Core protein mutations are key determinants of hepatitis B virus replication during the hepatitis B e antigen-negative stage.

The natural history of chronic hepatitis B virus (HBV) infection comprises distinct stages resulting from virus-host interactions. A late and pivotal event in this process is hepatitis B e antigen (HBeAg) seroconversion, marked by the abrogation of HBeAg expression, a significant reduction in viral load and the accumulation of mutations throughout the genome, particularly within the Core region. While HBeAg loss is associated with mutations in the basal core promoter and preCore regions, these alone do not account for the decreased viral load observed during this stage. To elucidate the contribution of Core variability to HBV replicative capacity, we engineered replication-competent chimeric genomes by reciprocally exchanging the core gene between a WT clone and three HBeAg-negative patient-derived isolates. These constructs were functionally characterized in Huh-7 cells to assess replication intermediates, antigen expression and viral transcriptional activity. Our findings demonstrate that mutations within the Core protein can either impair or enhance HBV replication, depending on their specific mutational patterns. Importantly, all viral replication intermediates were restored to WT levels when the WT Core protein was introduced into the HBeAg-negative genomes. Strong positive correlations between covalently closed circular DNA (CCC DNA) and other viral markers indicate that the Core protein exerts its regulatory effect primarily through regulation of CCC DNA levels. Notably, the absence of Core expression increased CCC DNA transcriptional activity, supporting a repressive role of the WT Core protein in gene expression. Collectively, these findings highlight the pivotal regulatory role of Core protein mutations in modulating HBV replication dynamics and gene expression during the HBeAg-negative phase and underscore its potential as a promising target for novel antiviral strategies.