Functional Study Of CMPK2 And FTR01/FTR42/FTR58 In Fish Cell Lines Antiviral Immunity

Innate immunity is the first line of defense against host viral infections,and intracellular antiviral factors play an important role in natural immunity.By analyzing the spleen RNA-Seq data of zebrafish infected with SVCV,it was found that CMPK2,FTR01,FTR42 and FTR58 were significantly up-regulated.Based on this result,we selected these four genes for further study.The results of this study have contributed to our better understanding of the antiviral innate immunity of teleost fish.1.Functional of fish CMPK2 in SVCV infectionCytidine/uridine monophosphate kinase 2(CMPK2)is known as a nucleoside monophosphate kinase in mitochondria to maintains intracellular UTP/CTP,and could be induced by immunostimulants LPS and Poly(I:C)in mammals,suggesting its potential antiviral and antibacterial role.In this study,CMPK2 was cloned and characterized in Fathead minnow(FHM)cells.In vivo analysis of tissue distribution revealed that CMPK2 transcript was detected in all the tissues of zebrafish(Danio rerio)examined in this study,particularly abundant in liver,spleen and kidney.In addition,indirect immunofluorescence showed that CMPK2 was localized in the cytoplasm of FHM cells.Expression of CMPK2 mRNA was significantly up-regulated following challenge with Spring viraemia of carp virus(SVCV),poly(I:C),or zebrafish IFN1 and IFN3 both in vitro and in vivo.Furthermore,overexpression and RNA interference of CMPK2 in SVCV-infected FHM cells showed significantly antiviral effect.In summary,this study for the first time shows the presence and distribution of CMPK2 in different tissues of zebrafish,but also demonstrates its antiviral potential against SVCV infection in vivo.Since little is known about the role of CMPK2 in SVCV infection,this study aimed to investigate the potential role of CMPK2 in SVCV infection and to understand the molecular mechanisms involved in fish immune responses to SVCV.These findings could contribute to explain the molecular mechanism of the CMPK2 mediated antiviral function.2.Molecular Characterization of FTR01,FTR42,and FTR58 in Zebrafish(Danio rerio)TRIM proteins were shown to play an important role in innate antiviral immunity.FinTRIM(FTR)is a new subset of TRIM genes that do not possess obvious orthologs in higher vertebrates.However,little is known about its function.In this study,we used bioinformatic analysis to examine the phylogenetic relationships and conserved domains of zebrafish(Danio rerio)FTR01,FTR42,and FTR58.Sequence analysis showed that the three finTRIMs were highly conserved,and all contain a RING domain,B-box domain,Coiled-coil domain(CCD)and SPRY-PRY domain.In addition,FTR42 and FTR58 had one CCD,whereas FTR01 had two CCDs.3.Expression Analysis of FTR01,FTR42,and FTR58 in SVCV-infected Zebrafish(Danio rerio)Qualitative real-time PCR was used to examine FTR01,FTR42,and FTR58 expression patterns in zebrafish embryonic fibroblast(ZF4)cells and zebrafish tissues.Tissue expression analysis revealed that the mRNA level of FTR01 was the highest in the liver,whereas those of FTR42 and FTR58 were the highest in the gill;the expression of these finTRIMs was clearly upregulated not in the eyes,but in the liver,spleen,kidney,gill,and brain of zebrafish following spring viremia of carp virus(SVCV)infection.Similarly,the expression of these three finTRIM genes also increased in ZF4 cells after SVCV infection.Our study revealed that FTR01,FTR42,and FTR58 may play an important role in antiviral immune responses,and these findings validate the need for more in-depth research on the finTRIM family in the future.