The rapid spread and strong immune evasion of the SARS-CoV-2 Omicron subvariants has raised serious concerns for the global COVID-19 pandemic. These new variants exhibit reduced fusogenicity and increased endosomal entry pathway utilization compared to the ancestral D614G variant, the underlying mechanisms of which remain elusive. Here we show that the C-terminal S1 mutations of the BA.1.1 subvariant, H655Y and T547K, critically govern the low fusogenicity of Omicron. Notably, H655Y also dictates the enhanced endosome entry pathway utilization. Mechanistically, T547K and H655Y likely stabilize the spike trimer conformation, as shown by increased molecular interactions in structural modeling as well as reduced S1 shedding. Importantly, the H655Y mutation also determines the low fusogenicity and high dependence on the endosomal entry pathway of other Omicron subvariants, including BA.2, BA.2.12.1, BA.4/5 and BA.2.75. These results uncover mechanisms governing Omicron subvariant entry and provide insights into altered Omicron tissue tropism and pathogenesis.

Determinants and Mechanisms of the Low Fusogenicity and Endosomal Entry of Omicron Subvariants