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 A. Plante, Xuping Xie, Patricia Aguilar, Scott C. Weaver, Pei Yong Shi, David H. Walker, Andrew L. Routh, Kenneth S. Plante, Vineet D. Menachery

Research output: Contribution to journalArticlepeer-review


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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