Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function

Abstract

HBV cccDNA, the template for transcription of all viral mRNAs, accumulates in the nucleus of infected cells as a stable episome organized into minichromosomes by histones and non-histone viral and cellular proteins. Using a cccDNA-specific chromatin immunoprecipitation (ChIP)-based quantitative assay, we have previously shown that transcription of the HBV minichromosome is regulated by epigenetic changes of cccDNA-bound histones and that modulation of the acetylation status of cccDNA-bound H3/H4 histones impacts on HBV replication. We now show that the cellular histone acetyltransferases CBP, p300, and PCAF/GCN5, and the histone deacetylases HDAC1 and hSirt1 are all recruited in vivo onto the cccDNA. We also found that the HBx regulatory protein produced in HBV replicating cells is recruited onto the cccDNA minichromosome, and the kinetics of HBx recruitment on the cccDNA parallels the HBV replication. As expected, an HBV mutant that does not express HBx is impaired in its replication, and exogenously expressed HBx transcomplements the replication defects. p300 recruitment is severely impaired, and cccDNA-bound histones are rapidly hypoacetylated in cells replicating the HBx mutant, whereas the recruitment of the histone deacetylases hSirt1 and HDAC1 is increased and occurs at earlier times. Finally, HBx mutant cccDNA transcribes significantly less pgRNA. Altogether our results further support the existence of a complex network of epigenetic events that influence cccDNA function and HBV replication and identify an epigenetic mechanism (i.e., to prevent cccDNA deacetylation) by which HBx controls HBV replication.

Fig. 1.

HBx is involved in viral replication, and it is required for HBV pregenomic RNA transcription. (A) HepG2 cells were transfected with monomeric linear full-length WT or HBx mutant HBV adw genomes, and cytoplasmatic HBV core particles were isolated at the indicated time points after transfection. (Upper) Southern blot analysis of HBV DNA replicative intermediates. (Lower) Densitometric quantification of HBV replicative intermediates. Signal intensity of the single-strand (SS) band underneath the linear double-stranded (DS) HBV DNA band was quantified with Quantity One 1-D Analysis Software (BioRad Laboratories). The band corresponding to the DS HBV DNA was not included in the quantitative analysis, because this DNA may be partially derived from transfected input DNA. The optical density value of the SS band at 48 h in WT replicating cells is set at 1.0. Data are expressed as relative arbitrary units (mean + SD) from three independent experiments. OC, open circular duplex HBV DNA; DS, double-strand; and SS, single-strand HBV DNA replicative intermediates. (B) cccDNA accumulation in HepG2 cells transfected with WT or HBx mutant HBV genomes. Real-time quantitative PCR analysis was performed using selective cccDNA primers to amplify cccDNA and beta-globin primers to normalize the DNA samples. Results are expressed as number of cccDNA copies per cell (mean + SD) from four independent experiments. (C) HepG2 were transfected with WT or with HBx mutant HBV genomes and mRNAs were extracted 48 and 96 h posttransfection as described in the Materials and Methods section. Specific primers were used to quantitate the HBV pregenomic RNA, and GAPDH amplification was used to normalize for equal loading of each RNA sample. Results are expressed as number of cccDNA copies per cell (mean + SD) from three independent experiments. pgRNA, HBV pregenomic RNA.

Fig. 2.

The viral transactivator HBx is recruited onto the cccDNA in HBV replicating cells. (A, Upper) Cross-linked chromatin from HepG2 cells transfected with monomeric linear full-length HBV DNA was immunoprecipitated with the relevant control IgG or specific anti-HBx, anti-AcH3, and anti-AcH4 antibodies and analyzed by PCR with HBV cccDNA selective primers. (Lower) ChIPed samples were analyzed by PCR using primers specific for the cyclin A2 coding region as a negative control. (B) Kinetics of HBx recruitment onto the HBV cccDNA. (Upper) ChIP analysis was performed on chromatin from HepG2 cells cotransfected with monomeric linear full-length WT HBV DNA and an HA-tagged HBx expression vector using the relevant control IgG or specific anti-HA tag and anti-AcH3 specific antibodies. (Lower) Exogenously expressed HBx is detected by anti-HA immunoblotting. Tubulin levels detected by immunoblotting were used to normalize equal loadings from lysate samples.

Fig. 3.

HBx modulates the epigenetic control of cccDNA function by affecting the recruitment of chromatin modifying enzymes. (A) HepG2 cells were transfected with monomeric linear full-length HBV DNA. (Upper) Chromatin prepared 48 h after transfection was immunoprecipitated with the relevant control IgG or specific anti-H4, anti-AcH3, anti-AcH4, anti-p300, anti-CBP, anti-PCAF, and anti-E2F1 antibodies and analyzed by PCR with HBV cccDNA selective primers. (Lower) Chromatin prepared 96 h posttransfection was immunoprecipitated with the relevant control IgG or specific antibodies to the class I HDAC1 and class III hSirt1 histone deacetylases. ChIPed DNA was analyzed by PCR with HBV cccDNA selective primers. (B–D) Chromatin was prepared from HepG2 cells transfected with WT or HBx mutant monomeric linear full-length genomes and immunoprecipitated with the relevant control IgG or a specific anti-HBx (B), anti-AcH4 (C), anti-p300, anti-HDAC1, anti-hSirt1, and anti-E2F1 (D) antibodies. Immunoprecipitated chromatin was analyzed by semiquantitative PCR (Upper) and real-time quantitative PCR (Lower) with HBV cccDNA selective primers. Light gray columns, mock; gray columns, WT HBV; black columns, mt HBx. Results are expressed as RT-PCR arbitrary units (mean + SD) from four independent experiments.

Fig. 4.

Schematic representation of cccDNA-bound histones acetylation status and the recruitment of chromatin modifying enzymes onto the viral minichromosome in relation to viral replication and HBx status. In cells replicating an HBx mutant HBV, cccDNA-bound histones are hypoacetylated, the recruitment of the p300 acetyltransferase is severely impaired, whereas the recruitment of the histone deacetylases hSirtl and HDAC1 is increased. This behavior is similar to what is observed in the later phase (96–144 h) of the in vitro viral replication cycle in our model system and mimicks the situation found in the liver of anti-HBe patients with low viremia (30).