Convergent evolution in nucleocapsid facilitated SARS-CoV-2 adaptation for human infection

During the early stages of the COVID-19 pandemic, several SARS-CoV-2 variants of concern (VOCs) independently acquired mutations in the highly variable 203–205 amino acid region of the nucleocapsid (N) protein including R203K + G204R (found in the Alpha, Gamma, and Omicron variants), R203M (in Delta), and T205I (in Beta). In previous research, we demonstrated that the R203K + G204R mutation significantly enhances SARS-CoV-2 N phosphorylation, which subsequently increases viral fitness and pathogenesis. In this study, we investigated the effects of the R203M and T205I mutations on SARS-CoV-2 infection. Using reverse genetics, we introduced these mutations into the early pandemic Washington-1 (WA-1) strain and observed that both the R203M and T205I mutants enhanced replication and viral fitness. However, unlike the R203K + G204R mutant, the R203M and T205I mutants caused only moderate changes in lung pathology. Notably, each mutation—R203K + G204R, R203M, and T205I—induced distinct patterns of N phosphorylation, which likely contribute to the observed phenotypic differences between the mutants. Interestingly, when bat cells expressing human ACE2 were infected with these mutants, we observed a reduction, rather than an enhancement, in both SARS-CoV-2 replication and N phosphorylation. Collectively, our findings suggest that the R203K + G204R, R203M, and T205I mutations are a result of convergent evolution and reflect how SARS-CoV-2 has adapted for human infection.