RNA Decapping Enzyme DDX41 Targets STING To Regulate The Role Of IFN-β In Inhibiting FAdV-4

Fowl adenovirus type 4(FAd V-4)belonged to the adenovirus family,a member of the genus Adenovirus.The infection of FAd V-4 caused fowl pericardial effusion hepatitis syndrome with a fatality rate of up to 80%,second only to avian influenza virus.As a member of the RNA Helicase family,DDX41(DEAD-box Helicase 41)plays an important role in the regulation of RNA metabolism.In recent years,DDX41 is increasingly studied as a cytoplasmic DNA receptor.At present,it has been proved that Chicken DDX41(ch DDX41)interacts with Chicken STING(interferon stimulator gene),activating natural immune signaling pathway to produce cytokines such as IFN-β,which affect viral replication.As a DNA virus,it has not been reported whether FAd V-4 can activate the active ch DDX41-ch STING-IFN-β signaling pathway to inhibit viral replication.Therefore,this study aims to explore how ch DDX41 can induce downstream cascade immune response against FAd V-4infection by activating the ch STING-IFN-β signaling pathway.The main research contents are as follows:1、Clonal expression,biological characterization and multiple antibody preparation of ch DDX41 geneIn this study,ch DDX41 gene was cloned from chicken hepatocellular carcinoma cells(LMH).Through a series of bioinformatics analysis,it was found that ch DDX41 was most closely related to birds and most closely related to fish.The SMART online website predicts that ch DDX41 consists of only one DEXDc,one HELICc and one Zn F-C2 HC domain.RTq PCR showed that ch DDX41 was highly expressed in thymus,muscle,kidney and lung.In order to investigate the expression of ch DDX41 protein,the soluble ch DDX41 protein was successfully expressed by the expression system of Escherichia coli for the first time and immunized into mice to obtain murine ch DDX41 polyclonal antibody with titer of 51200.2、The role of ch DDX41 in the replication of FAd V-4 infectionIn order to explore the effect of ch DDX41 on FAd V-4 replication,this study transfected ch DDX41 into LMH cells and infected with FAd V-4.After transfected with FAd V-4,IFA,RT-q PCR and Western blot were used to detect the effect of ch DDX41 on FADV-4replication at fluorescence,m RNA and protein levels,respectively.The results showed that the fluorescence intensity of FAd V-4 virus in LMH cells overexpressing ch DDX41 was significantly decreased.The virus copy number of FAd V-4 decreased significantly after overexpression of ch DDX41.The expression of Hexon protein decreased significantly after overexpression of ch DDX41,suggesting that FAd V-4 infection can induce CHDDX41-mediated innate immune response and inhibit viral replication.In this study,ch DDX41 was transfected into LMH cells,and RT-q PCR detection showed that,Overexpression of ch DDX41 can promote the expression of IFN-β,Mx-1,My D88,IRF7,MDA5,IL-1β,IL-6,IL-8,NF-κB,MAV5,PKR,TBK1 and other cytokines and antiviral proteins,thereby affecting viral replication.3、Preliminary study of ch DDX41 targeting ch STING to inhibit FAd V-4 replicationIn order to further explore the relationship between the CHDDX41-CHSTING signaling pathway and FAd V-4 replication,in this study,it was found that ch DDX41 was mainly located in the nucleus and ch STING was located in the cytoplasm under laser confocal microscopy.The results of Co-IP experiments showed that ch DDX41 and ch STING had interaction.In order to explore how ch DDX41 interacts with ch STING,this study found that the transfer of ch DDX41 from the nucleus to the cytoplasm after FAd V-4 infection is consistent with the localization of ch STING in cells and induces downstream cascade immune responses.In addition,this study also screened the DEXDc domain of ch DDX41 through Co-IP to interact with ch STING,which is necessary to inhibit FAd V-4 infection.In conclusion,this study was the first to analyze the biological characteristics of ch DDX41 gene and to prepare murine ch DDX41 polyantibody.The nucleoplasmic translocation of ch DDX41 after FAd V-4 infection interacts with ch STING located in the cytoplasm to enhance the ch DDX41-ch STNG signaling pathway,induce the expression of IFN-β,inflammatory factors and antiviral proteins,and inhibit FAd V-4 replication.In addition,the direct interaction between the DEXDc domain of ch DDX41 and ch STING was confirmed by Co-IP.This study provides a theoretical basis for further exploring the role of DDX41-STING signaling pathway in avian antiviral innate immune system.