新型免疫分子CCDC134的抗病毒免疫功能及其分子机制研究

Type I interferon (type I IFN) plays a central role in immune responses against viral infection. The production of type I IFN must be tightly regulated. The expression and regulation of Type I IFN is a hot research field of natural immunity. CCDC134 was discovered by the scholars of our country independently. To explore the function of CCDC134 in vitro, we performed siRNA to knock down the expression of CCDC134 transiently in mice primary macrophages. The Results showed that CCDC134 deficiency abrogated the production of type I IFN, upon VSV（vesicular stomatitis virus） and HSV-1 (herpes simplex virus 1) stimulation. We also found that CCDC134 transgenic mice were resistance to lethal VSV challenge by increasing the serum level of type I IFN, compared to wild type mice. These data showed that CCDC134 promoted the expression of type I IFN. However, the precise molecular mechanisms have not to be understood. Next we will explore the RNA virus activated signaling pathways in CCDC134 deficiency macrophage compared to wild type macrophage. We will look for the molecules interaction with CCDC134 with Massspectrum. Co-immunoprecipitation assay will help us confirm the association between proteins and interaction domain of each protein. Systemic analysis of transcriptome and modification of chromosome with CHIP sequencing and RNA-sequencing would be helpful to understand immune response in the absence of CCDC134. The Immunofluorescent Microscopy assay will reveal the localization of CCDC134 with RNA and DNA virus stimulation. Further the efficacy of recombinant CCDC134 protein against viral infection would be observed systemically. This topic would provide the landscape of antiviral immune responses of CCDC134 and a new therapeutic approach for viral infection.

I型干扰素在机体抗病毒免疫过程中发挥着关键作用，其表达调控的机制是天然免疫领域研究的热点。本课题组初步的预实验结果发现我国学者自主发现的新型免疫分子CCDC134转基因小鼠的抗病毒感染能力明显增强，该效应与该分子对于I型干扰素产生的调控密切相关。本项目拟在前期研究工作的基础上，利用已构建的基因缺陷小鼠，通过体内外实验确证CCDC134通过促进I型干扰素产生而增强机体的抗病毒感染效应。进一步通过Western Blot和免疫共沉淀-质谱等生化分析技术、CHIP sequencing和RNA sequencing 等系统生物学技术分析CCDC134在I型干扰素产生信号通路中的作用环节。在此基础上确认CCDC134作用的靶分子及其作用的机制。本项目的完成不仅可深入认识新型免疫分子CCDC134的功能，也可为抗病毒免疫治疗提供新的思路和靶点，并可为该分子在临床的应用提供理论支持。

本课题以Coiled-coil家族分子CCD134作为研究对象，探究其在天然免疫特别是抗病毒天然免疫应答过程中发挥的效应和分子机制。结果表明，CCDC134在天然免疫细胞-巨噬细胞中高表达，并且病毒感染能够显著诱导CCDC134的上调表达，增加CCDC134蛋白在小鼠血清中的含量。进一步研究发现，CCDC134对I型干扰素的产生具有明显的促进作用，进而增强巨噬细胞的抗病毒天然免疫功能，抑制病毒复制；CCDC134转基因小鼠相较于野生型小鼠对病毒感染表现出更强烈的抵抗作用。对I型干扰素产生的信号通路进行检测，发现过表达CCDC134显著增强了信号分子IRF3和NF-κB的磷酸化水平，而对其上游相关信号分子没有影响，因此推测CCDC134作用于IRF3的活化环节。进一步通过免疫沉淀和质谱对CCDC134相互作用蛋白进行检测，结果提示，CCDC134与E3泛素链接酶TRAF2、TRAF6，以及模式识别受体LRRFIP1具有潜在的相互结合关系。进一步研究发现，TRAF2/6能够促进CCDC134形成K63泛素化链修饰，第218位赖氨酸泛素化位点是CCDC134促进IRF3活化的关键位点。另一方面，CCDC134与LRRFIP1相互关系的研究结果表明，CCDC134能够与LRRFIP1直接结合，并且促进LRRFIP1- TBK1复合体组装。本课题对CCDC134抗病毒免疫效应及其作用机制的认识，可为病毒相关疾病的治疗提供新的策略和思路。本课题已发表相关SCI收录论文3篇，培养博士2名。课题负责人被评为2019年度上海市领军学者并与团队一起获得首届全国创新争先奖牌（排名第二）。