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

SARS-CoV-2 has a remarkable ability to respond to and evolve against the selection pressure by host immunity exemplified by the emergence of Omicron lineage. Here, we characterized the functional significance of mutations in Omicron (BA.1 and BA.5) spike. By systematic transfer of mutations in wild type (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 a highly resistant variant. Strikingly, the mutations in Omicron S2 modulated the neutralization sensitivity to N-terminal domain and receptor binding domain 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 the greatest impact on neutralization sensitivity and rendered the WTvirus > 100-fold resistant to S309, COVA2-17,and 4A8. The S2 mutations rendered the WT virus >100-fold resistant to S309, COVA2-17, and 4A8. The S2 mutations greatly reduced the antigenicity to neutralizing antibodies due to reduced exposure of epitopes. In addition to the effect on potency, S2 mutations increased antigenic heterogeneity of Omicron spike protein and neutralization of pseudoviruses plateaued below 100%. In terms of the entry pathway, S1 or S2 mutations only partially altered the entry phenotype of WT, and both sets of mutations were required for a complete switch to the endosomal route and loss of syncytia formation. In particular, N856K and L981F in Omicron BA.1 compromised fusion capacity which helps explain why subsequent Omicron variants lost them in order to regain fusogenicity. IMPORTANCE The Omicron subvariants have substantially evaded host-neutralizing antibodies and adopted an endosomal route of entry. The virus has acquired several mutations in the receptor binding domain and N-terminal domain of S1 subunit, but remarkably, also incorporated mutations in S2 which are fixed in Omicron sub-lineage. Here, we found that the mutations in the S2 subunit affect the structural and biological properties such as neutralization escape, entry route, fusogenicity, and protease requirement. In vivo, these mutations may have significant roles in tropism and replication. A detailed understanding of the effects of S2 mutations on Spike function, immune evasion, and viral entry would inform the vaccine design, as well as therapeutic interventions aiming to block the essential proteases for virus entry. Thus, our study has identified the crucial role of S2 mutations in stabilizing the Omicron spike and modulating neutralization resistance to antibodies targeting the S1 subunit.