Different Subtypes of Influenza Viruses Target Different Human Proteins and Pathways Leading to Different Pathogenic Phenotypes
H5N1 NP and H1N1 NP have different impacts on TNF-α-induced NF-κB activation. 293 T cells in 24-well plates were cotransfected with 125 ng pNF-κB-luc, 25 ng pRL-TK and indicated amount of H5N1 NP (a) and H1N1 NP (b) expression plasmid, or empty vector for 24 h. Cells were then mock-treated or treated with TNF-α (10 ng/ml) for 6 h. Reporter activity was determined by dual-luciferase reporter assays. The resultant ratios were normalized to the fold-change value by that of TNF-α-untreated cells cotransfected with empty vector, pNF-κB-luc and pRL-TK. Data shown represent three 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 transfected with empty vector, H5N1 NP or H1N1 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-IκBαantibody or the anti-phospho-IκBαantibody (c–f). HeLa cells were transfected with H5N1 NP, H1N1 NP expression plasmid, or empty vector for 30 h. The cells were then mock-treated or treated with TNF-α (10 ng/ml) for 30 min. HeLa cells were subjected to immunofluorescence staining for detection of p65 subcellular localization by using rabbit anti-p65 and FITC-conjugated secondary Ab (green). H5N1 NP and H1N1 NP expression levels were detected using a mouse anti-Flag tag and Texas Red-conjugated secondary Ab (red). Nuclei were stained by Hoechst 33258 (blue) (g, h).
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