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

To confirm knockdown of enzymatically active TMPRSS2 expression, Calu-3 cells were 234 treated with PPMO or remained untreated for 24 h, after which TMPRSS2-specific mRNA 235 was isolated and analysed by RT-PCR as described previously (Böttcher-Friebertshäuser et 236 al., 2011) . Total RNA was analysed with primers designed to amplify nucleotides 108 to 1336 237 of TMPRSS2-mRNA. A full-length PCR product of 1228 bp was amplified from untreated and 238 scramble PPMO treated Calu-3 cells, whereas a shorter PCR fragment of about 1100 bp was 239 amplified from T-ex5 PPMO-treated cells (Fig. 3C ). Sequencing revealed that the truncated 240 TMPRSS2-mRNA lacked the entire exon 5 (data not shown). To further confirm that T-ex5 241 PPMO single dose treatment prior to infection still interferes with TMPRSS2-mRNA splicing 242 at 72 h p.i., total RNA was isolated from infected cells at 72 h p.i. and amplified as described In sum, our data demonstrate that inhibition of either TMPRSS2 or furin strongly inhibits 301 SARS-CoV-2 in Calu-3 human airway cells, indicating that both proteases are critical for S 302 activation. In contrast, endosomal cathepsins are dispensable or not involved at all in SARS-303