Summary auto-generated
This study investigates how hepatitis B virus (HBV) mutations in the basal core promoter (BCP) region affect viral gene expression and replication. The researchers compared a frequently observed double mutation (positions 1762 and 1764) with its two putative single-mutation precursors. Using electrophoretic mobility shift assays, they identified that the liver-enriched factor (LEF) binding to the BCP involves the transcription factor COUP-TF or a closely related protein. The double mutation and the single mutation at position 1762 both prevent LEF binding, suppress precore RNA transcription (reducing HBeAg expression), and increase viral progeny production. However, the single mutation at position 1764 retains some LEF binding capacity, does not suppress precore RNA transcription, and actually reduces virus replication efficiency. These findings suggest that while the position 1762 mutation alone enhances viral replication similarly to the double mutation, factors beyond intracellular replication rates determine which variant dominates during chronic infection, explaining why the double mutation is preferentially selected over its single-mutation intermediates in infected patients.
Key findings
- The basal core promoter double mutation (nt 1762 A→T and nt 1764 G→A) prevents binding of a liver-enriched factor (LEF) containing COUP-TF, suppressing only precore RNA transcription and HBeAg expression while enhancing viral replication.
- The single mutation at position 1762 alone mimics the double mutation's effects on LEF binding, precore RNA suppression, and enhanced virus production, yet is rarely detected in patients.
- The single mutation at position 1764 retains LEF binding activity and does not suppress precore RNA transcription or enhance viral replication, providing no selection advantage.
- COUP-TF or a closely related protein is identified as a component of the LEF complex that regulates precore RNA transcription.
- Factors beyond intracellular replication efficiency appear to drive selection of the double mutation variant over single-mutation intermediates in chronic HBV infection.
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Abstract
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.