E3泛素连接酶斑点型锌指结构蛋白调控RLR信号通路抑制肠道病毒71型复制

E3 ubiquitin ligase SPOP regulates RLR signaling pathway and inhibits enterovirus 71 replication

目的探讨斑点型锌指结构蛋白[speckle-type POZ(pox virus and zinc finger protein)protein,SPOP]在肠道病毒71型(EV71)感染中的作用。方法免疫共沉淀分析SPOP对EV71非结构蛋白2A蛋白酶(2Apro)泛素化水平的影响,Western blot检测干扰素调节因子3(IRF3)蛋白磷酸化水平,过表达或敲低细胞中SPOP后感染EV71,RT-qPCR分析IFN-β转录水平,RT-qPCR和Western blot检测EV71结构蛋白VP1的转录水平及蛋白质水平。结果过表达HA-SPOP后感染EV71,发现EV71感染RD细胞受抑,同时EV71-2Apro的泛素水平呈HA-SPOP梯度依赖增多。转染shSPOP质粒进行内源性SPOP敲低后,黑色素瘤分化相关基因5(MDA5)、接头蛋白线粒体抗病毒信号蛋白(MAVS)、磷酸化干扰素调节因子3(p-IRF3)水平呈剂量依赖地减少,而转染HA-SPOP质粒后,MDA5、MAVS、p-IRF3蛋白质水平呈剂量依赖地增加。SPOP高、低表达促进或降低EV71感染的细胞表达IFN-βmRNA,同时抑制和增加VP1在mRNA或蛋白质水平表达。结论SPOP可提高EV71-2Apro的泛素化水平从而促进2Apro降解,推测SPOP可通过抑制2Apro对视黄酸诱导基因蛋白-Ⅰ(RIG-Ⅰ)样受体(RLR)信号通路中关键分子MAVS和MDA5的降解,从而上调IRF3磷酸化水平促进IFN-β释放,最终活化宿主细胞抗病毒固有免疫抑制EV71复制。

ObjectiveTo investigate the role of speckle-type POZ(pox virus and zinc finger protein)protein(SPOP)in enterovirus 71(EV71)infection.MethodsImmunoprecipitation analysis was employed to examine the impact of SPOP on the ubiquitin level of EV71 non-structural protein 2A protease(2A pro),while the phosphorylation level of IFR3 protein was assessed through Western blot.Cells were either overexpressed or knockdown of SPOP,followed by infection with EV71.RT-qPCR was utilized to analyze the transcription level of IFN-β,and the transcription level and protein level of EV71 structural protein VP1 were determined using RT-qPCR and Western blot,respectively.ResultsThe inhibition of EV71 infection in RD cells was observed following transfection with HA-SPOP.Additionally,it was found that the ubiquitin level of EV71-2A pro increased in a gradient-dependent manner.Subsequent transfection with shSPOP plasmid for endogenous SPOP knockdown resulted in a dose-dependent decrease in the levels of melanoma differentiation-associated gene 5(MDA5),mitochondrial antiviral signaling(MAVS),and p-IRF3.Conversely,transfection with HA-SPOP plasmid led to a dose-dependent increase in the levels of MDA5,MAVS,and p-IRF3.The expression of SPOP,whether high or low,had an impact on the expression of IFN-βin cells.Additionally,the levels of VP1 mRNA or protein were found to be inhibited or increased.ConclusionsSPOP plays a role in increasing the ubiquitination level of EV71-2A pro,which in turn promotes the phosphorylation level of IRF3 and secretion of IFN-β.This effect is achieved by inhibiting the cleavage of 2A pro against key molecules MAVS and MDA5 in the RLR signaling pathway,ultimately leading to the inhibition of EV71 replication.