FIG. 5.

RSV-induced TLR3 expression depends on RIG-I-induced IFN-β secreted from infected cells. (A) A549 cells were transfected with control siRNA (Con) and RIG-I siRNA for 48 h and RSV infected for 0, 9, or 18 h. QRT-PCR was performed using TLR3 probe. #, P is <0.01; *, P is <0.05 relative to control siRNA at the same time point. Error bars indicate standard deviations. The results shown here are representative of two independent experiments. (B) Noncontiguous genomic sequence of hTLR3 promoter. The location relative to the major transcription start site is shown at left. Underlines, two predicted ISRE sites (ISRE1 and ISRE2) and one STAT site; bold font, site-directed mutagenesis of each individual regulatory element was performed by rolling-circle PCR. (C) A549 cells were transfected with either wild-type hTLR3/LUC reporter gene or different site mutants. Twenty-four hours later, cells were RSV infected and normalized luciferase activity was measured 12 h later. WT, wild type; *, P is <0.001 relative to wild-type hTLR3/LUC activity at 12 h. Error bars indicate standard deviations. (D) Naïve A549 cells were treated with 20% (vol/vol) UV-RSV-CM taken from RSV-infected cells for the indicated times (in hours). Prior to its addition to A549 cells, UV-RSV-CM was preincubated with PBS, rabbit IgG (IgG), or neutralizing anti-IFN-β Ab for 2 h. An autoradiogram from Northern blot hybridization is shown. Top panel, hybridization using radiolabeled TLR3 cDNA; bottom panel, hybridization with β-actin as an internal control. α, anti. (E) IFN-β-deficient Vero cells were infected with RSV for 12 h or were treated with 20% (vol/vol) UV-RSV-CM for 12 h. The conditioned medium was collected from A549 cells 24 h after RSV infection. Top panel, 20 μg total RNA was isolated and Northern blot hybridization conducted using TLR3 cDNA probe; bottom panel, β-actin hybridization. −, absent; +, present.