Data sets used to generate the graphs.

The spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is activated by the host cell proteases cathepsin L or TMPRSS2. The ancestral virus circulating in Wuhan in 2020 and early variants mainly use TMPRSS2 for entry into Calu-3 lung cells while the Omicron subvariant BA.1 and most subsequently circulating Omicron subvariants employ both cathepsin L and TMPRSS2 for Calu-3 cell entry. Here, we investigated which amino acid residues in the S protein of the Omicron subvariant BA.1 control protease choice. We show that Y655 promotes S protein cleavage and cathepsin L-dependent entry while H655 jointly with N969 promotes TMPRSS2-dependent entry. These results define molecular signatures of SARS-CoV-2 protease choice and lung cell infection.