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.IMPORTANCEAfter its emergence, SARS-CoV-2 rapidly adapted to human infection, acquiring numerous mutations across its genome. Many of these mutations remain uncharacterized. This study examines a mutational hotspot among SARS-CoV-2 variants: residues 203-205 of the nucleocapsid (N) protein. We demonstrate that three unique mutations identified in this region among variants of concern enhance infection in human cells and animal models while eliciting distinct patterns of N protein phosphorylation. Intriguingly, these same mutations reduce both N protein phosphorylation and viral replication in bat cells. These findings suggest that each mutation represents independent adaptation by variants of concern for human infection. Importantly, this study underscores the critical role of these mutations in facilitating the expansion of SARS-CoV-2 into human populations and highlights the potential for similar mutations to drive future zoonotic coronavirus outbreaks.