule Formation and Function · 2021
  No notice
• A novel cell culture system modeling the SARS-CoV-2 life cycle · 2021
  No notice
• The SARS-CoV-2 Nucleocapsid Protein and Its Role in Viral Structure, Biological Functions, and a Potential Target for Drug or Vaccine Mitigation · 2021
  No notice
• GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein · 2021
  No notice
• The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA · 2021
  No notice
• Liquid–liquid phase separation underpins the formation of replication factories in rotaviruses · 2021
  No notice
• SARS‐CoV‐2 nucleocapsid protein phase‐separates with RNA and with human hnRNPs · 2020
  No notice

Reads

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.