Fig 1.
IL-6 inhibits HBV replication and cccDNA transcription in HepG2 cells transfected with HBV monomers.
HepG2 cells were transfected with monomeric linear full length wild type HBV adw (genotype A) genomes and were harvested after 48 hours. A) HepG2 transfected cells were left untreated or exposed to 5, 10, 20, 50 ng/ml of rIL6. The 3.5Kb/pgRNA species were quantified using selective primers and probes that do not detect the S [2.1 Kb], pre-S [2.4 Kb] or HBx [0.7 Kb] HBV RNAs. B) HBV DNA was extracted from viral particles isolated from the medium of untreated and rIL6 treated HepG2 transfected cells and quantified by real time qPCR using primers annealing to the S region. C). Cytoplasmic HBV core particles were isolated from untreated and IL6 treated transfected cells and total core particles associated HBV DNA was quantified as in B). The β-globin housekeeping gene was used to normalize the DNA samples. D) RNAs were isolated from untreated and IL6-treated HepG2 transfected cells. The 3.5Kb/pgRNA species were quantified as described in A). To evaluate total HBV RNA levels (corresponding to S [2.1 Kb], pre-S [2.4 Kb], and 3.5Kb-pregenome mRNAs) we used primers and probes that anneal to the S region and detect the S [2.1 Kb], pre-S [2.4 Kb] and 3.5Kb-pregenome but not the HBx [0.7 Kb] HBV RNAs. The amount of pre-S/S RNA was estimated by subtracting the 3.5Kb/pgRNA quantity from the total HBV RNA amount. hG6PDH mRNA amplification was used to normalize for equal loading of each RNA sample. All results in A-D) are expressed as arbitrary units and the histograms show the mean from three independent experiments; bars indicate S.D. E) Northern blot analysis of untreated and IL6-treated HepG2 transfected cells (48 hours; 20 ng/ml). 28S/18S rRNAs were used as an internal control for sample loading. 5 and 1.25 μg of RNAs extracted from the HepG2-derived 2.2.15 cell line were used as HBV positive loading controls. Figures represent the relative intensity of the 3.5Kb/pgRNA band normalized to the 28S rRNA. Right Panel: Densitometric quantification of HBV 3.5Kb/pgRNA was performed using the open source ImageJ software. F) cccDNA was extracted from the nuclei of untreated and IL6-treated HepG2 transfected cells. qPCR analysis was performed using cccDNA selective primers and β-globin primers to normalize the DNA samples. Results are expressed as in A-D). F-H) Cross-linked chromatin is extracted from HepG2 cells transfected with monomeric linear full-length HBV DNA and treated for 48 hours with rIL6. The cross-linked chromatin was immunoprecipitated with a relevant control IgG or specific anti-H3 (Fig 1G), anti-AcH3 (Fig 1H, left panel), anti-HDAC1 (Fig 1H, right panel) antibodies. Immunoprecipitated chromatin samples were analyzed by real time PCR with HBV cccDNA selective primers. ChIP results are expressed as Fold Induction (FI) of the % of Input and the histograms show the mean from three independent experiments; bars indicate S.D. * 0,01 ≤ P < 0,05; ** 0,001 ≤ P < 0.01; *** P < 0,001.
Fig 2.
IL-6 inhibits cccDNA transcription activity in HBV infected NTCP-HepG2 cells.
HepG2-NTCP cells were infected with 6 × 102 genome equivalents/cells of HBV in and treated with rIL6 for 48 hours at day 10 post-infection. A) Cytoplasmic HBV core particles were isolated from untreated and IL6 treated infected cells and total core particles associated HBV DNA was quantified as described in the Legend to Fig 1C). B) RNAs were isolated from untreated and IL6-treated HepG2-NTCP infected cells. The 3.5Kb/pgRNA and the pre-S/S RNA were quantified and described in the legend to Fig 1D. C) cccDNA was extracted from the nuclei of untreated and IL6-treated infected cells. qPCR analysis was performed using cccDNA selective primers and β-globin primers to normalize the DNA samples. All results in A-C) are expressed as arbitrary units and the histograms show the mean from three independent experiments; bars indicate S.D. D) Cross-linked chromatin is extracted from the nuclei of NTCP-HepG2 cells treated or not with with rIL6 for 48 hours at day 10 post-infection. The cross-linked chromatin was immunoprecipitated with a relevant control IgG or specific anti-AcH3 and anti-HDAC1 antibodies. ChIPs were analyzed and the results expressed as described in the Legend to Fig 1F. * 0,01 ≤ P < 0,05; ** 0,001 ≤ P < 0.01; *** P < 0,001.
Fig 3.
IL6 decreases HNF1α and HNF4α occupancy on the cccDNA.
A) Anti-HNF1α and anti-HNF4α cccDNA ChIP assay. HepG2 transfected cells were treated with rIL6 for 48 hours. Immunoprecipitated chromatin samples were analyzed by real time qPCR with HBV cccDNA selective primers (see Legend to Fig 1). ChIP results are expressed as Fold Induction (FI) of the % of Input and the histograms show the mean from three independent experiments; bars indicate S.D. B) Cellular RNAs were extracted from untreated and IL6 treated HepG2 cells. HNF1α and HNF4α were quantified by SYBR Green real time PCR. C) Nuclear extracts were analyzed by western blot. 30μg of nuclear proteins were analyzed for HNF1α, HNF4α, and β-lamin as control. D) Haptoglobin gene expression was measured by SYBR Green real time PCR. β-actin amplification was used in B) and D) to normalize the RNA samples. E) HepG2 cells were transfected with control or HNF1α or HNF4α siRNA pools. After 48 hours pgRNA was extracted and quantified as in Fig 1D.
Fig 4.
Modulation of P-STAT3 chromatin binding by IL6.
In upper panel, A) Anti-STAT3 and anti-P-STAT3 chromatin immuno-precipitations were performed as in Fig 2A and analyzed with cccDNA specific primers (see Legend to Fig 1). B) HepG2 cells were transfected with monomeric linear full-length HBV DNA in combination with the indicated siRNA pools. After 48 hours, total RNA was extracted and pgRNA levels analyzed by qPCR as described in Legend to Fig 1D. C) Anti-STAT3 and anti-P-STAT3 immuno-precipitates were analyzed with primers specific for the Haptoglobin (HP) promoter. All ChIP results are expressed as Fold Induction (FI) of the % of Input and the histograms show the mean from three independent experiments; bars indicate S.D. D) 30 μg of nuclear proteins were analyzed by immunoblot with anti-STAT3, anti-P-STAT3 and anti-lamin B (loading control) antibodies (left panel). Densitometric quantitation of STAT3, P-STAT3 and lamin B immunoblots are shown in the right panel.
Fig 5.
Schematic model of IL6 modulation of cccDNA transcription.
STAT3, HNF1α and HNF4α bind the cccDNA and contribute to activate the transcription of cccDNA (middle). IL6 treatment results in the hypo-acetylation of cccDNA-bound histones and inhibits HBV transcription through the combined effect on HNF1α and HNF4α protein levels and a lower recruitment of P-STAT3 to the cccDNA, as compared to cellular promoters (i.e. Haptoglobin).