Phenomenon And Mechanism Research On LINE-1 Suppression Mediated By Hepatitis B Virus X Protein

Background and aims:Human hepatitis B virus(HBV)is a world-widely spreading pathogen.HBV infects human liver cells and causes chronic infection,which may lead to hepatitis,cirrhosis,and hepatocellular carcinoma(HCC).Chronic hepatitis B(CHB)is a global public health burden.Hepatitis B virus X protein(HBx),encoded by HBV,is a trans-regulatory protein,which has pleiotropic functions including supporting viral gene expression and replication,interfering with several cellular signaling pathways,activating a number of signal transduction pathways,medicating protein degradation etc.These properties of HBx have important biological implications in HBV infection,chronic disease,and the occurrence of tumor.Accordingly,studies on HBx protein are attractive and promising.Long interspersed element type 1(LINE-1)is the only known active non-LTR endogenous retroelement that can replicate autonomously in human genome,and has impact on genome evolution,genome stability and the innate immune system.During the process of LINE-1 retrotransposition,LINE-1 can introduce damages to genomic(or simply double-stranded)DNA and activate the interferon signal system,which could have negative impacts on HBV infection and replication.HBV may use its own protein(s)to suppress LINE-1 retrotransposition.As a multifunctional regulator,HBx can bind RNA and DNA via its C-terminal region,suggesting that HBx protein may interact with LINE-1.The study focuses on HBx and LINE-1,to investigate whether HBx could regulate LINE-1 retrotransposition,and to further explore the mechanisms and biological significance of the interaction.Methods:1.To confirm the interaction between HBx and LINE-1 by using LINE-1retrotransposition assay,Flow cytometry and Western blotting.2.To explore the mechanisms of the interaction between HBx and LINE-1 by using Western blotting,Luciferase,co-IP,quantitative real-time PCR to confirm whether HBx regulate LINE1 the promoter activity,LINE-1 RNA,LINE-1 protein,LINE-1 RNP or the ability of integration.3.Identify the key functional regions of HBx that affect LINE-1 activity by construct different truncated mutants of HBx and the methods of above.4.To observe the impact of HBx and LINE-1 interaction on genome integrity by using Comet assay.And to explore the interplay among HBx,LINE-1 and the innate immune system by using ELISA,Western blotting,Luciferase,qRT-PCR etc.Results:1.HBx suppresses LINE-1 retrotranspositionVia a widely used,EGFP-based LINE-1 retrotransposition assay,it was determined that HBx potently suppresses LINE-1 replication activity in a dosage-dependent manner.2.HBx interacts with LINE-1 RNA and sabotages its stabilityMechanism research on HBx-mediated LINE-1 suppression suggested that,HBx does not affect the promoter activity of LINE-1 5’-UTR.Instead,HBx interacts with and destabilizes LINE-1 RNA,leading to reduced LINE-1 protein expression and LINE-1retrotransposition potency.3.HBx 120-141 is important for HBx-mediated LINE-1 suppressionComparing to wild type HBx,HBx Δ120-141 could no longer interact with LINE-1RNA,which results in compromised potency of HBx Δ120-141 on LINE-1 regulation.4.HBx-mediated LINE-1 inhibition is of potential biological significanceResults from the COMET assay indicated that,by regulating LINE-1 activity,HBx protects genomic DNA from LINE-1-induced damaging;meanwhile,examination on endogenous IFN levels suggested that,HBx also represses LINE-1-mediated innate immune activation.Conclusion:As reported in this study,through the recognition of both ORF1 and ORF2 regions,HBx interacts with and destabilizes LINE-1 RNA,leading to compromised expression of LINE-1 proteins and suppressed retrotransposition of LINE-1.HBx 120-141 is important for HBx to regulate LINE-1 activity,as HBx Δ120-141 could no longer recognize or interact with LINE-1 RNA.Further examinations indicated that,by inhibiting LINE-1,HBx protects the stability of genomic(or other double-stranded)DNA,and lowers endogenous IFN levels during HBV replication.Therefore,this study has revealed LINE-1 suppression as a novel function of HBx,uncovered associated mechanisms,unveiled its potential biological significance,and discussed its possible impact on HBV infection and replication.Subsequent investigation will further expose HBx’s function(s)in HBV replication,and thus provide new insights or even new perspective on drug development targeting HBx and the optimization of anti-HBV treatment strategies.