Adaptive advantage of deletion repair in the N-terminal domain of the SARS-CoV-2 spike protein in variants of concern

Mutations within the N-terminal domain (NTD) of the spike (S) protein play a pivotal role in the emergence of successful SARS-CoV-2 viral lineages. This study investigates the influence on viral success of novel combinations of NTD lineage-defining mutations found in the Alpha, Delta, and Omicron variants. We performed comparative genomics of more than 10 million public SARS-CoV-2 samples to decipher the transmission success of different combinations of NTD markers. Additionally, we characterized the viral phenotype of such markers in a surrogate in vitro system. Alpha viruses bearing repaired deletions S:ΔH69/V70 and S:ΔY144 in Alpha background were associated with increased transmission relative to other combinations of NTD markers. After the emergence of the Omicron BA.1 lineage, Alpha viruses harbouring both repaired deletions still showed increased transmission compared to their BA.1 analogues. Moreover, repaired deletions were more frequently observed among older individuals infected with Alpha, but not with BA.1. In vitro biological characterization of Omicron BA.1 spike deletion repair patterns also revealed substantial differences with Alpha. In BA.1, S:ΔV143/Y145 repair enhanced fusogenicity and susceptibility to neutralization by vaccinated individuals’ sera. In contrast, the S:ΔH69/V70 repair did not significantly alter these traits but reduced viral infectivity. Simultaneous repair of both deletions led to lower fusogenicity. These findings highlight the intricate genotype-phenotype landscape of the spike NTD in SARS-CoV-2, which impacts viral biology, transmission efficiency, and susceptibility to neutralization. Overall, this study advances our understanding of SARS-CoV-2 evolution, carrying implications for public health and future research. ### Competing Interest Statement The authors have declared no competing interest.