TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

Dorothea Bestle; Miriam Ruth Heindl; Hannah Limburg; Thuy Van Lam van; Oliver Pilgram; Hong Moulton; David A. Stein; Kornelia Hardes; Markus Eickmann; Olga Dolnik; Cornelius Rohde; Stephan Becker; Hans-Dieter Klenk; Wolfgang Garten; Torsten Steinmetzer; Eva Böttcher-Friebertshäuser

Proteolytic processing of CoV S is a complex process that requires cleavage at two different 378 sites and is yet not fully understood. The amino acid sequence at the S1/S2 and S2' 379 cleavage sites varies among CoVs (Fig. 1B) , suggesting that partially different proteases 380 may be involved in activation. Sequence analyses of the S protein of the novel emerged 381 SARS-CoV-2 suggested that the R-R-A-R motif at the S1/S2 site may by sensitive to 382 cleavage by furin, while the S2' site contains a single R residue that can be cleaved by Hoffmann et al., 2020). In the present study, we demonstrate that the SARS-CoV-2 S protein 385 is cleaved by furin and by TMPRSS2. Furthermore, we show that multicycle replication of 386 SARS-CoV-2 in Calu-3 human airway cells is strongly suppressed by inhibiting TMPRSS2 387 and furin activity, demonstrating that both proteases are crucial for S activation in these cells. 388