Figure 3. WT and mutant SV5 do not activate TLR3 pathways, but they are also not able to block dsRNA-mediated TLR3 signaling.

A) Overexpression of TLR3. A549 cells were co-transfected with a plasmid expressing TLR3 or its empty vector (as a control) along with a plasmid encoding luciferase under the control of the IFN-beta promoter. At 16 h pt, cells were infected with indicated viruses at an moi of 10. At 24 h pi, lysates were harvested and luciferase activity was determined. B) Infection of 293 cells. 293 cells stably expressing TLR3 or LacZ (as a control) were mock infected or infected at an moi of 10 with WT SV5-GFP, or the mutants P/V-CPI-. At 24 h pi, cells were examined for GFP expression. C) 293 cells stably expressing TLR3 or LacZ (as a control) were mock infected or infected at an moi of 10 with WT SV5-GFP, or the mutants P/V-CPI- or Le-(U5C, A14G). As a positive control, cells were treated with exogenous dsRNA (PolyI:C; 5 ug/ml). At 24 h pi, media were analyzed by ELISA for levels of IL-8. D) 293-TLR3 were mock infected or infected at an moi of 10 with the indicated viruses and 6 h later were left untreated or challenged with exogenous dsRNA (PolyI:C; 5 ug/ml). At 24 h pi media was harvested and analyzed by ELISA for levels of IL-8. For all panels, results are expressed as mean values from triplicate samples with error bars representing standard deviation. IL-8 values are normalized to 10⁶ cells. Le Mut; Le-(U5C, A14G).