Analysis of upstream region of hepatitis B virus core gene using in vitro transcription system

Transcription of the core (C) gene of hepatitis B virus DNA (HBV-DNA) was studied by an in vitro transcription system using nuclear extracts of human liver cell (HepG2) and non-liver cell (HeLa) origins. RNA polymerase II-dependent run-off transcription of 3.5-kb (C) mRNA was observed in both nuclear extracts; but the efficiency was much higher in the HepG2 nuclear extract. Analysis of run-off transcripts using upstream deletion mutants of HBV-DNA demonstrated that there are two transcription start sites located at approximately nucleotide numbers (nt) 1,797 +/- 5 and 1,815 +/- 5. This analysis also suggested that the minimum core promoter sequence and a cis-acting and liver-specific element for C mRNA transcription are located in the downstream region from -80 and -110 (HincII site) of transcription start sites, respectively. DNA-binding protein assays using synthetic double-stranded oligonucleotide probes corresponding to three regions in the upstream region (nt from 1,401 to 1,760) of transcription start sites revealed that there are some liver cell-specific and non-specific DNA-binding proteins in both nuclear extracts. The amount of those proteins was generally higher in the HepG2 nuclear extract. However, no obvious correlation was observed in the present study between the presence of DNA-binding proteins and transcription activity of nuclear extracts in our system. The possible causes of this discrepancy are discussed.