Fig. 7

OPTN is targeted by the NS3 protein of the Bluetongue virus to dampen IRF3 signaling. a HeLa cells were transfected with a plasmid encoding NS3-GFP; 16 h later, the NS3-GFP localization was assessed by immunofluorescence analysis. The Golgi apparatus was stained with an antibody against GM130. Scale bars, 10 μm. b HEK293T cells were transfected with either an IFNβ promoter reporter or with a NF-κB reporter, together with the Renilla luciferase gene as an internal control. In parallel, the cells were also transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were either left unstimulated (Unstim) or infected with Sendai virus (+ SeV) for 7 h. Luciferase assays were then performed and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with GFP-transfected cells in Student’s t test). RLU, relative luminescence units. c HEK293T cells were transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were infected with SeV for the indicated times. Cell lysates were analyzed by immunoblotting with antibodies against the indicated proteins. d HEK293T cells were transfected with a plasmid encoding OPTN and a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cell lysates (Lys.) were subjected to immunoprecipitation (IP) with an antibody against GFP. Samples were analyzed by immunoblotting with antibodies against the indicated proteins. e HEK293T cells were transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were either left unstimulated or infected with Sendai virus for 7 h. Cell lysates (Lys.) were then subjected to immunoprecipitation (IP) with an antibody against OPTN. Samples were analyzed by immunoblotting with antibodies against the indicated proteins. f MEFs were transfected with a plasmid encoding GFP or NS3-GFP. Then, 16 h after transfection, cells were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was assessed by immunofluorescence staining and aggregate counting in GFP-positive cells. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). ***P < 0.001 versus cells transfected with GFP alone (Student’s t test)