| Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC). Approximately 60-80% of world's HCC is related to HBV, and it is the third most common cause of cancer death in Asia-Pacific region. Almost 400 million people are chronically infected with HBV and one-third was likely to die of complications of cirrhosis, including liver failure and HCC. As there is a shortage of effective curative treatments, detection and prognosis of the risk of cancer development will be essential to improve survival of patients with chronic HBV infection.;A case-control study was previously carried out to identify HCC-associated genomic markers on HBV. Some of them are clustered at the preS1 and X promoter regions of HBV genotype B and core promoter of HBV subgenotype Cs. The functional significance of these markers to the virus was investigated in our study. Our result showed that one of those markers, the G1613A mutation on core promoter, can significantly increase the promoter activity in a genotype-dependent manner and the effect is reversible by the A-to-G back mutation. We have established an in vitro full-length HBV genome transfection system and the result suggested that the G1613A mutation suppressed the e antigen (HBeAg) secretion and enhanced virus DNA production by downregulating the precore (preC) mRNA transcription. In consistence to the clinical study, the mutation was associated to serum HBV DNA level higher than 6 log copies/1M in female HBV carriers in a univariate analysis. In addition, we demonstrated that the G1613A mutation is a hot spot mutation situated on the negative regulatory element (NRE) on the core promoter in an alignment analysis. To further investigate the molecular mechanism of the mutation, two unknown protein complexes had been shown to bind on the NRE. They showed different binding affinity to the G1613-wild-type and A1613-mutant NRE sequence. Moreover, we showed that in vitro synthesized RFX1 protein could bind to the mutated NRE probe at a higher affinity than that to wild-type NRE probe. Overall, our result suggests that the G1613A mutation exerts its effect by differential binding to some proteins via the NRE region. Studying the mechanism of the mutations may provide insights to the viral pathogenesis and HBV-associated HCC, which has long been a health burden in Asia-Pacific countries. |
