Mechanisms Of Action Of Interferon-induced Protein IFIT5 In Regulating Innate Immune Response To Duck Hepatitis A Virus

Duck viral hepatitis(DVH)is one of the most important infectious diseases affecting the duck industry.DVH in China is mainly caused by Duck hepatitis A virus(DHAV)genotype 1(DHAV-1)and genotype 3(DHAV-3).DHAV-1 causes severe liver hemorrhage and necrosis,neurological symptoms,and high mortality in ducklings.Natural immunity,also known as intrinsic immunity,is a natural immune defense against invasion of foreign microorganisms or pathogens developed by the body during genetic evolution and constitutes the first line of defense against pathogenic microorganisms.The immune system includes intrinsic immunity and adaptive immunity,through which the body can recognize itself and non-self-foreign substances and can expel non-self foreign substances from the body,thus playing the role of immune defense,stabilization and surveillance.Among them,type Ⅰ Interferon(IFN-Ⅰ)signaling pathway is an important part of the natural immune signaling pathway,which plays an important role in regulating the relationship between host and virus,and the process mainly consists of pattern recognition receptor-mediated signaling pathway cascade reaction to induce interferon production,activation of Jak-STAT signaling pathway,upregulation of interferon-stimulated genes IFN-stimulated genes(ISGs)expression,thus achieving direct or indirect pathogen clearance.The family of Interferon-induced proteins with tetratricopeptide repeats(IFITs)is a conserved group of ISGs that play an important role in the regulation of pathogen-mediated immune responses and maintenance of immune homeostasis.This protein family consists of four main members: IFIT1,IFIT2,IFIT3 and IFIT5.IFIT5 is different from IFIT1,IFIT2 and IFIT3 in the regulation of natural immunity,especially the role and molecular mechanism of IFN signaling pathway response needs to be studied.In this study,we further explored the role of IFIT5 in regulating the response of IFN-Ⅰ signaling pathway and the potential regulatory mechanisms based on the significant differences in the expression of duck IFIT5 gene after DHAV-1 infection obtained by transcriptomic high-throughput sequencing in the previous laboratory.To explore the role of IFIT5 in the IFN-Ⅰ signaling pathway induced by DHAV-1,the promoter activity of IFN-β and ISRE and the m RNA levels of IFN-β,IFITM1 and Mx induced by IFIT5 in DEF cells were examined by using a dual luciferin reporter enzyme system and real-time fluorescence quantitative PCR.The results indicated that overexpression of IFIT5 significantly suppressed the promoter activity of IFN-β and ISRE and the m RNA levels of IFN-β and ISGs.In contrast,knockdown of IFIT5 promoted the promoter activity of IFN-β,ISRE and the m RNA levels of IFN-β and ISGs.Simultaneous overexpression of IFIT5 contributed to DHAV-1 replication.To determine the target of IFIT5 in the IFN-Ⅰ signaling pathway,the eukaryotic expression vector of duck-derived IFIT5 was constructed in this study,and the laboratory-constructed eukaryotic expression vector of each molecule of the IFN-Ⅰ signaling pathway was used to transfect DEF cells,and the effect of IFIT5 overexpression on the IFN-βpromoter activity induced by each factor was detected using a dual fluorescent reporter gene.The results showed that IFIT5 could inhibit the IFN-β promoter activity induced by IRF7 and its upstream molecules,but had no effect on the IFN-β promoter activity induced by IRF7,which tentatively locked that IFIT5 might interact with IRF7.To further explore the interaction between IFIT5 and IRF7,we verified it by immunoprecipitation,prior to which polyclonal antibodies to duck-derived IFIT5 as well as IRF7 were constructed to facilitate subsequent experiments.The test results showed that exogenous expression of IFIT5 protein with HA tag and IRF7 protein with Flag tag interacted with each other,and further addition of IRF7 polyclonal antibody and IFIT5 polyclonal antibody were detected by immunoprecipitation under DHAV-1 stimulation and non-stimulation,respectively,and both results showed that IFIT5 and IRF7 interacted with each other.In this assay,we found that IFIT5 competitively binds to the IRF7 Subdomain III structural domain after constructing a truncator of IRF7 and transfecting cells with IFIT5,and we verified that IFIT5 inhibits the dimerization of IRF7 using immunoprecipitation and Native Page.This suggests that IFIT5 inhibits IFN-Ⅰ signaling pathway by interacting with IRF7 and inhibiting the dimerization of IRF7 and its entry into the nucleus.Taken together,this study reveals the mechanism by which IFIT5 negatively regulates DHAV-1-induced IFN-Ⅰ natural immune response.