title: The G1613A mutation in the HBV genome affects HBeAg expression and viral replication through altered core promoter activity.
abstract: Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC). Previously, we demonstrated that the G1613A mutation in the HBV negative regulatory element (NRE) is a hotspot mutation in HCC patients. In this study, we further investigated the functional consequences of this mutation in the context of the full length HBV genome and its replication. We showed that the G1613A mutation significantly suppresses the secretion of e antigen (HBeAg) and enhances the synthesis of viral DNA, which is in consistence to our clinical result that the G1613A mutation associates with high viral load in chronic HBV carriers. To further investigate the molecular mechanism of the mutation, we performed the electrophoretic mobility shift assay with the recombinant RFX1 protein, a trans-activator that was shown to interact with the NRE of HBV. Intriguingly, RFX1 binds to the G1613A mutant with higher affinity than the wild-type sequence, indicating that the mutation possesses the trans-activating effect to the core promoter via NRE. The trans-activating effect was further validated by the enhancement of the core promoter activity after overexpression of RFX1 in liver cell line. In summary, our results suggest the functional consequences of the hotspot G1613A mutation found in HBV. We also provide a possible molecular mechanism of this hotspot mutation to the increased viral load of HBV carriers, which increases the risk to HCC.
authors.affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China.
authors.forename: Man-Shan
authors.initials: MS
authors.lastname: Li
authors.forename: Terrence Chi-Kong
authors.initials: TC
authors.lastname: Lau
authors.forename: Sophie Ka-Ping
authors.initials: SK
authors.lastname: Chan
authors.forename: Chi-Hang
authors.initials: CH
authors.lastname: Wong
authors.forename: Patrick Kwok-Shing
authors.initials: PK
authors.lastname: Ng
authors.forename: Joseph Jao-Yiu
authors.initials: JJ
authors.lastname: Sung
authors.forename: Henry Lik-Yuen
authors.initials: HL
authors.lastname: Chan
authors.forename: Stephen Kwok-Wing
authors.initials: SK
authors.lastname: Tsui
mesh_terms: D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000175:diagnosis / Q000209:etiology / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000235:genetics* / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D006801:Humans; D008113:Liver Neoplasms / Q000175:diagnosis / Q000209:etiology / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008297:Male; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D000071701:Regulatory Factor X Transcription Factors; D000071716:Regulatory Factor X1; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D019562:Viral Load; D014771:Virion; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; C000605537:RFX1 protein, human; D000071701:Regulatory Factor X Transcription Factors; D000071716:Regulatory Factor X1; D015534:Trans-Activators; D014157:Transcription Factors; D008156:Luciferases