DDX1在猪传染性胃肠炎病毒非结构蛋白nsp14激活NF-κB信号通路中的作用机制

Transmissible Gastroenteritis Virus (TGEV) is the major pathogen causing diarrhea in piglets. Previous studies by our group and others have demonstrated that nuclear factor kappaB (NF-κB), a critical transcription factor in innate and adaptive immune response as well as cell proliferation and survival, was activated after TGEV infection. To explore the mechanisms behind this event, we investigated the role of TGEV structural proteins and non-structural proteins in the regulation of NF-κB. In our previous work, we found that non-structural protein 14 (nsp14) of TGEV mainly contributed to NF-κB activation. Futher study demenstrated that DDX1 significantly influenced NF-κB activation induced by nsp14. In the present proposal, luciferase reporter system, western blot and immunofluorescence technique will be used to evaluate the NF-κB activation by nsp14 and the functional domain of the nsp14 protein reponsible for NF-κB activation will be further identified by mutagenesis experiments. The role of DDX1 in nsp14 activeted NF-κB signal pathway will be determined by over-expression or siRNA of the DDX1 gene. Futhermore, co-immunoprecipitation and confocal microscopy will be used to analyze the interaction among DDX1, nsp14 and (or) p65. Finally, the impact of DDX1 on the viral multiplication will be investigated. This study would add more clues to elucidate the molecular mechanism of DDX1 involved in NF-κB activation triggered by TGEV nsp14 protein and provide us with useful information to better design vaccines against TGEV infection.

猪传染性胃肠炎病毒（TGEV）是引起仔猪腹泻的主要病原。以前的研究表明TGEV感染能显著激活炎症相关转录因子NF-κB，但其具体机制尚不清楚。在我们的前期研究中，通过对TEGV编码的所有结构蛋白和非结构蛋白进行扫描，发现非结构蛋白nsp14显著激活NF-κB，进一步研究还发现DEAD-box解旋酶1（DDX1）直接影响nsp14对NF-κB的激活。本项目拟采用荧光素酶报告系统、siRNA和免疫荧光等技术，进一步探讨nsp14对NF-κB的激活作用并确定其关键功能域，分析DDX1在nsp14激活NF-κB中的作用以及nsp14对DDX1表达和亚细胞定位的影响，揭示nsp14、DDX1和NF-κB p65亚基之间的相互关系以及DDX1对TGEV增殖的影响，从而阐明DDX1参与nsp14激活NF-κB信号通路的分子机制，为揭示TGEV的致病和免疫机理及进一步预防和控制猪传染性胃肠炎提供理论依据。

猪传染性胃肠炎病毒（Transmissible Gastroenteritis Virus, TGEV）属于冠状病毒科冠状病毒属成员，该病毒感染对两周龄的仔猪致死率可达到100%，给养猪业造成了严重的经济损失。我们的前期研究表明TGEV感染能显著激活核因子kappa B（Nuclear factor-kappa B，NF-κB），但其具体机制尚不清楚。本项目通过NF-κB荧光素酶报告系统对TEGV编码的所有结构蛋白和非结构蛋白进行扫描，发现非结构蛋白nsp14显著激活NF-κB；进一步的Western blotting和间接免疫荧光实验显示，nsp14能诱导IκBα（inhibitor of kappa B）降解，促进p65的磷酸化和转核，表明nsp14能显著激活NF-κB，且呈剂量依赖性；突变体实验显示，3’-5’外切核酸酶活性和N7-甲基转移酶活性都在一定程度上参与了nsp14对NF-κB的激活。.DEAD-box解旋酶1(DEAD-box RNA helicase 1, DDX1）是ATP依赖的RNA解旋酶，参与许多RNA的代谢过程和抗病毒免疫反应。研究证实DDX1能与SARS冠状病毒和鸡传染性支气管炎病毒编码的nsp14蛋白相互作用，又能增强NF-κB介导的多种细胞因子的转录。DDX1与TGEV nsp14蛋白是否存在相互作用及DDX1在nsp14激活NF-κB中的作用尚不清楚。本研究通过免疫共沉淀和间接免疫荧光实验对nsp14与DDX1是否存在相互作用进行了检测，证实两种蛋白确实存在相互作用；进一步发现超表达DDX1能够协同nsp14激活NF-κB，而干扰DDX1的作用则相反，表明TGEV nsp14通过宿主蛋白DDX1诱导NF-κB活化。此外，DDX1对TGEV的增殖有一定的抑制作用，而干扰DDX1后TGEV诱导的NF-κB活化和ISGs等炎症因子产生水平均显著下调，提示DDX1可能通过增强TGEV诱导的抗病毒免疫应答，从而抑制TGEV增殖。本研究首次对TGEV的编码蛋白激活NF-κB的作用进行研究，阐明了DDX1在nsp14和TGEV激活NF-κB信号通路中的重要作用，揭示了DDX1具有抑制TGEV复制的新功能，为进一步阐明TGEV的免疫机理和致病机制及有效预防和控制TGEV感染提供理论依据。

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