Molecular Characteristics Of RIG-I And MAVS Of The Lamprey And Their Signaling Pathways In Antiviral Immunity

At present,the viral diseases of aquatic animals have caused huge economic losses to the aquaculture,which seriously affects the sustainable development of China's aquaculture industry.Innate immunity is an important part of the immune system and the first barrier of the fish against virus invasion.Therefore,innate immunity plays an important role in the virus defense of aquatic animals.Therefore,it has important practical significance of in-depth study on the anti-viral immune defense mechanism of the lamprey for exploring the methods of the control of fish viruses.Lampetra japonica is one of the most primitive vertebrates,so studying their innate immunity is important for understanding the origin of the immune system.In the innate immune system,retinoic acid-inducible gene-I(RIG-I)is one of the main recognition receptors of viral dsRNA(double-stranded RNA).In recent years,researchers have been a large number of RIG-I receptors and related genes were cloned and identified in low vertebrates,which greatly promoted the development of antiviral immunity in lower vertebrates.This paper studied the RIG-I-like receptors and related genes and their antiviral immune signaling pathways.First,the lamprey not only have an immune response to the stimulationthe of poly(I:C)(polyinosinic-polycytidylic acid),but also have RIG-I-like receptors and their signaling pathway-related molecules.It was revealed that the most abundant genes were concentrated in protein translation modification and degradation,and were enriched by KEGG(Kyoto Encyclopedia of Genes and Genomes)though the data analysis of the poly(I:C)-stimulated lampreys by high-throughput transcriptome sequencing.The most abundant genes are concentrated on the signal pathways of signal transduction,transport and catabolism,and the immune system,indicating that lamprey produced immune response to poly(I:C)stimulation.19 genes of poly(I:C)stimulation-related signaling pathways in further analysis,mainly are related to RIG-I-like receptor signaling pathways,and the expression levels of interleukin-8(IL-8)and NF-kappa-B inhibitor alpha(NFKBIA)were up-regulated in the comparisons of different groups.The comprehensive analysis showed that the lamprey not only produced an immune response to dsRNA virus stimulation,but also there are related molecules of RIG-I-like receptors and their signaling pathways,which laid a foundation for the later study of the lamprey-RIG-I receptors.Secondly,there are not only RIG-I-like receptors and their related molecules,but also triggers the antiviral immune signaling pathway by nuclear regulation though RIG-I/MAVS and TLR3/TRIF stimulated by poly(I:C)and Grass Carp Reovirus(GCRV)in the lamprey,thereby producing effector molecules of IL-8 and Mx to inhibit viral proliferation.It is confirmed that grass carp ovary cell line(CO)is resistant to GCRV infection by over-expressing RIG-I or mitochondrial antiviral signaling protein(MAVS)with MTT.Furthermore,it was showed that there are up-regulate the expression of NF-?B and interferon-sensitive responsive element(ISRE)in HEK293 by overexpression of RIG-I or MAVS of lampreys.detected by the dual luciferase reporter system,thereby activating interferon(IFN)antiviral signaling pathways;moreover,it was found that lampreys cells by both stimulation(transfection)of poly(I:C)in vitro and intraperitoneal injection with GCRV in vivo could activated both RIG-I/MAVS and TLR3/TRIF-dependent antiviral signaling pathways though nuclear regulatory by QPCR.At the same time,the activation of the antiviral immune signaling pathway in the lamprey can also induce mitochondria-dependent apoptosis in cells,thereby achieving antiviral purposes.It was revealed that the lamprey MAVS was not only localized to mitochondria,but also accompanied by the production of reactive oxygen species(ROS)by overexpressing the lamprey MAVS protein in the cells with laser confocal;simultaneously,the QPCR method showed that,compared with the control group,the expression of Bax was significantly increased,and that of Bcl-2 was significantly decreased,with being consistent with the results of immunoblotting,and it was also found to be accompanied by the release Cytochrome C(Cyt C);the results in the experiment showed that overexpression of the lamprey MAVS can activate intracellular mitochondria-dependent apoptosis,thereby inhibiting virus proliferation.The results of this study preliminarily clarified that stimulation of the lamprey in vitro and in vitro can not only activate RIG-I/MAVS and TLR3/TRIF to initiate antiviral immune signaling pathway by nuclear regulation to inhibit viral proliferation,but also activate intracellular mitochondria-dependent apoptotic signaling pathways in order to exert antiviral effects;This study is conducive to understanding of the evolutionary history of vertebrate antiviral immunity,and provides data support for later scholars in the research of anti-viral immunity of the jawless vertebrate.