Abstract
The hepatitis B X protein (HBx) plays a role in the epigenetic regulation of hepatitis B virus (HBV) replication. This study investigated the effects of HBx mutations on HBV transcription and the recruitment of HBx, histone acetyl-transferase P300 and histone deacetylase 1 (HDAC1) to circularized HBV DNA (which resembles covalently closed circular DNA [cccDNA]). Compared with wild type, majority of mutants had lower levels of intracellular HBV RNA (44–77% reduction) and secretory HBsAg (25–81% reduction), and 12 mutants had a reduction in intracellular encapsidated HBV DNA (33–64% reduction). Eight mutants with >70% reduction in HBV RNA and/or HBsAg were selected for chromatin immunoprecipitation analysis. Four HBx mutants with mutations in amino acid residues 55–60 and 121–126 had a lower degree of HBx-cccDNA association than wild type HBx (mean % input: 0.02–0.64% vs. 3.08% in wild type). A reduced association between cccDNA and P300 (mean % input: 0.69–1.81% vs. 3.48% in wild type) and an augmented association with HDAC1 (mean % input: 4.01–14.0% vs. 1.53% in wild type) were detected. HBx amino acid residues 55–60 and 121–126 may play an important role in HBV transcription regulation, via their impeded interaction with cccDNA and altered recruitment of histone modifying enzymes to cccDNA.
Highlights
The hepatitis B virus (HBV) covalently closed circular DNA is the template for transcription, which produces HBV pregenomic RNA and mRNAs
The detectability of HBV RNA at the time points studied, with its peak at 72 hours post-transfection (Fig. 1B), suggesting that this circularized HBV DNA may act as a “closed circular DNA (cccDNA)-like” template for HBV transcription in our system[7,8]
HBxStop mutation resulted in a significant reduction in HBV RNA levels, which were restored to almost wild type level when complement with pcHBxWT
Summary
The hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is the template for transcription, which produces HBV pregenomic RNA and mRNAs. From the HBV mRNAs, all viral proteins, including the hepatitis B surface antigen (HBsAg), hepatitis B core protein, polymerase, and hepatitis B X protein (HBx), are synthesized. Belloni and colleagues have shown that HBx is associated with the cccDNA minichromosome and other histone modifying enzymes and plays a role in the regulation of HBV transcription[8]. In an HBx-null strain, HBV transcription level is reduced, but can be restored by complementation with an HBx expression plasmid, suggesting that HBx is required for efficient HBV transcription[3] Another earlier study has identified that the HBx C-terminal trans-activation domain is responsible for efficient HBV replication[2]. We attempted to identify the HBx aa residues that play an important role in HBV replication and its association with cccDNA. The effect of HBx mutations on the recruitment of histone acetyl-transferases and histone deacetylases towards cccDNA was studied
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