FIG 3.

NV RNA replication does not induce an IFN response. (A) 293FT-ISRE-Luc reporter cells have strong IFN responses to various stimuli and detect both IFN induction and signaling. Cells were pretreated with B18R protein (125 ng/ml) or carrier protein BSA (control) for 1 h and stimulated with the indicated reagents for 18 h. Doses of stimuli were as follows: SeV, 40 HA/ml; poly(I·C), 100 μg/ml; IFN-α and IFN-β, 1,000 U/ml; IL-29, 100 ng/ml. Luciferase activity was normalized to that of BSA-pretreated and unstimulated (mock) cells and is presented as fold induction. *, P < 0.05; **, P < 0.01, compared to results with BSA pretreatment. (B) 293FT-ISRE-Luc reporter cells were untransfected (control) or transfected with carrier RNA or NV RNA and incubated for 48 h. As a positive control, 100 μg/ml poly(I·C) was added to the medium of untransfected cells for 6 h. Luciferase activity was normalized to that of untransfected cells (control) and is presented as fold induction. (C) Western blotting of NV VP1 and ISG56 in the same cell lysates from the experiment shown in panel B. NV VP1 was concentrated by IP prior to Western blotting. Actin and two nonspecific protein bands (marked by asterisks) served as equal loading controls. (D) Time course of 293FT-ISRE-Luc reporter assay. 293FT-ISRE-Luc reporter cells were untransfected (control) or transfected with carrier RNA or NV RNA and incubated for 8, 24, 48, and 72 h. As a positive control, 100 μg/ml poly(I·C) was added to the medium of untransfected cells for 6 h before each time point. Luciferase activity was normalized to that of untransfected cells (control) and is presented as fold induction. Note that the vertical axis is in logarithmic scale. pIC, poly(I·C).