FIG 6.

Construction of Tet on-BiLC stable cell lines for detecting the interactions between influenza virus polymerase subunits. 293T stable cell lines expressing GN-PB1 and PA-GC (A), PB1-GN and PB2-GC (B), and PA-GC, PB2-GN, and Flag-PB1 (C) were constructed using the Tet on system. The stable cell lines were induced by doxycycline, and RLU values of cell lysates were determined by the use of a microplate reader at 24 h postinduction. The levels of protein expression were detected by Western blotting as indicated. (D) The inhibitory functions of interferon alpha (IFN-α), IFN-β, and IFN-γ with respect to the influenza virus polymerase assembly were detected with the stable cell line expressing PA-GC, PB2-GN, and PB1-Flag. The stable cell lines were incubated with the indicated interferons (IFN-α and IFN-γ, 1,000 U/ml; IFN-β, 10 ng/ml) for 24 h and then induced by doxycycline (300 ng/ml) for another 24 h. The RLU values of cell lysates were determined by the use of a microplate reader at 24 h postinduction. The levels of protein expression were detected by Western blotting as indicated. (E) The dose-dependent effects of IFN-β on the influenza virus polymerase assembly were detected with the stable cell line expressing PA-GC, PB2-GN, and PB1-Flag. Data are shown as means ± SEM. **, P < 0.01; ***, P < 0.001 (Student's t test).