Effects of a frequent double-nucleotide basal core promoter mutation and its putative single-nucleotide precursor...

The basal core promoter (BCP) of hepatitis B virus (HBV) directs the transcription of both precore RNA and core RNA which code for e antigen (HBeAg) and core antigen, respectively. A double mutation in the BCP which converts nucleotide (nt) 1762 from A to T and nt 1764 from G to A is frequently observed in patients with chronic hepatitis B. We recently demonstrated that this double mutation prevented the binding of a liver-enriched factor (LEF) to the BCP, suppressed only precore RNA transcription (and hence HBeAg expression), and enhanced progeny virus production. In order to understand the mechanism for the selection of this frequent double mutation, we have extended our previous studies to further characterize LEF and to compare the effects of this double-nucleotide mutation (M1) with each single-nucleotide mutation at nt 1762 (M2) and nt 1764 (M3). Our results indicate that LEF is likely composed of a heterodimer formed between the transcription factor chicken ovalbumin upstream promoter-transcription factor (COUP-TF) and an unidentified liver-enriched factor. Further studies reveal that both M1 and M2 prevent the binding of LEF to the BCP, suppress only precore RNA transcription, and increase the efficiency of progeny virus synthesis. In contrast, M3 retains some LEF binding activity, does not suppress HBV RNA transcription, and reduces slightly the efficiency of virus progeny synthesis. The reduced ability of M3 to replicate indicates that it has no selection advantage in itself at the level of the infected hepatocyte. In spite of its enhanced replication rate, M2 is rarely detected in HBV patients. This indicates the involvement of factors other than intracellular replication rates in the selection of these virus variants in the infected individual.