| TLRs(Toll-like receptors)are pivotal pattern recognition receptors in initiating innate immunity and triggering adaptive immunity.TLRs recognize the conserved molecular structure of pathogens,known as pathogen-associated molecular patterns(PAMPs),activating immune cells in response to pathogen infection.TLR pathways have been comprehensively investigated in mammals.However,the functions of many TLRs in fish remain largely unknown,and the teleost-specific TLR19 is one of them.In this study,we identified TLR19 from grass carp(Ctenopharyngodon idella),and analysed its role in resistance to grass carp reovirus(GCRV).We then explored its ligand,adaptor and signaling pathways.The main results obtained are showed as follow:1.TLR19 inhibited proliferation of GCRV.Constructed TLR19-HA vector,synthesized small interfering RNA(siRNA),and then transfected the CIK cells.Under GCRV infection,we detected the content of virus in CIK cells and the damage of the virus to the cells.The results showed that overexpression of TLR19 inhibited the proliferation of the virus in CIK cells,and the cell survival rate was significantly higher than that of the control group,whereas knocking down TLR19 obatained opposite effects.2.TLR19 localized in early endosome.Constructed TLR19-eGFP,Rab5-RFP(early endosome protein marker),Rab7-RFP(late endosome protein marker)fusion expression vectors,transfected into CIK cells,and labeled with ER-Tracker and Hoechst 33342.Images were collected by confocal laser scanning microscope.The results demonstrated that TLR19 localized in the eraly endosome.3.TLR19 recognized double strand RNA(dsRNA)and recruited TIR-domain-containing adaptor protein(TRIF).The TLR19-LRR protein was expressed and purified.And four representative PAMPs(polyinosinic-polycytidylic acid(poly(I:C)),Lipopolysaccharides(LPS),peptidoglycan(PGN),double strand DNA(dsDNA))were employed.The ligand of TLR19 was explored by enzyme linked immunosorbent assay(ELISA).Subsequently,dual luciferase reporter assays were performed to test these results upon overexpression of TLR19.The results indicated that TLR19 recognized and responded to dsRNA analog poly(I:C).Constructed TRIF,myeloid differentiation factor 88(MyD88),TIR-associated protein(TIRAP),sterile alpha,TIR motif containing 1(SARM1)and SARM1s2 Flag-tagged fusion vectors,and TRIF-RFP,MyD88-RFP,TIRAP-RFP,SARM1s2-RFP fusion vectors,respectively.TLR19-HA was transfected with Flag-tagged fusion vectors and TLR19-eGFP was transfected with red fluorescence fusion vectors,respectively.Fluorescence co-localization and co-immunoprecipitation experiments were performed to investigate the adaptor of TLR19.The results showed that TLR19 colocalized and interacted with adaptor TRIF,but not other adaptors.4.TLR19 triggered both interferon(IFN)and nuclear factor ?B(NF-?B)pathways via interferon regulatory factor(IRF3).Overexpressed TLR19 and detected protein and phosphorylation of IRF3 and IRF7 by western blotting(WB).It was found that TLR19 facilitated protein and phosphorylation levels of IRF3,and inhibited phosphorylation of IRF7.Subsequently,qRT-PCR and dual luciferase reporter assays were used to detect mRNA levels and promoter activities of IFNs and NF-?Bs,which were downstream molecules of IRF3 and IRF7.The results indicated that TLR19 enhanced the mRNA expressions and promoter activities of major IFNs and NF-kBs.However,TLR3,another antiviral molecules,only induced the expressions of IFN1,indicating that TLR19 triggered a stronger IFN and NF-?B response against GCRV infection.Collectively,these results demonstrate that teleost-specific TLR19 recognizes dsRNA,recruits adaptor molecule TRIF,enhances IRF3 protein and phosphorylation levels,triggers both IFN and NF-?B pathways and prevents viral proliferation.This is first time to systematically clarify TLR19 signaling pathway,which is the third TLR member recognizing dsRNA.The results will provide a theoretical basis for the antiviral immune mechanisms and evolutionary immunology in teleost. |
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