流感病毒非结构蛋白对TBK-1的抑制作用

The inhibitory function of influenza nonstructural protein 1 to TBK-1

目的 流感病毒非结构蛋白1(nonstructuralprotein 1,NS1)抑制干扰素调节因子(inter feronregulatoryfactors,IRF) 3的机制未明,TANK结合激酶1(TANK bindingkinase 1,TBK- 1)能使IRF 3活化,研究NS1是否对TBK -1有抑制作用。方法 亚克隆IRF 3、NS1和TBK- 1至pcDNA3.1 flag构建flag IRF 3、flag NS1和flag TBK- 1质粒;用TBK- 1对TBK- 1+NS1以及IRF 3+TBK- 1对IRF 3+TBK- 1+NS1两组实验,分别共转染人胚胎肾上皮2 93细胞,用抗flag抗体作Westernblot分析,鉴定IRF 3、NS1和TBK- 1的表达,观测NS1对TBK 1活化IRF 3的抑制作用;荧光素酶功能分析方法观测NS1对TBK 1诱导的干扰素β(IFN β)启动子pGL- 2B荧光素酶活性的影响。结果 IRF- 3、NS1和TBK- 1均有高表达。TBK 1能使IRF 3活化,Westernblot分析显示:TBK -1转染的细胞出现迁移较慢的IRF 3Ⅲ和Ⅳ型,NS1共转染可使IRF- 3Ⅲ和Ⅳ型几乎消失;荧光素酶功能分析显示,NS1能抑制TBK- 1活化内源性IRF 3所诱导的IFN -β启动子活性,约为对照组的1 4 ,TBK 1+IRF- 3共转染可使IFN- β启动子活性增高近10 0 0倍,NS1可使TBK -1活化外源性IRF 3所诱导的IFN- β启动子的活性降至对照组的约1 2至1 3。结论 流感病毒NS1可抑制TBK 1所致的IRF 3活化。

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.