A Functional Variant Of CD40 Modulates Clearance Of Hepatitis B Virus In Hepatocytes Via Regulation Of The ANXA2/CD40/BST2 Axis

Background and objection:Hepatitis B virus(HBV)infection is one of the most serious and prevalent health problems worldwide.More than 250 million people are chronic hepatitis B patients who are chronically infected with HBV,which gives rise to increased risk of developing into liver failure,cirrhosis,and hepatocellular carcinoma.Persistent HBV infection or HBV clearance has been considered a multifactorial event that is generally attributed to viral factors,environmental factors,and host genetic factors.Our previous genome-wide association study identified a single-nucleotide polymorphism(SNP)——rs1 883832,in the 5’untranslated region of CD40 predisposing to chronic HBV infection,but the underlying mechanism remains undefined.This study aimed to investigate whether rs1883832 was the real functional SNP(fSNP)of CD40 and how it modulated HBV replication and transcription in hepatocytes.Methods:We screened and determined the 10 candidate fSNP(rs1883832 itself and those in high LD with rs1883832[r2>=0.8]in Asian people)that resided within～17 kb of the 5’UTR,promoter,or within intron 1 of CD40 via dual luciferase assay,electrophoretic mobility shift assay(EMSA)and online public database.Flanking restriction enhanced pulldown(FREP),chromatin immunoprecipitation(ChIP)and TA cloning experiments were used to identify fSNP associated regulatory protein.The potential anti-HBV activity of CD40 and its downstream molecule BST2 was assessed in HBV-transfected and HBV-infected cell models.The specific role of BST2 in interferon(IFN)-driven inhibition of HBV transcription was explored by western blot(WB)and quantitative real-time polymerase chain reaction(qPCR).Meanwhile,the association of a potential fSNP rs9576 of BST2 with treatment response was evaluated in 954 chronic hepatitis B(CHB)patients treated by pegylated IFN-alpha(PegIFN-α).Results:1.Rs1883832 exhibited significant allele-imbalanced luciferase activity between the risk allele and non-risk allele;rs1883832 displayed allele-inequal gel shifts between risk allele and non-risk allele.Data from the WashU Epigenome Browser further demostrated that rs1883832 was an fSNP in HepG2 cell and liver tissue,for DNase I hypersensitivity and active histone methylation.2.FREP assay suggested that ANXA2 was a regulatory protein on rs1883832 region.WB and qPCR results showed that ANXA2 was negatively correlated with CD40.ChIP and TA cloning experiments noted that ANXA2 preferentially bound to the risk allele T of rs1883832 compared to the non-risk allele C.3.Down-regulation of CD40 was found to promote HBV RNA,including total RNA and 3.5kb RNA,as well as HBsAg and HBcAg.On the contrary,ectopic overexpression of CD40 restricted HBV replication and transcription.4.KEGG pathway analysis suggested that the anti-HBV effect of CD40 might be related to the JAK-STAT signaling pathway.Knockdown of CD40 expression down-regulated the expression of p-STAT1,p-STAT3 and IRF9,as well as a reduction in nuclear IRF9,while overexpression of CD40 had the opposite effect.Dual luciferase assays demonstrated that silencing of CD40 expression inhibited the luciferase activity of IFN-stimulated response elemen(ISRE).However,a higher level of luciferase activity was observed in CD40-overexpressed cells.5.qPCR and WB results showed that interferon-stimulated gene BST2 was downstream of CD40.GTEx database indicated that CD40 was positively associated with BST2 in liver tissue.Similar results were also observed in HBV-infected liver tissues from published data.Suppression of JAK/STAT pathway or BST2 expression abrogated CD40-induced inhibitory effect of HBV.6.Cells transfected with BST2 plasmid responded better to IFN-α than the control cells.Consistently,the potential fSNP rs9576 of BST2 was significantly associated with respond to PegIFN-α treatment for CHB patients.Conclusion:We verified rs1883832 as the causal fSNP of CD40,predisposing to chronic HBV infection,and elucidated its unique mechanism in modulating HBV clearance in hepatocytes via regulation of the ANXA2/CD40/BST2 axis,which may shed new light on HBV therapy.