Study On The Mechanism Of ADAR1 Affecting HBV Expression By RNA Editing Of COX18 And MAVS Genes

ObjectiveHepatitis B virus infection is an important health problem worldwide,and China is a high prevalence country.There are nearly 100 million HBV carriers,of whom about 30 million are chronic hepatitis B patients,which is a heavy economic,social and health burden for China.IFNa has antiviral and immunomodulatory effects,and is currently the first-line drug for the treatment of hepatitis B.However,individual differences in response to IFNa are the main problem affecting the clinical use of IFNα.ADAR1 has a wide range of RNA editing functions as an important downstream regulator of interferonα,and it has editing regulatory effects on genes in the host and virus.In our laboratory study,single nucleotide polymorphisms of ADAR1 were found to be associated with the regulation of HBV replication.The aim of this study was to investigate the molecular mechanisms of ADAR1 editing function and regulation of HBV replication,with a view to providing new potential targets for improving HBV treatment efficacy.MethodsFirstly,qPCR and WB were used to detect the expression changes of related genes and proteins in HepG2.2.15 cells after treatment with IFNa and ADAR1 plasmid.Then,the mechanism of ADAR1 regulating the expression of COX18 and MAVS in HepG2.2.15 cells was studied by RIP assay and double luciferase reporter assay.The antiviral effect of MAVS and sh-cox18 was confirmed in HepG2.2.15 cells and hbv-tg mice.The effects of COX 18 on the antiviral signaling pathway of MAVS were studied by Co-IP,fluorescence co-localization and ELISA.ChIP,dual luciferase reporter gene experiment and Co-IP experiment were used to explore the molecular mechanism of HBc regulating MAVS expression.Transcription factor databases were used to predict binding factors in the MAVS promoter region.In CHB patients,the association between MAVS gene polymorphism and IFNa response was confirmed by association analysis.Results1)ADAR1 could enhance the expression of COX 18 at mRNA and protein levels(P<0.05).2)The site of chr4:73057732 in 3’UTR of COX18 edited by ADAR1 enhanced the mRNA stability of COX18 via HuR mediated post-transcriptional regulation(P<0.05).3)COX18 promoted the replication of HBsAg,HBV-DNA and other markers;the expression of COX18 decreased the HBV markers in HepG2.2.15 cells and full-length HBV-tg mice(P<0.05).4)The interaction between COX18 and MAVS inhibited the binding of MAVS to TRAF3,resulting in the phosphorylation attenuation of IRF3 and the reduction of IFNβ production(P<0.05).5)IFNa down-regulated the expression of MAVS at mRNA and protein levels by improving the RNA editing function of ADAR1 through HuR mediated post-transcriptional regulation(P<0.05).6)MAVS inhibited the replication levels of HBsAg,HBV-DNA and other markers in C57BL/6j-tg(A1b1HBV)44Bri/J mice and full-length hbv-tg mice,and the replication effect of HBsAg,HBV-DNA and other markers was significantly enhanced by the combination of IFN blocking(P<0.05).7)HBc inhibits the activity of MAVS promoter by interacting with transcription factor SP1 to regulate the expression of MAVS(P<0.05).8)CHB patients with the A allele at rs3746662 on the 3’UTR of the MAVS gene had higher MAVS expression in liver paraneoplastic tissues and a higher response rate to IFNa treatment for hepatitis B(P<0.05).Conclusions1)RNA editing of ADAR1 influences the mRNA stability of COX18 and MAVS through HuR mediated post-transcriptional regulation and influences the expression levels of COX18 and MAVS.2)Effective interference with COX 18 mRNA can reduce the HBV replication level in HepG2.2.15 cells and full-length hbv-tg mice.COX18 inhibits the mavs-mediated antiviral signaling pathway in cells by interacting with MAVS.3)The expression of MAVS was inhibited by IFN alpha through ADAR1 in HepG2.2.15 cells,the combination of MAVS and IFN alpha in two HBV-transgenic mouse models showed a more significant anti-HB V response than that of IFN alpha alone.4)HBc regulates the activity of MAVS promoter through transcription factor SP1 and inhibits the expression of MAVS;5)Polymorphisms in MAVS gene affects the response of interferon therapy to chronic hepatitis B in Han Chinese.The above studies showed that ADAR1 editing enzyme can affect HBV replication by regulating the expression of COX18 and MAVS,providing a new understanding for the treatment of hepatitis B based on MAVS and COX18.