FIG 2.

Proteolytic activation of IAVs of different HA subtypes in human Calu-3 airway cells. (A) Multicycle replication of IAV and IBV in PPMO-treated Calu-3 cells. Cells were treated with 25 μM T-ex5 or scramble PPMO for 24 h or remained untreated (w/o). Cells were then inoculated with viruses at an MOI of 0.01 to 0.001 and incubated in the absence of PPMO for 24 h. Cells were fixed and immunostained for NP (green). The nuclei were stained using DAPI (blue). Representative images from two or three independent experiments are shown. Scale bars indicate 100 μm. (B) RT-PCR analysis of TMPRSS2 mRNA in PPMO-treated Calu-3 cells. Cells were treated with T-ex5 PPMO (25 μM) or remained untreated (w/o) for 24 h, the medium was replaced, and the cells were incubated in the absence of T-ex5 for 24 h. Total RNA was isolated and amplified with TMPRSS2-specific primers to amplify a full-length (filled arrowhead) and truncated Δex5 (open arrowhead) mRNA fragment. (C) Analysis of HA cleavage in PPMO-treated Calu-3 cells. Cells were treated with PPMO for 24 h as described above, inoculated with virus at an MOI of 1, and incubated for 24 h. Cell lysates were subjected to SDS-PAGE and Western blotting with HA-specific antibodies. Beta-actin served as a loading control. HA2 was not detected by H4- and H5-specific antibodies. (D) Virus growth kinetics of H4, H5, and H16 IAV in PPMO-treated Calu-3 cells. Calu-3 monolayers were treated with PPMO or remained untreated for 24 h as described above and then inoculated with H4, H5, or H16 IAV at an MOI of 0.01 to 0.001 and incubated without PPMO for 72 h. At the indicated time points, virus titers were determined by plaque assay or focus-forming assay (H16-SC35M). Data are mean values ± SD from two or three independent experiments.