Fig. 3.

Effects of cell-surface proteases on HR2 peptide antiviral activities. (A) Calu3 and Vero81 cells were incubated with MERS pps in the presence of increasing concentrations of HR2 peptides. (B) Total cellular RNA was isolated from Calu3and Vero81 cells, and evaluated for the expression of DPP4, TMPRSS2, furin, cathepsin L (Cat. L) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) transcripts by reverse transcription – quantitative PCR. Expression levels were plotted relative to HPRT expression levels. ND = Not Detected. (C) Calu3 and Vero81 cells were transduced with MERS pps in the presence of increasing concentrations of HR2 peptide or camostat (Camo). Camo was present from 1 h pre-transduction and HR2 was present at the time of MERS pp inoculation. (D) Calu3 cells were incubated with or without 10 μM camostat (Camo) for 1 h, then transduced with MERS pps in the presence of increasing concentrations of HR2 peptide. (E) Vero81 cells were transfected with either TMPRSS2 or vector control plasmids. At 2 d post-transfection, cells were transduced with MERS pps in the presence of increasing concentrations of HR2 peptide. For (A), (C), (D) and (E), unbound MERS pps and entry inhibitors were removed at 1 h post-transduction. Virus entry was quantified by measuring luciferase levels at 48 h post-transduction and data were normalized to control conditions lacking inhibitors. Error bars present SD from the mean (n = 3). Statistical significance was assessed by student's t-test. ***, P < 0.001.