1. Volume 130, Issue 3, Pages 823-837 (March 2006)
Hepatitis B Virus Replication Is Regulated by the Acetylation Status of Hepatitis B Virus cccDNA-Bound H3 and H4 Histones 
Teresa Pollicino, Laura Belloni, Giuseppina Raffa, Natalia Pediconi, Giovanni Squadrito, Giovanni Raimondo, Massimo Levrero 
Gastroenterology 
Volume 130, Issue 3, Pages (March 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
Linear monomeric HBV genomes after transfection into HuH7 cells start a complete viral replication cycle. (A) Southern blot analysis of HBV replicative intermediates. Lanes correspond to DNA extracted from cytoplasmic HBV core particles derived from HuH7 cells which were transfected with 200 ng of linear monomeric HBV DNA, 10 μg of pUC19 as a mock control, 2 μg of linear monomeric HBV DNA, and 10 μg of pHBV (adwR9) plasmid carrying a head-to-tail HBV dimer DNA.23 Cells were harvested 48 hours posttransfection for Southern blotting. Membranes were exposed to an x-ray film for autoradiography at −80°C for 4 hours. (B) Real-time PCR quantification of cccDNA accumulation in HuH7 HBV-transfected cells. Results are expressed as number of cccDNA copies per cell (mean ± SD) from 3 independent experiments. (C) Southern blot analysis of HBV cccDNA selectively extracted from HBV-transfected cells and from liver tissue of a chronic hepatitis B patient used as a positive control. Abbreviations: OC, open circular duplex HBV DNA; DS, double-strand HBV DNA replicative intermediates; SS, single strand HBV DNA replicative intermediates.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
cccDNA-bound H3 and H4 histones are acetylated in vivo. (A) ChIP on HBV-transfected cells was performed by using specific antibodies to HBV core protein (HBc), histone H4 (H4), acetylhistone H3 (AcH3), acetylhistone H4 (AcH4), p300, CBP, or control IgG. Specific primers were used to amplify selectively the precore–core promoter of HBV cccDNA forms (35 PCR cycles). (B) The ChIP assay was applied (left panel) to nuclear (nuc) and, as a control, to proteinase K–treated cytoplasmic (cyto) extracts from HuH7 cells transfected with the linear monomeric HBV DNA. Selective HBV cccDNA primers were used to detect, by PCR (35 cycles), HBV cccDNA in anti-AcH3, anti-AcH4 and IgG immunoprecipitates from nuclear and cytoplasmic extracts. As a positive control, a PCR with primer pairs covering the S region (forward position : 5′- CCTGTATTCCCATCCCATC-3′; reverse position : 5′-TAGGGTTTAAATGTATACCC-3′) was conducted both on nuclear and cytoplasmatic fractions (right panel). NC, negative control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
HBV replication parallels the acetylation status of HBV cccDNA-bound H3 and H4 histones and the decline of viral replication associates to the recruitment onto cccDNA of HDAc1 at later time points. (A) HuH7 cells were transfected with monomeric linear full-length HBV genomes, and cytoplasmic HBV core particles were isolated at the indicated time points after transfection. Left panel: HBV DNA replicative intermediates were detected by Southern blot hybridization. Right panel: 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). 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. Data are expressed as fold increase (mean ± SD) from 3 independent experiments. (B) ChIPs were performed on HBV-transfected cells by using antibodies specific to H4, AcH4, AcH3, the class I deacetylase HDAc1, or control IgG, at the indicated time points after transfection, followed by amplification (35 cycles) with PCR primers specific for HBV precore–core promoter and HBV cccDNA. (C) Kinetics of cccDNA accumulation in HuH7 HBV-transfected cells assessed by real-time PCR quantification. Results are expressed as number of cccDNA copies per cell (mean ± SD) from 3 independent experiments. (D) Kinetic quantitative evaluation of H4- and AcH4-bound cccDNA was performed by real-time PCR on the immunoprecipitated chromatin as detailed in the Materials and Methods section and expressed as a percentage of input. Data (mean ± SD) from 3 independent experiments are shown. OC, open circular duplex HBV DNA; DS, double-strand HBV DNA replicative intermediates; SS, single-strand HBV DNA replicative intermediates.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
Influence of HBV genotype, YMDD mutation, and nucleoside analogs treatment on cccDNA levels and cccDNA-bound histone acetylation in HuH7 cell transfected with linear monomeric HBV DNAs. (A) HuH7 cells were transfected with linear monomeric wild-type HBV genomes of genotype D, genotype A, and with a genotype D genome carrying the YMDD mutation. (B) Southern blot analysis of cytoplasmic HBV replicative intermediates isolated from HuH7 cells transfected with wild-type and YMDD genotype D HBV DNAs. (C) HBV DNA transfected HuH7 cells were treated with the indicated amounts of lamivudine (LAM) or adefovir dipivoxil (ADV) for 72 hours. In (A) and (C), the upper panels show the real-time PCR quantification of cccDNA, expressed as number of cccDNA copies per cell (mean ± SD), from 3 independent experiments. Lower panels show the quantitative evaluation of AcH4-bound cccDNA by real-time PCR on the immunoprecipitated chromatin (see Materials and Methods section) expressed as percentage of input. Data (mean ± SD) from 3 independent experiments are shown. OC, open circular duplex HBV DNA; DS, double-strand HBV DNA replicative intermediates; SS, single strand HBV DNA replicative intermediates. P values were calculated using the nonparametric Wilcoxon rank sum test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
The class I/II histone deacetylase inhibitors VPA and TSA increase HBV replication, transcription, and HBV cccDNA-bound acetylated H3 and H4 histones. (A) ChIP performed by the use of antibodies specific to AcH3 and to AcH4 or control IgG and with PCR primers specific for HBV precore–core promoter and HBV cccDNA. HBV-transfected HuH7 cells were left untreated (NT) or treated for 16 hours with 5 mmol of VPA or 300 nmol of TSA. (B) Northern blot analysis of HBV transcripts isolated from HuH7 cells 48 hours after transfection with monomeric linear HBV genomes. Cells, 24 hours after transfection, were left untreated (HBV NT) or treated for 16 hours with either 5 mmol of VPA or 300 nmol of TSA (top). Densitometric quantitation of pregenomic HBV mRNA (3.5 kb) from nontreated and from VPA- or TSA-treated HuH7 cells transfected with HBV genomes (bottom). (C) Southern blot analysis of cytoplasmic HBV replicative intermediates isolated from HuH7 cells transfected with monomeric linear HBV genomes and left untreated (HBV NT) or treated for 16 hours with either 5 mmol of VPA or 300 nmol of TSA (top). Densitometric quantitation of HBV replicative intermediates (single-strand band) from nontreated and from VPA- or TSA-treated HuH7 cells, which were transfected with HBV genomes (bottom). (D) Southern blot analysis of HBV DNA encapsulated in HBV viral particles secreted into the cell medium of HuH7 cells transfected with monomeric linear HBV genomes and left untreated (HBV NT) or treated for 16 hours with either 5 mmol of VPA or 300 nmol of TSA (top). Densitometric quantitation of open circular (OC) HBV DNA from nontreated and from VPA- or TSA-treated HuH7 cells, which were transfected with HBV genomes (bottom). In B, C, and D, the signals were quantified with Quantity One 1-D Analysis Software (BioRad); data are from 3 independent experiments and values are expressed as mean ± SD. OC, open circular duplex HBV DNA; DS, double-strand HBV DNA replicative intermediates; SS, single-strand HBV DNA replicative intermediates.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

7. Figure 6
The acetylation status of HBV cccDNA-bound H3 and H4 histones correlates to HBV viremia levels in chronic hepatitis B patients. (A) ChIP of liver nuclear extracts from 10 HBsAg-positive patients with chronic hepatitis by using specific antibodies to AcH3, AcH4, HDAc1, or control IgG, and specific PCR primes for HBV precore–core promoter and HBV cccDNA. (B) HBV DNA quantification in the serum samples from 10 HBsAg-positive patients with active (AcH3-AcH4 positive/HDAc1 negative, 4 cases; AcH3-AcH4 positive/HDAc1 positive, 2 cases) or suppressed (AcH3-AcH4 negative/HDAc1 positive, 4 cases) HBV replication was performed with the Roche Amplicor HBV Monitor Kit. P values were calculated using the nonparametric Wilcoxon rank sum test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions