Impact of a naturally occurring hepatitis B virus genotype D-specific core-promoter mutation on viral replication

Hepatitis B virus (HBV) infects human populations worldwide. HBV strains are classified into 10 genotypes, of which the HBV genotype D (HBV/D) infection is particularly prevalent in several countries. The HBV core promoter regulates viral replication and transcription, and the naturally occurring A1762T/G1764A double mutation (CP1) in the core promoter accelerates HBV replication. Previous clinical studies showed that a new core-promoter mutation, G1764T/C1766G (CP2), is frequently observed in genomes containing the G1757A substitution, which is unique to HBV/D; however, CP2 is not observed in genomes containing the 1762T/1764A double mutation. In this study, we found that the CP2 mutation dramatically increased viral replication and transcription efficiency in two cell lines; the degree of stimulation was comparable to that induced by CP1. Introduction of the 1757A substitution reduced the increase in viral replication induced by the CP1 mutation. By contrast, the addition of the 1757A substitution significantly increased the effect of the CP2 mutation. The transcriptional activity of CP1 was decreased by the 1757A substitution, due to a reduction in HNF1 binding affinity, suggesting that 1757G is an important component of the HNF1 binding consensus sequence. The HBV/D-specific CP2 mutation creates a binding site for the transcription factor HNF3, thereby increasing its transcriptional activity. HBX proteins containing substitutions reflecting the two types of core-promoter mutations did not affect the efficiency of viral replication. Therefore, we hypothesize that the introduction of the CP2 mutation represents a survival strategy for HBV/D, allowing it to escape the effect of the 1757A substitution.