FIG. 4.

Inhibition of HA cleavage in T-ex5-treated Calu-3 cells. (A) Confluent Calu-3 monolayers were treated with PPMO (25 μM) for 24 h prior to infection. Cells were inoculated with Memphis/96 (H1N1) at an MOI of 0.0001 for 1 h, and the inoculum was then removed and the cells incubated in the presence of PPMO (25 μM). At 72 h p.i., virus-containing supernatants were pelleted by ultracentrifugation and analyzed by SDS-PAGE and Western blotting using H1-specific antibodies (upper panel). Total cellular RNA was isolated and analyzed by RT-PCR using TMPRSS2-specific primers to amplify a full-length fragment of 1,228 bp. Full-length products and truncated PCR products are indicated by arrows (lower panel). The full-length PCR product in PPMO T-ex4-treated cells is indicated by a white arrow. (B) Dose-dependent inhibition of HA cleavage by PPMO T-ex5. Calu-3 monolayers were treated with different concentrations of PPMO (5, 10, or 25 μM) for 24 h prior to infection. Cells were inoculated at an MOI of 0.0001 for 1 h, and the inoculum was then removed and the cells further incubated in the presence of PPMO (5, 10, or 25 μM). At 72 h p.i., virus-containing supernatants were subjected to SDS-PAGE and Western blot analysis with H1-specific antibodies (upper panel). Total RNA was isolated and analyzed by RT-PCR using TMPRSS2-specific primers as described above (lower panel).