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

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

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 SARS-CoV-2 nucleocapsid protein. Recreating the alpha variant mutation in an early pandemic (WA-1) background, we found that the R203K/G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. Importantly, the R203K/G204R mutation increases nucleocapsid phosphorylation, providing a molecular basis for these phenotypes. Notably, an analogous alanine substitution mutant also increases SARS-CoV-2 fitness and phosphorylation, suggesting that infection is enhanced through ablation of the ancestral ‘RG’ motif. Overall, these results demonstrate that variant mutations outside spike are also key components in SARS-CoV-2’s continued adaptation to human infection. One-Sentence Summary A mutation in the nucleocapsid gene of the SARS-CoV-2 alpha variant is found to enhance replication, fitness, and pathogenesis.