FIG. 5.
AG879 and A9 strongly inhibit the synthesis of all three viral RNA species independently of the NF-κB pathway. (A) AG879 and A9 inhibit viral RNA transcription from the cRNA or vRNA promoter, based on the FLuc-based 5-plasmid assay. A549 cells were transfected with expression plasmids of NP, PA, PB1, and PB2, together with a vNA-LUC or cNA-LUC reporter construct. DMSO or inhibitor was added to the cells at 8 h posttransfection and FLuc activity determined 16 h later. FLuc, firefly luciferase. (B) Synthesis of all three RNA species (vRNA, cRNA, and mRNA) was decreased in the virus-infected cells treated with AG879 and A9. The viral RNA level was quantified by real-time RT-PCR and is shown as CT value. (C) Overexpression of NF-κB molecule p65 cannot reverse the inhibition of viral RNA transcription from the cRNA promoter by AG879 or A9. The 5-plasmid assay was conducted as described for panel A in cells transfected with empty vector plasmid or p65 expression vector. Compound treatment and LUC assay were similarly performed. Results shown are the averages from at least three independent experiments. Error bars indicate SD. Statistical analysis was conducted with Student's t test (**, P < 0.01).