The Mechanism Of DTMUV Regulates Host LGP2 Protein In Escaping Innate Immunity

Duck Tembusu virus（DTMUV）is an acute infectious disease caused by Tembusu virus in almost all duck species.In addition to ducks,the virus can infect birds such as chickens,geese and sparrows.The disease has the characteristics of wide spread,fast spread and strong infectivity.The research achievements of Tembusu virus are increasingly abundant,but the specific pathogenesis and immune regulation of DTMUV against the host are still unclear and need to be explored.The RIG-I like receptor pathways（RLRs）are not as abundant in birds as they are in mammals.We demonstrate that DTMUV regulates immune escape of the RLRs signaling pathways in its native host ducks.Due to the lack of RIG-I gene,the mechanism by which DTMUV regulates the RLRs signaling pathway to achieve immune escape in chickens remains unclear.In this study,DTMUV-infected DF-1cells were used as models to investigate the effects of host chLGP2 protein and DTMUV non-structural protein NS1 on viral replication.The results of this study are mainly divided into the following three aspects.1.DTMUV induces the innate immune response of DF-1 cells at the early stage of infection.DF-1 cells were infected with DTMUV at the dose of 10⁴TCID₅₀.The cells were collected at 3 h,6 h,12 h and 24 h after virus infection.The mRNA transcription levels of RLRs pathway cytokines（LGP2,MDA5,MAVS,STING,TRAF3,TRADD,TBK1,IKKα,IRF3,IRF7,NF-κB,IFN-α,IFN-βand STAT1）were determined by qRT-PCR.The test results show that:Compared with the control cells,DTMUV infected DF-1 cells at 3 h,6 h,12 h and 24 h,the detected cytokine mRNA transcription levels all showed different degrees of increase,and at 3 h after infection,cytokine mRNA transcription levels were upregulated to the highest level.The mRNA transcription levels of LGP2,MDA5,MAVS,STING,TRAF3,TRADD,TBK1,IKKα,IRF3,IRF7,NF-κB,IFN-α,IFN-βand STAT1 were up-regulated by1.69 times,2.34 times,2.38 times,2.23 times,1.49 times,respectively.1.10 times,2.92 times,1.75 times,1.84 times,1.32 times,1.58 times,1.63 times,2.40 times and1.34 times.The results showed that DTMUV infected DF-1 cells can activate the host RLRs signaling pathway and induce the innate immune response of DF-1 cells.2.Effect of chLGP2 protein on DTMUV replication.The plasmid pEGFP-N1 and PEGFP-N1-chLGP2 were transfected into DF-1cells,and 24 h after transfection,the cells were collected for qRT-PCR and Western blot detection,respectively.The results showed that chLGP2 inhibited the expression of NF-κB protein.siRNA knockdown of chLGP2 promoted the expression of NF-κB protein.After DTMUV infection with DF-1 cells for 3,6,12 and 24 h,qRT-PCR results showed that the mRNA transcription levels of RLRs pathway cytokines（MDA5,MAVS,STING,TRAF3,TRADD,TBK1,IKKα,IRF3,IRF7,NF-κB,IFN-α,IFN-βand STAT1）were significantly down-regulated by chLGP2.3 h after infection,cytokine mRNA transcription levels were most significantly down-regulated.The mRNA transcription levels of MDA5,MAVS,STING,TRAF3,TRADD,TBK1,IKKα,IRF3,IRF7,NF-κB,IFN-α,IFN-βand STAT1 were downregulated by 0.63 times,0.22 times,0.36 times,0.67 times,0.40 times and 0.37times,respectively Times,0.61 times,0.48 times,0.27 times,0.25 times,0.25 times,0.28 times and 0.26 times.The mRNA transcription levels of MDA5,TRADD,TBK1,IKKα,IRF7,NF-κB and IFN-βwere significantly improved by siRNA knockdown of chLGP2.Western blot results showed that the expression of NF-κB protein was significantly inhibited by chLGP2 protein after DTMUV infection with DF-1 cells,and the opposite result was obtained when chLGP2 protein was knocked down.Together,these results suggest that chLGP2 enhances the replication of DTMUV in DF-1 cells by inhibiting the NF-κB mediated signaling pathway.3.Effect of DTMUV NS1 protein on DTMUV replication.DF-1 cells were transfected with pEGFP-N1 and PEGFP-N1-NS1 plasmids,respectively.24 h after transfection,the cells were collected for qRT-PCR detection.The results showed that DTMUV NS1 protein significantly inhibited the mRNA transcription levels of NF-κB and IFN-βby 0.84 and 0.61 times,respectively.Western blot showed that DTMUV NS1 significantly inhibited the expression of NF-κB protein.After DTMUV infection with DF-1 cells for 3,6,12 and 24 h,qRT-PCR results showed that the mRNA transcription levels of RLRs pathway cytokines（MDA5,MAVS,STING,TRAF3,TRADD,TBK1,IKKα,IRF3,IRF7,NF-κB,IFN-α,IFN-βand STAT1）were significantly down-regulated by NS1 protein.3 h after infection,cytokine mRNA transcription levels were most significantly down-regulated.The mRNA transcription levels of MDA5,MAVS,STING,TRAF3,TRADD,TBK1,IKKα,IRF3,IRF7,NF-κB,IFN-α,IFN-βand STAT1 were downregulated by 0.30 times,0.22 times,0.20 times,0.59 times,0.40 times,0.45times,respectively Times,0.52 times,0.39 times,0.45 times,0.26 times,0.83 times,0.19 times and 0.28 times.Western blot results showed that DTMUV infected DF-1cells,the expression of NF-κB protein was significantly inhibited by DTMUV NS1protein.Together,these results suggest that DTMUV NS1 protein enhances the replication of DTMUV in DF-1 cells by inhibiting the NF-κB mediated signaling pathway.In conclusion,this study revealed the role of chLGP2 in the anti-DTMUV innate immune response in DF-1 cells,and DTMUV NS1 protein escaped the innate immune response by regulating the RLRs signaling pathway.The results provided a theoretical basis for further understanding of the pathogenesis of DTMUV in chickens and the RLRs signaling pathway in chickens.