Molecular Mechanism Of Helicase DHX9 Promoting Hepatitis B Virus Replication

Objective:Hepatitis B virus(HBV)infection is a major cause of acute and chronic liver diseases.During the HBV life cycle,HBV hijacks various host factors to assist viral replication.In this research,we find that the HBV regulatory protein X(HBx)can induce the up-regulation of DExH-box RNA helicase 9(DHX9)expression by repressing proteasome-dependent degradation mediated by MDM2 Furthermore,we demonstrate that DHX9 contributes to viral DNA replication in dependence on its helicase activity and nuclear localization.In addition,the promotion of viral DNA replication by DHX9 is dependent on its interaction with Nup98Methods:PART 1:Mechanism of HBx up-regulation of DHX9 expression:(1)Western blot was used to detect the changes of DHX9 expression in human and HBV transgenic mouse liver samples and liver cancer cell models in the presence of HBV infection.To investigate the effect of HBV replication on the expression level of DHX9.HBx protein expression plasmids were transfected in HBV replication cell models to explore the effect and mechanism of HBx protein on DHX9 expression.The CHX(Cycloheximide)time node experiment explores the effect of HBx on the stability of DHX9 protein;the ubiquitination experiment explores whether HBx can inhibit the ubiquitination level of DHX9;the Co-IP experiment explores whether HBx and DHX9 are combined with each other.HBx and MDM2 plasmids were co-transfected in a cell model to investigate whether HBx could inhibit the degradation of DHX9 by MDM2 through the proteasome pathway.Co-IP experiments explored whether HBx can inhibit the regulation of DHX9 ubiquitination by MDM2PART2:Phenotypic and functional study of DHX9 promoting HBV replication:transfer DHX9 siRNAs or expression plasmids into HBV replicated human liver cancer cell models;qPCR and Southern blot were used to detect the effects of knockdown and overexpression of DHX9 on HBV core DNA levels,ELISA assays were performed to measure the levels of HBsAg and qPCR assays were used to detectHBV 3.5 kb RNA levels DHX9 helicase site mutant K417R and nuclear localization deletion mutation NLS were constructed and transfected intoHepAD38 and Huh7 cells.QPCR and Southern blot were used to detect changes in HBV replication level to investigate the effect of helicase activity and nuclear localization on HBV replication levels.In HBV replicated hepatocellular carcinoma model,siRNAs were used to knock down Nup98 or transfected into its overexpression plasmids,and then transfected with DHX9.QPCR and Southern blot were used to detect the changes of HBV core DNA level The effect of Nup98 on DHX9 to promote HB V replication was explored The Nup98 498-920aa mutant was constructed and co-transfected with DHX9 in HepAD38 and Huh7(co-transfected with HBV replication plasmids)cells,and qPCR was used to detect changes in HBV replication levels.The effects of Nup98 on DHX9's promotion of HBV replication were explored after Nup98 lost interaction with DHX9Results:PART 1:Mechanism of HBV up-regulation of DHX9 expression level DHX9 protein levels were significantly up-regulated in liver tissues of HBV transgenic mice,and HBV-infected cell models.HBx up-regulated the expression level of DHX9 among the four proteins expressed by HBV in the liver cancer cell model;and the regulation of DHX9 by HBx occurred at the post-transcriptional level.The CHX protein stability test showed that HBx can extend the half-life of DHX9;ubiquitination experiments showed that HBx can inhibit the ubiquitination of DHX9 Co-IP experiments confirmed that HBx and DHX9 proteins interact with each other and that HBx can inhibit the ubiquitination regulation of DHX9 by MDM2PART2:Phenotype and mechanism of DHX9 promoting HBV replication:In the model of HBV-replicated liver cancer cells,and over-expressing DHX9 can promote HBV replication without affecting the levels of HBV 3.5kb RNA and HBsAg.Similarly,knocking down DHX9 can inhibit HBV replication levels in Huh7 and HepG2-NTCP cells.DHX9 promotes HBV replication depending on its helicase activity and localization in the nucleus.The promotion of viral DNA replication by DHX9 is dependent on its interaction with Nup98Conclusion:After infecting liver cells,HBV activates DHX9 expression to promote virus replication.The specific molecular mechanism is:HBx protein degrades DHX9 by inhibiting the MDM2-mediated ubiquitination pathway and the upregulated DHX9 facilitates viral DNA replication which mainly occurs during the DNA replication.This promotion of viral replication depends on DHX9 helicase activity and localization in the nucleus and the promotion of viral DNA replication by DHX9 is dependent on its interaction with Nup98.