RIG-I样受体（RIG-I like receptors，RLRs）是细胞质内识别病毒病原相关分子模式（Pathogen-associated molecular patterns，PAMPs）的一类模式识别受体（Pattern recognition receptors，PRRs）。RLR天然免疫通路是胞质内抗病毒的主要信号通路。RLRs识别并结合病毒后，通过定位在线粒体膜上的VISA蛋白传递信号，最终激活转录因子NF-κB和IRF3/7等，启动天然免疫反应。RLR通路在哺乳动物甚至鱼类中已有较深入研究，然而在无脊椎动物中的研究却很少。本研究利用分子生物学和免疫学等方法，首次对长牡蛎（Crassostrea gigas）中RLR抗病毒天然免疫信号通路上的关键基因的进行克隆鉴定并进行功能分析。初步确认了VISA依赖RLR天然免疫通路在长牡蛎中存在，探讨了这些关键基因在牡蛎天然免疫防御过程中的作用。

首先，我们首次成功克隆了长牡蛎RIG-I-1及VISA基因。序列比对和同源分析显示CgRIG-I-1和CgVISA分别为无脊椎动物RIG-I家族和VISA家族的新成员。结合实验室以往研究数据，CgRIG-I-1和CgVISA mRNA在不同组织中均有表达，并且其表达受poly(I:C)，poly(dA:dT)和牡蛎疱疹病毒（OsHV-1）的诱导。CgRIG-I-1蛋白作为模式识别受体能与poly(I:C)直接结合且其CARD结构域能与CgVISA蛋白相互作用，CgVISA蛋白的跨膜结构域TM在介导互作过程中起到重要作用。此外，CgVISA蛋白可以通过跨膜结构域TM发生自身二聚化反应，这可能是长牡蛎VISA蛋白招募下游信号分子的一个必须路径。

其次，首次克隆了长牡蛎CgTRAF2，CgTRAF3和CgTRAF6基因，qRT-PCR结果显示，CgTRAF2，CgTRAF3和CgTRAF6 mRNA的表达受病毒 PAMP刺激的诱导。对CgVISA干扰后样品CgTRAF2/3/6 mRNA的表达数据分析结果显示，CgTRAF2和CgTRAF3可能是长牡蛎RLR免疫通路中位于VISA下游的信号分子；而酵母双杂交与免疫共沉淀结果显示CgTRAF6蛋白与CgVISA蛋白的直接相互作用。另外CgTRAF2定位在细胞质中；CgTRAF3存在可变剪切形式，可变剪切形式间可能存在互相的调控；CgTRAF6能激活转录因子NF-κB的启动子。

最后，我们利用RACE技术首次在长牡蛎中克隆了无脊椎动物IRF家族新成员CgIRF2和CgIRF8。CgIRF2和CgIRF8 mRNA在不同组织中均有表达，并且表达量受poly(I:C)和OsHV-1的诱导。对CgVISA干扰后样品中CgIRF2/8 mRNA的表达数据分析结果显示，CgIRF2和CgIRF8可能是长牡蛎RLR天然免疫通路中位于VISA下游的信号分子。此外，萤光素酶双报告基因实验结果显示，高表达CgIRF2和CgIRF8蛋白能显著激活IFNβ以及ISRE启动子。亚细胞定位结果显示，CgIRF2 和CgIRF8均为细胞核，细胞质双定位的蛋白。这些研究初步揭示了长牡蛎IRF2和IRF8在长牡蛎抗病毒天然免疫中的作用，丰富了人们关于无脊椎动物IRF功能的了解。也可以为进一步更深入的低等动物IRF功能的研究打下基础。

综上所述，本研究首次在无脊椎动物长牡蛎中确认了VISA依赖的RLR天然免疫信号通路的存在。克隆鉴定了CgRIG-I-1，CgVISA，CgTRAF2/3/6，CgIRF2和CgIRF8等参与RLR通路的关键基因并初步研究了其在天然免疫过程中起到的作用。丰富了人们关于无脊椎动物RLR免疫通路的理解，为深入研究无脊椎动物天然免疫机制打下基础，也为开发牡蛎抗病毒药物，预防长牡蛎大规模死亡提供理论支持。

 

RIG-I-like receptors (RLRs) are cytoplasmic pattern recognition receptors (PRRs) which are known to play an important role in sensing RNA virus pathogen-associated molecular patterns (PAMPs). Firstly, RLRs recognize the virus RNA. Then RLRs bind to the mitochondrial-membrane-located protein VISA, trigger the host immune response and ultimately activate the transcription factor NF-κB and IRFs. Much more in-depth study has been executed on mammals and even bony fishes’ RLR immune pathways. However, there are few researches on invertebrates RLR signaling pathway. In this study, we set up identification and functional study on key genes of the Pacific oyster RLR signaling pathway through molecular biology, bioinformatics and immunology technologies.

Firstly, we cloned oyster CgRIG-I-1 and CgVISA genes. Sequence alignments and homology analysis showed that CgRIG-I-1 and CgVISA are new members of invertebrate RIG-I family and VISA family respectively. Considering our previous studies, CgRIG-I-1 and CgVISA mRNA were expressed in all oyster tissues, and their expression were induced by poly(I:C), poly(dA:dT) and oyster herpesvirus 1 (OsHV-1) challenges. CgRIG-I-1 protein, as a pattern recognition receptor, can bind to poly(I:C) directly and interact with the CgVISA protein through the N-terminal CARD domains. The transmembrane domain of CgVISA plays an important role in mediating CgRIG-I-1_CARD and CgVISA interaction. Furthermore, CgVISA protein could dimerize through its transmembrane domain, which may be essential for the recruitment of downstream signaling moleculars.

Secondly, we cloned and characterized CgTRAF2, CgTRAF3 and CgTRAF6 from C. gigasa for the first time. The results of qRT-PCR showed that,  the mRNA expression of CgTRAF2, CgTRAF3 and CgTRAF6 was all induced by the virus PAMP stimulation. The mRNA expression pattern of CgTRAFs after the RNAi of CgVISA showed the similar expression style with that of CgVISA, indicating that CgTRAF2/3/6 might be a signal molecule involved in Crassostrea gigas RLR/VISA immune pathway. And yeast two-hybrid and co-immunoprecipitation result proposed that CgTRAF6 could interact with CgVISA protein. In addition, CgTRAF2 located in the cytoplasm; CgTRAF3 presences an alternative splicing form; CgTRAF6 can activate the NF-κB promoter. 

Finally, we obtained the full-lengh cDNA sequences of oyster CgIRF2 and CgIRF8 by RACE. CgIRF2 and CgIRF8 mRNA expression were found in all tested tissues and induced by poly(I:C) and OsHV-1 challenges. CgIRF2 and CgIRF8 might function downstream of CgVISA. And Overexpression of CgIRF2 and CgIRF8 protein can significantly activate IFNβ and ISRE promoters. Furthermore, we found  that CgIRF2 and CgIRF8 could locate both in cell nucleus and in cytoplasmic.

In summary, we firstly confirmed the presence of VISA-dependent RLR signaling pathways in a invertebrate C. gigas. We identified key genes of oyster RLR pathway such as CgRIG-I-1, CgVISA, CgTRAF2/3/6, CgIRF2 and CgIRF8 and studied the roles they played in oyster innate immunity. Our studies would broad people’s understanding of invertebrate RLR immune pathway and would facilitate the further research on invertebrates RLR signaling.