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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,功能分析提供了NS1显著抑制TBK
Objective The mechanism by which nonstructural protein 1 (NS1) inhibits interferon regulatory factor (IRF) 3 is unclear. TANK binding kinase 1 (TBK-1) activates IRF 3 and studies NS1 Does it have an inhibitory effect on TBK-1? Methods Flag IRF 3, flag NS1 and flag TBK-1 plasmids were constructed by subcloning IRF 3, NS1 and TBK-1 to pcDNA3.1 flag; TBK-1 + NS1 and IRF 3 + TBK- + TBK-1 + NS1 cells were co-transfected into human embryonic kidney 293 cells with anti-flag antibody for Western blot analysis to identify the expression of IRF 3, NS1 and TBK-1 observed NS1 TBK 1 activated IRF 3 The effect of NS1 on TBK1-induced pGL-2B luciferase activity of interferon beta (IFN beta) promoter was observed by luciferase functional assay. Results IRF-3, NS1 and TBK-1 were highly expressed. TBK 1 could activate IRF 3. Western blot analysis showed that IRF 3 and IV were almost disappeared when co-transfection with TBK-1 transfected cells showed IRF 3 Ⅲ and Ⅳ with slow migration. Luciferase function analysis Showed that NS1 could inhibit the activity of IFN-β promoter induced by endogenous IRF 3 activated by TBK-1, about 14% of the control group. The co-transfection of TBK 1 + IRF-3 could increase the activity of IFN-β promoter Nearly 100-fold, NS1 decreased the activity of the IFN-β promoter induced by TBK-1-activated exogenous IRF 3 to about 12 to 13 in the control group. Conclusion The influenza virus NS1 inhibits the activation of IRF 3 induced by TBK 1, and functional analysis provides a significant inhibition of TBK by NS1