Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis

Bryan A. Johnson, Yiyang Zhou, Kumari G. Lokugamage, Michelle N. Vu, Nathen Bopp, Patricia A. Crocquet-Valdes, Birte Kalveram, Craig Schindewolf, Yang Liu, Dionna Scharton, Jessica Plante, Xuping Xie, Patricia Aguilar, Scott C. Weaver, Pei-Yong Shi, David H. Walker, Andrew Routh, Kenneth S. Plante, Vineet D. Menachery

Research output: Contribution to journalArticlepeer-review

59 Scopus citations


While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203–205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo. Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral ‘RG’ motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2’s continued adaptation to human infection.

Original languageEnglish (US)
Article numbere1010627
JournalPLoS pathogens
Issue number6
StatePublished - Jun 2022

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Molecular Biology
  • Genetics
  • Virology


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