The Role Of Duck LGP2 In RLR-mediated Innate Immune Of Anti-Tambusu Virus

Laboratory 2 of genetics and physiology(LGP2)and retinoic acid induced gene-I(RIG-I)and melanoma differentiation associated gene 5(MDA5)are members of retinoic acidinducible gene-I-like receptor(RLR)in pattern recognition receptors(PRRs).They play an important role in the host’s innate immunity.Due to the lack of caspase activation and recruitment domain(CARD),LGP2 is controversial as a negative or positive regulator in antiviral response.Duck Tembus virus(DTMUV)is a kind of flavivirus,which can cause egg drop syndrome and fatal encephalitis in diseased ducks,which brings serious damage to the duck industry in China.As RLR signaling pathway plays an important role in antiviral immune response,its family members are potential research objects in the future antiviral therapy research and development.At present,the research on RLR is mainly focused on mammals,while the research on duck is relatively few.It has been proved that duck RIG-I(du RIG-I)and duck MDA5(du MDA5)are involved in the innate immunity of host against DTMUV,but the specific mechanism of duck LGP2(duLGP2)as a regulator of RLR signaling pathway is not completely clear.In this study,duLGP2 overexpression and knock-down duck embryo fibroblast(DEF)cells were constructed to determine whether duLGP2 is involved in duck innate immunity and its role in duck innate immunity.By infecting duLGP2 overexpressed and knock-down DEF cells with DTMUV and editing ducks with duLGP2 overexpression gene constructed by lentiviral vector,the role of duLGP2 in host anti-DTMUV innate immunity was determined in vivo and in vitro.Co-transfection of duLGP2 with du RIG-I or du MDA5 was used to determine whether there was interaction between duLGP2 and du RIG-I or du MDA5.The main research results are as follows:1.The role of duLGP2 in duck innate immunityIn duLGP2 overexpressed DEF cells,the expression levels of type I IFNs(IFN-α and IFN-β),MAVS,antiviral proteins(PKR,OAS),MHCs(MHC-Ⅰ and MHC-Ⅱ)were significantly decreased,while the changes of cytokines in duLGP2 knock-down cells were roughly opposite,indicating that duLGP2 participates in duck innate immunity and inhibits the expression of type I IFNs,ISGs and downstream cytokines MAVS of du RIG-I and du MDA5.2.The role of duLGP2 in the innate immunity of duck anti-DTMUVAfter duLGP2 overexpression DEF cells were infected with DTMUV,the DTMUV viral copy number was significantly up-regulated,the expressions of pro-inflammatory cytokines(IL-1β,IL-2,IL-8 and IFN-γ)and MHC-Ⅱ were significantly up-regulated,and the expressions of type I IFNs(IFN-α and IFN-β),antiviral proteins(PKR and OAS),TNF-α and MAVS were down-regulated in the early stage,and then up-regulated with time.The expression of MHC-Ⅰ was down-regulated.After duLGP2 knockdown DEF cells were infected with DTMUV,the DTMUV viral copy number was significantly down-regulated and the expression of IL-2 was up-regulated.The expression of type I IFNs(IFN-α and IFN-β),IL-1β,PKR,MAVS and MHC-Ⅱ was up-regulated in the early stage,and returned to or lower than that of the control group at 24 hpi or 36 hpi,while the expression of IL-8,IFN-γ,OAS and MHC-Ⅰ was down-regulated.DEF cells were infected with duLGP2 lentivirus expression vector Lv-LGP2 and control lentivirus Lv-NC,respectively.The expression level of duLGP2 in DEF cells infected with Lv-LGP2 was increased,and the expression was further upregulated after DTMUV infection.After Lv-LGP2 infection,the m RNA expression levels of duLGP2 in spleen,lung,kidney,brain,thymus and bursa of Fabricius were all up-regulated,and the up-regulation level was higher in spleen,lung and kidney,but lower in brain,thymus and bursa of Fabricius.The viral load in lung and brain tissue of duLGP2 overexpression gene editing ducks infected with DTMUV was significantly up-regulated.The expression of IFN-γ,IL-8,antiviral proteins(PKR and OAS)and MHCs(MHC-Ⅰ and MHC-Ⅱ)in lung tissue was up-regulated,and the expression of type I IFNs(IFN-α and IFN-β),IL-1β,IL-2,TNF-α and MAVS was down-regulated at 1 dpi,and returned to the expression level similar to or higher than that of the control group at 3 dpi or 5 dpi.The expressions of pro-inflammatory cytokines(IL-1 β,IL-2,IL-8,IFN-γ and TNF-α),OAS and MHC-Ⅰ in brain tissue were upregulated,while the expressions of type I IFNs(IFN-α and IFN-β),PKR,MAVS and MHC-Ⅱwere down-regulated at 1 dpi,up-regulated at 3 dpi,and returned to the level similar to or lower than that of the control group at 5 dpi.Combined with in vivo and in vitro experiments,it can be concluded that DTMUV can promote the expression of duLGP2,and the overexpression of duLGP2 can promote the replication of DTMUV,and the up-regulation of DTMUV copy number can cause more intense inflammatory response and antiviral response mediated by type I IFNs.3.Interaction between duLGP2 and du RIG-I or du MDA5duLGP2 and du RIG-I or du MDA5 plasmids were co-transfected into HEK293 cells.The results of co-immunoprecipitation showed that complexes could be formed between duLGP2 and du RIG-I or du MDA5 proteins,and DTMUV infection could promote the formation of complexes,which indicated that there was interaction between duLGP2 and du RIG-I or du MDA5,and DTMUV could promote this interaction.