Figure 2
ORF4b can inhibit IFN-β expression by targeting MDA5, TBK1 and IKKε.
(A) Association of ORF4b protein with MDA5, TBK1 and IKKε. The 293T cells were co-transfected with expression plasmids for HA-tagged ORF4b protein and the indicated transducer proteins including GFP-tagged MAVS and IRF3, FLAG-tagged IKKε, TBK1, MDA5 and RIG-I. Input cell lysates and immunoprecipitates were analyzed by Western blotting (WB) with anti-GFP (α-GFP), anti-FLAG (α-FLAG) and anti-HA (α-HA) antibodies. (B) Co-localization of ORF4b protein with MDA5, TBK1 and IKKε. HeLa cells were co-transfected with expression plasmids for HA-tagged ORF4b protein and the indicated expression plasmids for Flag-tagged MDA5, TBK1 and IKKε. Cells were then stained for ORF4b and MDA5/TBK1/IKKε with anti-HA and anti-FLAG antibodies, respectively. The green (ORF4b) and red (MDA5/TBK1/IKKε) fluorescent signals were merged. Nuclei were stained with DAPI (blue). Cells were analyzed by confocal microscopy using a 100× objective and representative images are shown. Scale bar, 20 μm. (C) association of ORF4b protein with endogenous MDA5, TBK1 and IKKε. 293T cells were transfected with ORF4b expressing plasmid and immunoprecipitated by anti-HA monoclonal antibody and the co-immunoprecipitation was assessed by Western blotting using anti-MDA5, IKKε and TBK1 monoclonal antibody. (D) ORF4b could also inhibit interferon-inducing activity of IRF-3 and IRF7. Experiments were carried out as in Fig. 1D except that 293T cells were not stimulated with SeV but co-transfected with plasmids expressing MDA5, RIG-I, MAVS, TBK1, IKKε, IRF3 and IRF7. Data are representative of three independent experiments with triplicate samples.
