Different Subtypes of Influenza Viruses Target Different Human Proteins and Pathways Leading to Different Pathogenic Phenotypes

Figure 2

H5N1 NP inhibits the NF-κB signaling pathway by targeting IKKα. 293 T cells in 24-well plates were cotransfected with 125 ng pNF-κB-luc, 25 ng pRL-TK and either HA-TRAF2 (a), HA-TAK1+HA-TAB1 (b), HA-TAB2 (c), HA-IKKα (d), HA-IKKβ (e), together with the indicated amounts of H5N1 NP expression plasmids. Total amounts of transfected DNA were kept equal by adding empty vector. Reporter activity was determined 30-h post-transfection by the dual-luciferase reporter assays. The resultant ratios were normalized to the foldchange value by that of cells cotransfected with empty vectors, pNF-κB-luc and pRL-TK. Data represent at least 3 independent experiments, with each determination performed in duplicate (mean ± SD of fold-change). Asterisks indicate significant differences between groups (^∗∗, Student’s t-test). 293 T cells were transfected with Flag-H5N1 NP or either HA-TAK1 orSS HA-TAB1 or HA-TAB2 or HA-IKKαor HA-IKKβ HA-vector expression plasmids for 30 h. All cells were then treated with TNF-α (10 ng/ml) for 30 min. The cells were lysed and subjected to immunoprecipitation (IP) using the mouse anti-HA tag. IP products and 5% input samples were analyzed by immunoblotting (f), 293 T cells transfected with empty vector, H5N1 NP -expressing plasmid were stimulated with TNF-α (20 ng/ml) for indicated durations. Equal amounts of cell lysates were analyzed by immunoblotting with the anti-phospho-IKKαantibody (g).

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