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The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

Jasmine Cubuk, Jhullian J. Alston, J. Jeremías Incicco, View ORCID ProfileSukrit Singh, Melissa D. Stuchell-Brereton, Michael D. Ward, Maxwell I. Zimmerman, Neha Vithani, View ORCID ProfileDaniel Griffith, Jason A. Wagoner, View ORCID ProfileGregory R. Bowman, Kathleen B. Hall, View ORCID ProfileAndrea Soranno, View ORCID ProfileAlex S. Holehouse
doi: https://doi.org/10.1101/2020.06.17.158121
Jasmine Cubuk
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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Jhullian J. Alston
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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J. Jeremías Incicco
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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Sukrit Singh
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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  • ORCID record for Sukrit Singh
Melissa D. Stuchell-Brereton
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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Michael D. Ward
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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Maxwell I. Zimmerman
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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Neha Vithani
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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Daniel Griffith
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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  • ORCID record for Daniel Griffith
Jason A. Wagoner
3, Stony Brook, NY 11794
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Gregory R. Bowman
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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  • ORCID record for Gregory R. Bowman
Kathleen B. Hall
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
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Andrea Soranno
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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  • ORCID record for Andrea Soranno
  • For correspondence: soranno@wustl.edu alex.holehouse@wustl.edu
Alex S. Holehouse
1, 660 S. Euclid Ave., St. Louis, MO, 63110, USA
2, St. Louis, MO 63130, USA
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  • ORCID record for Alex S. Holehouse
  • For correspondence: soranno@wustl.edu alex.holehouse@wustl.edu
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Abstract

The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein that plays a variety of roles in the viral life cycle including replication, transcription, and genome packaging. Despite its critical and multifunctional nature, the molecular details that underlie how N protein mediates these functions are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to the function of SARS-CoV-2 N protein. N protein contains three intrinsically disordered regions and two folded domains. All three disordered regions are highly dynamic and contain regions of transient helicity that appear to act as local binding interfaces for protein-protein or protein-RNA interactions. The two folded domains do not significantly interact with one another, such that full-length N protein is a flexible and multivalent RNA binding protein. As observed for other proteins with similar molecular features, we found that N protein undergoes liquid-liquid phase separation when mixed with RNA. Polymer models predict that the same multivalent interactions that drive phase separation also engender RNA compaction. We propose a simple model in which symmetry breaking through specific binding sites promotes the formation of metastable single-RNA condensate, as opposed to large multi-RNA phase separated droplets. We speculate that RNA compaction to form dynamic single-genome condensates may underlie the early stages of genome packaging. As such, assays that measure how compounds modulate phase separation could provide a convenient tool for identifying drugs that disrupt viral packaging.

Competing Interest Statement

The authors have declared no competing interest.

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Posted June 18, 2020.
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The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA
Jasmine Cubuk, Jhullian J. Alston, J. Jeremías Incicco, Sukrit Singh, Melissa D. Stuchell-Brereton, Michael D. Ward, Maxwell I. Zimmerman, Neha Vithani, Daniel Griffith, Jason A. Wagoner, Gregory R. Bowman, Kathleen B. Hall, Andrea Soranno, Alex S. Holehouse
bioRxiv 2020.06.17.158121; doi: https://doi.org/10.1101/2020.06.17.158121
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The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA
Jasmine Cubuk, Jhullian J. Alston, J. Jeremías Incicco, Sukrit Singh, Melissa D. Stuchell-Brereton, Michael D. Ward, Maxwell I. Zimmerman, Neha Vithani, Daniel Griffith, Jason A. Wagoner, Gregory R. Bowman, Kathleen B. Hall, Andrea Soranno, Alex S. Holehouse
bioRxiv 2020.06.17.158121; doi: https://doi.org/10.1101/2020.06.17.158121

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