The effects and mechanisms of cholesterol 25-hydroxylase on the replication of Japanese encephalitis virus
Objective This study aims to explore the roles of interferon-stimulated gene cholesterol-25-hydroxylase(CH25H)on the replication of Japanese encephalitis virus(JEV)and its underlying mechanisms,providing potential targets for developing anti-JEV drugs and theoretical support for the research.Methods First,we investigated whether CH25H could be induced by typeⅠinterferons or JEV infection in human kidney HEK293T cells through real-time fluorescence quantitative PCR.The cells were stimulated with different concentrations of interferon α(IFN-α),and 12 hours later,CH25H expression was measured via real-time fluorescence quantitative PCR.HEK293T cells were infected with JEV at different multiplicities of infection(MOI),and 24 hours later,changes in CH25H mRNA expression levels were assessed.Eukaryotic expression plasmid pcDNA3.1-CH25H-3×HA was constructed for overexpression of CH25H in HEK293T cells,and its effect on JEV replication was analyzed using real-time fluorescence quantitative PCR and plaque formation assay.The reasonable use concentration of 25-hydroxycholesterol(25HC),the enzyme active product of CH25H,in HEK293T,BHK21,and Vero cells was obtained by CCK8 assay,and the roles of 25-hydroxycholesterol(25HC),the enzyme active product of CH25H,in JEV replication and its impact on JEV life cycle,were further studied by qPCR,viral plaque assay,immunofluorescence assay and adsorption assay.Point mutation was employed to construct an enzymatically inactive mutant,CH25HM,which was transfected into HEK293T cells.After 24 hours of JEV infection,the dependence of CH25H-mediated impact on JEV replication on its enzymatic activity was revealed through qPCR and plaque formation assay.Results CH25H could be induced by IFN-α in HEK293T cells,but after JEV infection,CH25H mRNA expression was downregulated.Overexpression of CH25H in HEK293T cells significantly inhibited JEV replication.25HC,the enzyme product of CH25H,can inhibit JEV replication in HEK293T,BHK21,and Vero cells.In terms of mechanism,25HC can inhibit JEV replication by affecting the virus adsorption process.Expressing the enzymatically inactive mutant CH25HM in HEK293T cells,also significantly inhibited JEV replication.Conclusions JEV infection down-regulates the expression of interferon-stimulated gene CH25H,while CH25H inhibits JEV replication in a manner dependent on enzyme activity and non-enzyme activity.
Japanese encephalitis viruscholesterol 25-hydroxylase25-hydroxycholesterolinterferon-stimulated genesviral replication
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