The inhibitory function of influenza nonstructural protein 1 to TBK-1
Objective To explore whether NS1 is able to in hi bit the activity of TBK-1. Methods IRF-3, NS1 and TBK- 1 were subcloned into pcDNA3.1-flag respectively. TBK-1 vs TBK-1+NS1 were tra nsfected into HEK 293 cells. Cell extracts were analysed by Western blot and the n probed with monoclonal flag antibody. Luciferase assay was carried out by co- transfection of reporter plasmid, interferon-β promoter-pGL-2B, with two gro ups of cDNA expression plasmids: TBK-1 vs TBK-1+NS1; and IRF-3+TBK-1 vs IRF -3+TBK-1+NS1. Cells were collected and assayed for luciferase activity 24 hour s after transfection. As positive control, cells were transfected with or withou t NS1 and then infected with Sendai virus for 8 hours after 16 hours′ transfect ion. Results IRF-3, NS1 and TBK-1 were strongly express ed. TBK-1 can activate IRF-3, NS1 inhibited the luciferase activity of IFN-β promoter. TBK-1 and IRF-3 co-transfection induced a 1000-fold stimulation o f IFN-β promoter. NS1 induced a 1/2 to near 1/3 inhibition of luciferase a ctivity of IFN-β promoter compared with control. Conclusion Influenza NS1 protein can inhibit the phosphorylation of IRF-3 activated by TBK-1. Functional analysis provides evidence that NS1 can significantly inhi bit the luciferase activity of IFN-β promoter induced by TBK-1. The results h ave important impact on antiviral therapeutic strategy and pharmaceutical develo pment.