IFIT3 Promotes Anti-HBV Efficacy Of IFN-α Through JAK-STAT Signaling Pathway

ObjectiveIncreasing evidence indicates that adjuvant interferon-α(IFN-α)therapy is an effective treatment option for a subgroup of patients with chronic hepatitis B(CHB).The activation of Janus kinase(JAK)-signal transducer and activator of transcription(STAT)signaling cascades,and the induction of downstream transcription of IFN-stimulated genes(ISGs)plays pivotal roles in IFN-α effects.It has been confirmed that IFIT3,a member of the IFITs gene family of classic ISGs,could inhibit the replication of various viruses,but its effect on HBV replication is unclear.Here,we analyzed the expression of IFIT3 in the peripheral blood of chronic HBV-infected patients,and then the effect of IFIT3 on the JAK-STAT signaling pathway under the dual intervention of IFN-α and HBV was explored in vitro.The aim of our study is to identify the role and mechanism of IFIT3 on IFN-α antiviral activities against HBV.MethodPeripheral blood was collected from untreated patients with HBV carriers or healthy controls.The level of IFIT3 m RNA was examined by real time PCR.Correlations between IFIT3 m RNA expression and clinical features were analyzed.The p HBV 1.2plasmid was transiently transfected in Hep G2 / HL-7702 cells to observe the effect of HBV infection on the expression of IFIT3.The role of IFIT3 in JAK-STAT signaling was investigated through gain of function and loss of function by real time PCR and Western blot simultaneously.We further performed q PCR and Western blot analysis using m RNA and protein extracts from Hep G2 cells treated with highly selective JAK inhibitor Ruxolitinib to analysis the mechanism of IFIT3.Finally,the content of HBs Ag and HBe Ag in the supernatant of p HBV1.2-Hep G2 cells stably transfected with IFIT3 was detected by chemiluminescence microparticle immunoassay(CMIA).ResultsOur results showed that untreated patients with chronic HBV infection exhibited elevated expression of IFIT3 than that in healthy cohort.Further,we observed that individuals under hepatitis B e antigen(HBe Ag)-positive phases had higher levels of IFIT3 than those under HBe Ag-negative phases of the disease.Consistently,IFIT3 was verified to be induced by HBV via transient transfection with the p HBV1.2plasmid in Hep G2/HL-7702 cells,and IFN-α enhanced the upregulation effect on IFIT3.Mechanistically,knockdown of IFIT3 inhibited the phosphorylation of STAT2,but not STAT1,whereas overexpression of IFIT3 produced an opposite effect in vitro.Meanwhile,overexpression of IFIT3 could enhance IFN-α triggered ISG expression,including myxovirus protein A(Mx A),2’,5’-oligoadenylate synthetases(OAS),and double stranded RNA-dependent protein kinase(PKR),while knockdown of IFIT3 suppressed the expression of these genes.Similarly,a weaker induction of IFN-αtriggered ISG expression was observed in Ruxolitinib treated cells compared with non-treated Hep G2 cells which IFIT3 were loss of function.After interference with IFIT3 expression,HBs Ag,HBe Ag and HBV DNA secreted by Hep G2 cells transiently transfected with p HBV1.2 plasmid increased.ConclusionsThese findings suggest some interaction mechanisms between IFIT3 and IFN-α in HBV treatment.IFIT3 works in a STAT2-dependent manner.IFIT3 may serve as a novel therapeutic target for promoting the effect of IFN-α by the JAK-STAT signaling pathway in HBV infection.