The Role Of Hepatitis E Virus ORF2 Interacting Protein VDAC1 In Type I Interferon Signaling Pathway

Hepatitis E virus(HEV)is a zoonotic pathogen causing hepatitis in both human and animal hosts,which is responsible for acute hepatitis E outbreaks worldwide.Due to the absence of a robust cell culture system and animal model,the mechanisms of viral life cycle and pathogenesis are still unclear.Therefore,it is of great significance to explore the interaction and mechanism between HEV and host cells.In this study,voltage-dependent anion channel 1(VDAC1)protein was obtained by bioinformatics analaysis and Mass spectrum assay,and then its role in type I interferon signaling pathway was explored.The results are listed as follows:1.From the Gene Expression Omnibus(GEO)database,we screened and downloaded a dataset of monocyte samples and whole blood samples from eight CHE patients and eight healthy controls.23 differentially expressed genes(DEGs)were obtained by Rlimma package analysis.Functional enrichment analysis showed that these genes were mainly involved in biological processes such as type I interferon response and type I interferon signaling pathway,etc.Combined with four machinelearning strategies(RF,LASSO,SVM and WGCNA),two key biomarkers(VDAC1and BATF2)related with chronic HEV infection were got.2.The recombinant plasmid of HEV ORF2 was constructed and transfected into human hepatocellular carcinoma cells(Huh-7)for co-immunoprecipitation and silver staining.After protein mass spectrum assay,37 specific proteins were obtained compared with the control group.One of them is VDAC1,which is one of the biomarkers obtained by bioinformatics analysis.And then,the interaction between HEV ORF2 and VDAC1 was verified by co-immunoprecipitation and indirect immunofluorescence.Besides,the expression level of VDAC1 was down regulated with the increase of ORF2 expression.3.To explore the role of VDAC1 on regulating the type I interferon signaling pathway,the VDAC1 recombinant plasmid was transfected into HEK293 cells and treated cells with Poly(I:C),and then the RT-q PCR results showed that the overexpression of VDAC1 induced a higher level of IFN-β.Further studies showed that VDAC1 negatively regulated the expression of adaptor molecules,MDA5,MAVS,TBK1 and IRF3 which are key proteins in type I interferon signaling pathway.And VDAC1 has specific interaction with MAVS,TBK1 and IRF3,thus determining the adaptor molecule IRF3 of VDAC1 regulating type I interferon pathway.4.In order to preliminarily clarify whether VDAC1 regulates the production of IFN-β by affecting the nuclear translocation of IRF3,nuclear-cytoplasmic separation and indirect immunofluorescence test were carried out,and we found that VDAC1 could interact with IRF3 and co-localize with IRF3.Overexpression of VDAC1 significantly inhibited the nuclear translocation of IRF3 protein,suggesting that VDAC1 inhibited the nuclear translocation of IRF3 by binding to IRF3.Above all,this study first discovered the role of VDAC1 in the regulation of innate immune response,which filled the gap for the related research of this protein and also provided important clues for studying the pathogenesis of HEV infection.