鸡MDA5-STING-IFNβ天然免疫通路中STING介导信号传导和抗病毒的分子机制

It was previously found that MDA5 (mammalian melanoma differentiation associated gene 5, MDA5) could interact with STING (stimulator of interferon genes, STING) to reconstitute a new MDA5-STING-IFNβ signal pathway in RIG-I(retinoic acid inducible gene I, RIG-I) and IRF3(IFN regulatory factor 3, IRF3) absent chicken cells,but not in mammalian cells. However, the mechanisms of signal transduction and antiviral effects of this pathway remains unclear. This proposal will focus on the analysis of typeⅠinterferon pathway mediated by chicken MDA5（chMDA5）and chicken STING (chSTING), and will highlight the function of chSTING. So, the molecular and structural basis of interaction between chMDA5 and chSTING will be solved, and the mechanism of posttranslation modification of chSTING will be analyzed. The IFN regulatory factor required for activation of beta IFN by chSTING will be investigated by using immunoprecipitation and mass spectrometry combined assay, RNA interference technique and protein-nucleic acid interaction technique,such as electrophoretic mobility shift assay (EMSA). To determine the roles of chSTING in inhibition of H9N2 avian influenza virus(AIV) replication, the virus titers will be measured in CRISPR-Cas9 mediated chSTING gene knocked out and mock chicken cells. Combined with gene chip and qRT-PCR technology, the antiviral immune genes related to chSTING in the process of H9N2 AIV infection will also be characterized. These findings will allow the chicken specific MDA5-STING-IFNβsignaling pathway to be clearly illustrated and the mechanism of chSTING in antivirus to be determined,which is crucial for the prevention and control of AIV and other viral infections.

课题组前期研究发现在免疫分子RIG-I和IRF3天然缺失的鸡细胞中，MDA5与STING互作构成了一条在哺乳动物中并不存在的MDA5-STING-IFNβ天然免疫信号通路，但对该通路具体的信号传导和抗病毒机制尚不清楚。本项目将聚焦此通路，以鸡STING为核心，通过免疫共沉淀、质谱分析、RNA干扰和蛋白核酸互作等技术，分析STING与MDA5等信号分子互作的分子基础；阐明其亚细胞定位依赖的功能域及其生物学意义；解析STING介导信号传导依赖的泛素化、磷酸化等修饰机制；揭示鸡STING激活IFNβ依赖的转录因子。应用CRISPR-Cas9技术构建STING基因敲除鸡细胞系，进而基于H9N2禽流感病毒感染后的复制水平和免疫分子表达差异，揭示鸡STING介导的抗病毒途径。通过研究，初步阐明鸡I型干扰素信号通路中STING介导信号传导和抗病毒的分子机制，为利用鸡天然免疫能力抗感染奠定理论和应用基础。

课题组发现在RIG-I和IRF3天然缺失的鸡细胞中，MDA5与STING互作形成哺乳动物中不存在的MDA5-STING-IFNβ天然免疫新通路。为了揭示该通路中STING介导信号转导和抗病毒的分子机制，本项目首先聚焦“鸡在天然缺失IRF3条件下，STING如何激活IFN”这一关键科学问题，利用CRISPR-Cas9基因编辑技术成功获得包括IRF7在内的一系列基因缺失细胞株，通过基因过表达、共表达、Co-IP、LC-MS/MS、Western blot等技术，明确IRF7是鸡STING激活IFN依赖的关键转录因子，结束了长期悬而未决的鸡IFN转录因子问题；同时，提出了鸡STING激活IRF7的“脚手架模型”，完美解释了鸡STING通过IRF7激活IFN的机制：STING接收上游激活信号后发生磷酸化，发挥脚手架功能，同时招募TBK1和IRF7，介导二者相互靠近，随后TBK1发挥蛋白激酶活性磷酸化IRF7上SLQxSyS基序内的S474位点，实现对IRF7精确调控。此外，项目重点研究了chSTING磷酸化激活调控机制，确定TBK1是催化鸡STING磷酸化的关键蛋白激酶，发现TBK1通过磷酸化鸡STING S366对其功能进行正向调控；同时发现，TBK1通过磷酸化鸡STING的S327/S311位点对其进行负向调控，这是关于TBK1负调控功能的首次发现。同时，我们还揭示了鸡STING泛素化调控机制，初步确定了鸡STING泛素化位点，明确了鸡STING泛素化依赖的E3泛素连接酶为TRIM25。本项目首次全面解析了鸡STING激活IFN的分子基础，揭示了鸡STING通过磷酸化和泛素化修饰精确控制通路启闭的分子机制。发表相关SCI论文6篇，中文论文2篇，完成博士学位论文1篇、硕士学位论文3篇。成果不仅为研究家禽天然免疫机制提供了基础数据，更为通过调控鸡天然免疫力防控病毒感染奠定应用基础。