Figure 2.

Efficiency of TMPRSS2 cleavage of SARS-CoV and MERS-CoV S. (A) P6-P3′ amino acid sequences of the synthesized and analysed FRET substrates representing the CoV S cleavage site motifs, which all contain an N-terminal o-aminobenzoyl fluorophore and a C-terminal Tyr(3-NO₂)-NH₂ as a quenching residue. The TMPRSS2 cleavage site is indicated by a red arrow. (B) The cleavage efficiency of the FRET substrates by recombinant TMPRSS2 was measured in an enzyme kinetic assay. The data shown are the mean values + SD based on three independent measurements with three independent weights of the substrates. (C) HeLa cells were transfected with plasmids encoding C-terminally FLAG-tagged SARS-CoV or MERS-CoV S, and TMPRSS2. Simultaneously, HeLa cells were treated with 50 µM MI-1851 or remained untreated. At 48 h after transfection and inhibitor treatment, the cell lysates were subjected to SDS-PAGE and Western blot analysis with an antibody targeting the S2 subunit of SARS-CoV S or the C-terminal FLAG-tag of MERS-CoV S. The data shown are the representative results of three independent experiments.