Roles of various regions of the HBV core promoter in the transcription of precore and pregenomic RNA

To determine the potential functions of different regions in the HBV core promoter, several mutants containing B3 and/or C deletion (p3.6IIΔB3, p3.5IIΔB3, p3.6IIΔC, p3.5IIΔB3C) have been constructed based on the two HBV transcription units (p3.6II and p3.5II) which contained ENII/Cp and Cp immediate upstream of the linear HBV genome, respectively. The functional effects of deletions were assessed with respect to antigen production, viral mRNA synthesis, and viral DNA replication. When HepG2 cells were transfected with C deletion mutants, HBeAg became undetectable in the supernatant while the expression of HBcAg in the cell lysate was retained. Consistently, precore RNA disappeared but the synthesis of pregenomic RNA and viral DNA replication could still be observed. On the other hand, deletions of B3 fragment reduced the expression of HBeAg and HBcAg significantly, while maintained the synthesis of precore RNA, pregenomic RNA and viral progeny DNA. The results strongly suggest that both fragment C and fragment D serve as minimal promoter elements for the transcription initiation of 3.5 kb RNAs. Notably, the D fragment which contains a novel nuclear factor binding site close to the start site of pregenomic RNA, is critical for the basal transcription of pregenomic RNA, which has not been reported previously. B3 plays an important role in the efficient transcription of precore and pregenomic RNAs, but is not indispensable for their basal transcription.