# Amino acid residues 655 and 969 in the spike protein of Omicron subvariant BA.1 control use of TMPRSS2 versus Cathepsin L dependent entry pathways and cell tropism

## TL;DR

This study identifies specific amino acid residues in the Omicron BA.1 variant that determine how the virus enters lung cells using different host proteases.

## Contribution

The study identifies residues Y655 and H655/N969 as key determinants of protease usage in the Omicron BA.1 spike protein.

## Key findings

- Y655 promotes cathepsin L-dependent entry into cells.
- H655 and N969 together promote TMPRSS2-dependent entry.
- These residues define molecular signatures for protease choice and lung cell infection.

## Abstract

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.