IFIT3 Promotes Anti-HBV Effect Of Interferon-alpha And Its Mechanism

【Objective】 At present,the mechanism of anti-HBV effects of interferon alfa(IFN-α)is still unclear.In this study,we explored the role of the IFIT family proteins in IFN-α against HBV replication,preliminary elucidation of its possible mechanism of action,and provide theoretical basis and experimental basis for improving the mechanism of IFN-α anti-HBV replication.【Method】 HBV-infected patients’ whole blood and the corresponding serum samples(72 cases)were collected.At the same time,the health check-ups that matched the age and gender of HBV-infected persons were collected as healthy control group(50 cases).HBV serum markers HBs Ag and HBe Ag were quantified by chemiluminescent microparticle immunoassay(CMIA),and ALT and AST were measured by a rate method.Real-time fluorescence quantitative PCR was used to detect the expression of IFIT family protein in peripheral blood mononuclear cells(PBMC).The correlation between IFIT3 and clinical indicators related to HBV infection was analyzed to explore the relationship between IFIT3 and HBV infection.Construction of small interfering RNA(si RNA)transiently interferes with the expression of IFIT3,constructs the IFIT3 expression vector,knocks down or overexpresses the expression of IFIT3 in Hu H7 cells,observes the replication of HBV,and knocks down or overexpresses IFIT3 of Hu H7 cells and PBMC to observe the antiviral effect Protein PKR,OAS1,Mx A expression levels,explore the mechanism of IFIT3 to promote IFN-α anti-HBV effect.Westren-Blot technique was used to detect the expression of STATs in Hu H7 cells after knockdown of IFIT3,and to explore the regulation of IFIT3 in JAK-STAT signaling pathway.【Results】 The age and gender of erolled cases of this experiment had no differences between groups.The expression of IFIT family proteins in PBMCs of HBV-infected patients was higher than that in healthy controls.The expression of IFIT1,IFIT3,and IFIT5 in PBMC of HBV-infected patients was positively correlated with the HBV DNA load in serum(P=0.0009,P=0.0003,P=0.0184),of which IFIT1 and IFIT3 were highly correlated with HBV DNA(R=0.3867,R=0.4206);there was no significant difference between IFIT family protein and ALT,AST,HBs Ag,and HBe Ag(P>0.05).Stimulation of PBMC and Hu H7 cells in vitro with IFN-α induced intracellular expression of the IFIT family proteins,among them,IFIT3 has the highest induced expression level.The transfection of HBV plasmid could induce the expression of IFIT family proteins in Hu H7 cells too.Stimulated PBMC with culture supernatant of Hep G2.2.15 which containing HBV can also increase the expression of IFIT family.However,PBMC stimulated with the serum of HBV-infected patient reduce the expression of IFIT family.With the addition of IFN-α,the levels of HBs Ag,HBe Ag,and HBV DNA in the supernatant of IFIT3 knockdown Hu H7 cells were higher than those of cells without IFIT3 knockdown,while the expression of the antiviral proteins of the interferon signaling pathway PKR,OAS,and Mx A was decreased,indicating that the ability of Viral clearance in cell is weakened;overexpression of IFIT3 decreases the supernatant HBs Ag,HBe Ag,and HBV DNA levels,and increases the expression of antiviral proteins PKR,OAS,and Mx A,confirming that IFIT3 can promote the HBV clearance ability of IFN-α,phosphorylation of STAT2 protein was suppressed after knockdown of IFIT3 in Hu H7 cells.Therefore,IFIT3 acts as an interferon-alpha stimulatory protein in the anti-HBV effect of IFN-α.【Conclusion】 The level of IFIT family protein expression is elevated in HBV-infected individuals and is positively correlated with HBV viral load in vivo.Interferon alpha promotes phosphorylation of STAT2 mainly through activation of JAK-STAT signaling pathway.Furthermore,up-regulation of downstream antiviral protein expression exerts anti-HBV effects,and IFIT3 acts as an interferon effector molecule to positively regulate the signaling pathway,thereby enhancing the anti-HBV effect of interferon alpha.