| Innate immunity is an important part of fish immunity,and interferon-mediated signaling pathways are very important in innate antiviral immunity.Interferon(IFN)regulatory factor(IRF2)can regulate a variety of important physiological processes in fish such as IFN-mediated host immune processes,and mitochondrial antivirus signaling protein(MAVS)activivates IFN expression through RIG/MAVS signaling in fish.Therefore,in this study,we cloned and studied the irf2(Olirf2)and mavs(Olmavs)genes that affect IFN expression from medaka(Oryzias latipes),studied their antiviral function in vitro,laying the foundation for fish antiviral immunity research.Furthermore,this experiment provides a theoretical basis for constructing virus-resistant fish through gene editing or genetic modification.The main results of Olirf2 and Olmavs are as follows:1.In this study,the Olirf2 from medaka was cloned and identified.Olirf2 encodes 320 amino acids which has 73.35% identity to bastard halibut(Paralichthys olivaceus).Multiple alignment analysis showed that Olirf2 shares a IRF association domain(IAD2)and a N-terminal DNA binding domain(DBD).The expression of Olirf2 was detected in various tissues of medaka.The CDS of Olirf2 was inserted into the plasmid of p Tol2/CMVIRF2/IE1-pr for transient or stable expression in fathead minnow line of epithelial cells(FHM).Furthermore,the results showed that overexpression of Olirf2 promoted the replication of Spring Viremia of Carp Virus(SVCV)and significantly reduced the expression of mx1,ifn and irf3 genes.Further studies by luciferase reporter system showed that Olirf2 inhibited promoter activities of SVCV-induced NF-κB and ISRE,indicating that Olirf2 may promote the replication of SVCV by inhibited cellular innate immune response.However,constant overexpression of Olirf2 enhanced the antiviral ability in cells and increased the expression of mx1,ifn and irf3 genes.Therefore,our research suggested that the Olirf2 had ‘double sides’ for regulating the antiviral effect,which depended on the expression condition.2.To better understand the multiple functions of Mavs in fish immune response,Olmavs was cloned and identified in this study.Olmavs encodes 504 amino acids which has 39.78% and 19.37% identity to tilapia(Oreochromis niloticus)and human(Homo sapiens).Multiple alignment analysis showed that Olmavs shares a N-terminal tandem caspase activation and recruitment domain(CARD),a central proline-rich region and a transmembrane(TM)domain at C-terminal.In healthy medaka,the transcript of Olmavs was predominantly detected in the kidney.Transient overexpression of Olmavs could significantly evoke the activity of NF-κb-containing promoter and ISRE-containing promoter,also increased the expression of mx1,ifn and irf3 genes,subsequently enhanced the antiviral ability in FHM,which demonstrated by the decrease of virus plaques,titers,together with transcripts of vital gene svcv-g.In contrast,constant overexpression of Olmavs inhibited the expression of interferon related genes,hspb8,ifn and irf3,also promoted the replication of SVCV in FHM,such as the significant increase of expression of svcv-g and viral titers.Therefore,the Olmavs regulated the viral titers from double sides which depended on its expression level in vitro.In conclusion,the Olirf2 and Olmavs genes both have ‘double sides’ for regulating the antiviral effect,which depended on the expression condition.Also,their roles in IFNmediated antiviral signaling pathways may be opposite. |
