SARS-CoV-2 Spike H655Y drives protease preference but does not dictate cellular tropism

Throughout the COVID-19 pandemic, SARS-CoV-2 has undergone rapid adaptation, with the Omicron variant exhibiting an unexpected shift towards upper airway infection and preference for membrane fusion activated by endosomal cathepsins, a reversion to ancestral sarbecovirus entry mechanisms. This phenotype coincides with convergent acquisition of the spike mutation H655Y, which reduces TMPRSS2-mediated activation. Here, using a comprehensive panel of 13 spikes of SARS-CoV-2 variants, we interrogated whether H655Y-mediated protease switching explains Omicron’s upper airway phenotype, challenging several existing hypotheses, from spike stability, shedding and acquired intra-molecular interactions by H655Y. Our findings reveal that protease preference and tissue tropism are mechanistically uncoupled. While spike pre-processing by furin determines protease preference in pre-Omicron variants, this relationship breaks down in H655Y-bearing viruses. Notably, mutations in the NTD and RBD of BA.2.86 can override the H655Y phenotype entirely, indicating that RBD-mediated interactions, rather than protease usage, represent the critical determinants of Omicron’s upper airway adaptation. This work reframes our understanding of coronavirus spike evolution, revealing that tissue tropism operates through mechanisms fundamentally distinct from those dictating protease preference.