Journal zyxwvutsrqp of Gastroenterology and zyxwvutsrqponm Heparology zyxwvutsrq (1993) 8, S110-113 ORIGINAL ARTICLE zyx Regulation of core promoter of hepatitis B virus C-H. YUH AND L-P. TING Graduate Institute of Microbiology and Immunology, National Yang-Ming Medical College, Shih-Pai, Taipei, Taiwan, Republic of China Abstract In order to understand the cycle and disease process of hepatitis B virus (HBV) at the molecular level, the regulation of viral gene expression was studied. This paper reports the characterization of HBV core promoter: two 3.5 kb transcripts, the precore and the pregenomic RNA are made from it. A short sequence (from nt1744 to 1851, referred to as the basic core promoter or BCP) is identified, that is sufficient to direct the correct initiations of both precore and pregenomic messages. Sequences upstream of BCP (from nucleotide, nt1636 to 1744, referred to as the core upstream regulatory sequence or CURS) have a strong stimulatory effect on BCP. Deletion analysis of CURS suggests that it contains multiple regulatory elements that control BCP in an interactive manner. The CURS stimulates BCP in a position- and orientation- dependent manner. Therefore, it is unlikely that the effect is mediated through the enhancer zy I1 that has been co-localized to the same sequence. Key words: hepatitis B virus, viral gene expression, transcriptional regulation. INTRODUCTION Hepatitis B virus (HBV) is a small DNA virus with a partially double-stranded 3.2 kilobase (kb) genome. The transcription of this genome produces the longer (pre- core) and shorter (pregenomic) 3.5 kb RNA. The produc- tion of these RNA is of special interest in that they have dual functions: the pregenomic RNA is packaged into nucleocapsids along with the viral polymerase and serves as the template for viral genome replication, while both of them encode such important viral proteins as core pro- tein, polymerase (by pregenomic RNA) and e antigen (by precore RNA).’-4 The regulation of expression of these messages is the pivotal event that governs the viral replication cycle. The longer precore RNA initiates at nucleotide (nt) 1785-1786 and 1791-1793, while the shorter pregenomic RNA starts at nt1818-1820.4.5 Both 3.5 kb RNA end at the one and only polyadenylation site in the HBV genome. In this study, the promoter region which controls the expression of precore and pregenomic RNA is examined. METHODS The human hepatoma cell line HepG2 weas cultured in Dulbecco’s modified Eagle’s medium (Flow Laborato- ries, North Ryde, NSW, Australia) supplemented with 10% fetal calf serum (Boehringer Biochemical, Man- nheim, Germany), 100 IU of penicillin/mL, 100 mg of streptomycin/mL, 1% non-essential amino acids, 25 mg of fungizone/mL and 2 mmol/L L-glutamine at 37°C in a z 5% CO, atmosphere. Cells were transfected with plas- mids containing the chloramphenicol acetyltransferase (CAT) gene constructs by the calcium phosphate precipi- tation method.6 Since the expression of transfected plas- mid depends on their supercoiled form, all plasmids used in one set of experiments were simultaneously prepared, checked for supercoil form, aliquoted in 100 pL and stored in 70% ethanol. Each aliquot was used only once. Each set of experiments was performed with two different preparations of plasmids and repeated two to three times for each preparation. Furthermore, 5 pg of target DNA and 1 pg of target with 4 pg of carrier DNA were used for the transfection, respectively, to compare the results. CAT assays were performed by the method of Gorman et al.’ with modifications as previously described.’ The CAT activity was normalized using the CAT activity value of pSV,CAT, in which the CAT gene is driven by the SV40 early promoter and enhancer, as 100%. Where CAT activity was high, the cell lysate was serially diluted before using the CAT assay. Furthermore, for different transfection experiments the CAT activities were propor- tional to the amount of the input DNA, suggesting that the transfected DNA templates manifest transcription activities that faithfully reflect their promoter strength. Correspondence: Ling-Pai Ting, Graduate Institute of Microbiology and Immunology, National Yang-Ming Medical College, Shih-Pai, Taipei 11221, Taiwan, Republic of China. Accepted for publication 17 November 1992.