Mutations in S2 subunit of SARS-CoV-2 Omicron spike strongly influence its conformation, fusogenicity and neutralization sensitivity

Abstract

SARS-CoV-2 has remarkable ability to respond to and evolve against the selection pressure by host immunity exemplified by emergence of Omicron lineage. Here, we characterized the functional significance of mutations in Omicron spike. By systematic transfer of mutations in WT spike we assessed neutralization sensitivity, fusogenicity, and TMPRSS2-dependence for entry. The data revealed that the mutations in both S1 and S2 complement to make Omicron highly resistant. Strikingly, the mutations in Omicron S2 modulated the neutralization sensitivity to NTD- and RBD-antibodies, but not to S2 specific neutralizing antibodies, suggesting that the mutations in S2 were primarily acquired to gain resistance to S1-antibodies. Although all six mutations in S2 appeared to act in concert, D796Y showed greatest impact on neutralization sensitivity and rendered WT virus >100-fold resistant to S309, COVA2-17, and 4A8. S2 mutations greatly reduced the antigenicity for NAbs due to reduced exposure of epitopes. In terms of the entry pathway, S1 or S2 mutations only partially altered the entry phenotype of WT and required both sets of mutations for complete switch to endosomal route and loss of syncytia formation. In particular, N856K and L981F in Omicron reduced fusion capacity and explain why subsequent Omicron variants lost them to regain fusogenicity.