Intramolecular loops control SARS-CoV-2 nucleocapsid protein self-association and nucleic acid binding dependent on phosphorylation

Nguyen, A.; Datta, S. A.; Trent, C.; Wu, D.; Saleem, Z.; Szczesna, E.; Owoborode, F.; Li, Y.; Adly, A. N.; Kalish, H. R.; Piszczek, G.; Morgan, D. O.; Schuck, P.; Zhao, H.

doi:10.64898/2026.01.10.698783

bioRxiv · 10.64898/2026.01.10.698783

Intramolecular loops control SARS-CoV-2 nucleocapsid protein self-association and nucleic acid binding dependent on phosphorylation

Nguyen, A.·Datta, S. A.·Trent, C.·Wu, D.·Saleem, Z.·Szczesna, E.·Owoborode, F.·Li, Y.·Adly, A. N.·Kalish, H. R.·Piszczek, G.·Morgan, D. O.·Schuck, P.·Zhao, H.

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Abstract

The nucleocapsid protein of SARS-CoV-2 scaffolds genomic RNA into ribonucleoprotein complexes (RNP) for assembly in the virion, but also fulfills critical intracellular functions in replication and the suppression of host defense. It is comprised of a folded nucleic acid binding domain (NTD) and a dimerization domain, connected by a disordered linker containing a serine/arginine-rich (SR) region and a leucine-rich sequence (LRS). The switch between intracellular and assembly functions of N-protein is controlled by phosphorylation of the SR region, but the molecular details are unclear. Here we describe a model in which two mutually exclusive intramolecular loops bind the NTD and dynamically control self-association and nucleic acid binding properties dependent on the SR linker phosphorylation state. The model is supported by biophysical properties and interactions of full-length protein, point mutants, and peptide fragments. We find SR linker phosphorylation compacts the protein and inhibits nucleic acid binding and RNP formation, while enhancing self-association through promotion of transient coiled-coils in the LRS of the linker. These changes shift the nucleocapsid protein to a configuration poised for multi-valent interactions that support intracellular functions.

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Nguyen, A., Datta, S. A., Trent, C., Wu, D., Saleem, Z., Szczesna, E., Owoborode, F., Li, Y., Adly, A. N., Kalish, H. R., Piszczek, G., Morgan, D. O., Schuck, P., Zhao, H.. 2026-01-12. Intramolecular loops control SARS-CoV-2 nucleocapsid protein self-association and nucleic acid binding dependent on phosphorylation. https://doi.org/10.64898/2026.01.10.698783

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