Functional Characterizations Of RIG-I To GCRV And Viral/Bacterial Pamps In Grass Carp Ctenopharyngodon Idella

Grass carp (Ctenopharyngodon idella) is considered one of the economically importantaquaculture species in China with the largest production among the “Great Four CulturedFish”. However, grass carp is susceptible to a wide variety of diseases, and grass carphemorrhage caused by grass carp reovirus (GCRV)(a dsRNA virus) was one of the mostserious epidemics in grass carp. As lower vertebrates, grass carp rely mainly on innateimmunity against pathogens infection for the defective adaptive immune system. To date, noeffective method is provided for the prevention and treatment of this disease.RIG-I (retinoic acid inducible gene-I) is one of the key cytosolic pattern recognitionreceptors (PRRs) for detecting pathogen associated molecular patterns (PAMPs) andmediating the induction of type I interferon and expreeeions of effector molecules (such asMx, ISG15(IFN-stimulated gene15) and ADAR (adenosine deaminases acting on RNA)).RIG-I consists of three distinct domains: N-terminal two tandem caspase activation andrecruitment domains (CARDs), central DExD/H box RNA helicase domain and C-terminalregulatory/repressor domain (RD). In mammals, CARDs of RIG-I mediate the interactionwith the CARD of interferon-β promoter stimulator1(IPS-1) and transmit downstreamsignaling molecules. The central DExD/H helicase domain is responsible for dsRNArecognition and ATPase binding, which leads to the dimerization and structural alterations ofRIG-I. The C-terminal RD inhibits RIG-I signaling cascade, however, recent report indicatesthat RD is responsible for RNA binding.Like mammals, RIG-I also possess three classical domains in teleosts. The mechanism iswell analysed in mammals, however,the study of the accurate function of RIG-I is still in itsinfancy in teleosts. Therefore, the research for RIG-I domains will contribute to the ofunderstanding signaling pathways and the detail effector regions in innate immunty inteleosts.Based on the full-length cDNA sequence of CiRIG-I (Ctenopharyngodon idella RIG-I)of the previous study, six representative overexpression plasmids were constructed for stablyexpressing recombinant proteins in CIK cells, respectively. The quantitative real-time RT-PCR (qRT-PCR) detected mRNA expressions of CiIPS-1, CiIFN-I and CiMx2post virus(GCRV) infection and dsRNA viral PAMP (poly(I:C), polyinosine-polycytidylic acid),gram-negative bacterial PAMP (LPS, lipopolysaccharide) or gram-positive bacterial PAMP(PGN, peptidoglycan) stimulation in the steadily transfected cells.After viral infection and PAMPs stimulation, repressor domain (RD) exerted inhibitoryfunction of signaling channels. After GCRV infection and poly(I:C) stimulation, CARDs ofCiRIG-I played positive and pivotal roles. The DExD/H helicase motif was crucial forsignaling pathway upon LPS and PGN stimulation. Virus titer test and96-well plate stainingassay showed that all constructs exhibited the antiviral activity more or less. Interestingly,pΔCARDs (pCMV-EGFP-CMV-SV40-CiRIG-I-ΔCARDs) transfected cells showed a postivemodulation in RIG-I signal transduction post GCRV infection. According to viral quantities, itdemonstrated that ΔCARDs of CiRIG-I (relative to the control) were moderately resistant toGCRV replication.The results show that the full-length RIG-I played a key role in RLRs (RIG-I-likereceptors) pathway mediating strikingly broad expressions in grass carp, responding to notonly dsRNA virus or synthetic dsRNA but also bacterial PAMPs. According to a series ofresearch, we preliminary evidence that the distinct domians of CiRIG-I exert differentantiviral and antibacterial functions, which provids a theoretical foundation for furtherrecognition of RNA viruses and the antiviral innate immunity. Ultimately, it will supply anovel sight for the defense of grass carp hemorrhage.