Analysis Of The Role Played By Immune-related Factors, ISG15and Mig1, In Cynoglossus Semilaevis’s Antiviral Immunity

Based on the cDNA library constructed from major immune organs (head kidneyand spleen) of Cynoglossus semilaevis challenged with the mixture of L. anguillarumC312and E. tarda TX1, two immue-related factors, ISG15(Interferon StimulatedGene15) and Mig1(Megalocytivirus induced gene1), were identified throughrandom sequencing.In mammals, ISG15is known to play a role in antiviral immunity mainly due toa process called ISGylation, which is ubiquitination alike. In this study, the newlyisolated ISG15homologue, CsISG15, is confirmed to share the same role. qRT-PCRplus Western Blot assay showed that CsISG15expression in head kidney and spleenwas drastically induced by RBIV-C1infection, while RNAi analysis showed that viralload in lymphocytes was greatly enhanced after CsISG15expression had beeninterfered. Both of these suggest a role of CsISG15in host’s antiviral immunity. Tofurther elucidate this role, recombinant proteins, rCsISG15and rCsISG15M, wereexpressed in E. coli and purified under native conditions—rCsISG15M is a mutationtype of the wild type rCsISG15, with the conserved motif LRGG mutated to LAAG.We found that rCsISG15activated macrophages and greatly reduced intracellularviral loading during megalocytivirus infection of HK lymphocytes, whereas antibodyblocking of CsISG15enhanced viral infection. The same effects induced byrCsISG15M were significantly weakened, thus suggested a relation between thefunction and LRGG motif. Further study showed that rCsISG15enhanced theexpression of immune genes in HK lymphocytes, which suggested an immueregulatory way of CsISG15as an extracelluar cytokine. As the recombinant proteinsused in this study have not been processed by host enzyme, that is, both rCsISG15 and rCsISG15M have a C-terminal extension beyond the LRGG motif, it isimpossible for them to bind the targets via ISGylation. So our results also suggest anew role for the LRGG motif other than that in ISGylation.Mig1was identified as a novel gene，and was named as ‘Megalocytivirusinduced gene1’(Mig1) because it can be induced by megalocytivirus infection. Noclose homologue of Mig1was found in the NCBI database, however, the N-terminalregion of CsMig1shares moderate sequence similarities with several virus inducedgenes isolated from other teleost species. qRT-PCR analysis showed that CsMig1expression in head kidney and spleen was drastically induced by RBIV-C1infection,and, over expression of CsMig1in HK lymphocytes significantly reduced viral loadduring RBIV-C1infection, both suggest a relation between CsMig1and host antiviralimmunity. Due to a lack of known IFN genes in tongue sole, we prepared the culturesupernatant of virus-stimulated HK lymphocytes, which was supposed to containIFNs. Following test showed that CsMig1expression in lymphocytes could be greatlyinduced by the prepared culture, thus suggest the possibility of CsMig1expressionbeing stimulated by IFNs.