Journal: Viruses

Article Title: H1N1 Influenza A Virus Protein NS2 Inhibits Innate Immune Response by Targeting IRF7

doi: 10.3390/v14112411

Figure Lengend Snippet: NS2 interacts with IRF3 and IRF7. ( A ) NS2 inhibits activation of the IFN-β promoter induced by RIG-IN, MDA5-Card, MAVS, TBK1, IKKε, IRF3, and IRF7. HEK293 cells were transfected with indicated plasmids along with increasing amounts (0, 200 ng, and 400 ng/mL) of Flag-NS2 expression plasmids. The activation of STAT1-luciferase triggered by IRF9 was used as a negative control. Reporter assays were performed 24 h after transfection. ( B ) Overexpressed NS2 interacts with IRF3 and IRF7. HEK293 cells were transfected with indicated plasmids for 24 h. Then, Co-IP and immunoblotting analyses were performed using indicated antibodies. ( C ) NS2 co-localizes with IRF3 and IRF7 in the cytoplasm. U2OS cells were transfected with Flag-NS2 and HA-IRF3/IRF7 plasmid for 24 h, and then stained with Flag antibody or HA antibody and secondary antibodies. Nuclei were counter-stained with DAPI. Scale bar, 20 μm. The data shown represent three independent experiments ( p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), ‘ns’ indicates no significant difference).

Article Snippet: Reagents used in the study included: anti-Flag agarose affinity beads (A2220) and protein A/G agarose affinity beads (P6486/E3403) (Sigma, Dallas, TX, USA); human IFN-β DuoSet ELISA kit (DY814-05) (R&D, Minneapolis, MN, USA); NP-40 (ST366), DAPI (C1002), and Passive Lysis 5× Buffer (E1941) (Promega, Madison, WI, USA).

Techniques: Activation Assay, Transfection, Expressing, Luciferase, Negative Control, Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Staining