Abstract
SARS-CoV-2 entry into host cells depends on proteolytic activation of the spike protein by host proteases, a process shaped by spike mutations that influence viral specificity and infectivity. Using human airway epithelial models, this study investigated how different SARS-CoV-2 variants interact with host serine proteases. The Delta variant exhibited enhanced and stable binding to Hepsin through stronger ionic and hydrophobic interactions, promoting efficient spike activation and cell entry. In contrast, Omicron BA.1 showed weaker Hepsin binding and relied more on TMPRSS2 or cathepsins, depending on the cellular context. These findings reveal how variant-specific differences in protease usage are linked to spike protein mutations and cleavage site evolution. By illuminating the dynamic interplay between viral adaptation and host protease specificity, this work provides insights into mechanisms that influence viral transmission and immune evasion, with implications for developing targeted antiviral strategies and understanding the evolution of emerging SARS-CoV-2 variants.