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Control of cccDNA function in hepatitis B virus infection

Open AccessPublished:June 10, 2009DOI:https://doi.org/10.1016/j.jhep.2009.05.022
      The template of hepatitis B virus (HBV) transcription, the covalently closed circular DNA (cccDNA), plays a key role in the life cycle of the virus and permits the persistence of infection. Novel molecular techniques have opened new possibilities to investigate the organization and the activity of the cccDNA minichromosome in vivo, and recent advances have started to shed light on the complexity of the mechanisms controlling cccDNA function. Nuclear cccDNA accumulates in hepatocyte nuclei as a stable minichromosome organized by histone and non-histone viral and cellular proteins. Identification of the molecular mechanisms regulating cccDNA stability and its transcriptional activity at the RNA, DNA and epigenetic levels in the course of chronic hepatitis B (CH-B) infection may reveal new potential therapeutic targets for anti-HBV drugs and hence assist in the design of strategies aimed at silencing and eventually depleting the cccDNA reservoir.

      Keywords

      Abbreviations:

      HBV (hepatitis B virus), CH-B (chronic hepatitis B), HCC (hepatocellular carcinoma), RC (relaxed circular), PF-RC DNA (protein free relaxed circular), cccDNA (covalently closed circular DNA), NHEJ (nonhomologous end joining), DLS (double-stranded linear (DSL)), pgRNA (pregenomic RNA), LS (viral large surface protein), ChIP (chromatin immuno-precipitation)
      Infection with hepatitis B virus (HBV) continues to be a major health problem with about 400 million people chronically infected worldwide who are at high risk of developing liver cirrhosis and hepatocellular carcinoma (HCC) [
      • Shepard C.W.
      • Simard E.P.
      • Finelli L.
      • Fiore A.E.
      • Bell B.P.
      Hepatitis B virus infection: epidemiology and vaccination.
      ]. HBV is a member of the Hepadnaviridae family, that includes small enveloped DNA viruses infecting primates, rodents, and birds. One common characteristic of these viruses is their high species and cell-type specificity, as well as a unique genomic organization and replication mechanism.

      1. cccDNA and HBV replication

      Upon infection of the hepatocyte, the core nucleocapsid is released into the cytoplasm and the genomic DNA is transferred to the cell nucleus, where the relaxed circular (RC), partially double-stranded DNA is converted into a covalently closed circular DNA (cccDNA) molecule [
      • Tuttleman J.S.
      • Pourcel C.
      • Summers J.
      Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells.
      ,
      • Newbold J.E.
      • Xin H.
      • Tencza M.
      • Sherman G.
      • Dean J.
      • Bowden S.
      • et al.
      The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes.
      ] (Fig. 1). Biochemically, the formation of cccDNA from RC DNA requires: (a) the removal of the viral polymerase covalently attached to the 5′ end of the negative DNA strand; (b) the removal from the 5′ end of the plus-strand DNA of the short RNA oligomer which is used to prime the plus-strand DNA synthesis; (c) the removal of precisely one copy of the short terminal redundancy (“r”) from the minus-strand DNA; (d) the completion of the positive strand, which is variable in length in the RC DNA, by the cellular replicative machinery and (e) the ligation of the two viral DNA strands (reviewed in [
      • Nguyen D.H.
      • Ludgate L.
      • Hu J.
      Hepatitis B virus–cell interactions and pathogenesis.
      ]). In hepatoma cell replication systems, the HBV cccDNA derives predominantly from RC DNA with a precise junction sequence and is accompanied, differently from DHBV replicating cells and human infected livers, by the accumulation of the “protein-free or PF-RC DNA” intermediates, from which the covalently attached viral reverse transcriptase (RT) protein has been removed [
      • Gao W.
      • Hu J.
      Formation of hepatitis B virus covalently closed circular DNA: removal of genome-linked protein.
      ]. PF-RC DNA, in contrast to RT-linked RC DNA contains almost exclusively, mature plus-strand DNA, suggesting that the RT protein is removed preferentially from RC DNA in mature core particles [
      • Gao W.
      • Hu J.
      Formation of hepatitis B virus covalently closed circular DNA: removal of genome-linked protein.
      ]. A small amount of cccDNA can also be generated from the so-called double-stranded linear (DSL) form of HBV-DNA [
      • Staprans S.
      • Loeb D.D.
      • Ganem D.
      Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA.
      ] by intramolecular ligation of the linear DNA ends via nonhomologous end joining [NHEJ] [
      • Yang W.
      • Summers J.
      Illegitimate replication of linear hepadnavirus DNA through non-homologous recombination.
      ]. However, this process of “illegitimate replication” leads to the formation of a cccDNA that harbors small deletions and/or insertions that preclude viral DNA replication.
      Disguised as a stable non-integrated minichromosome, the cccDNA is not replicated by the host’s semi-conservative DNA synthesis machinery, but it utilizes the cellular transcriptional machinery to produce all viral RNAs necessary for protein production and viral replication, which takes place in the cytoplasm after reverse transcription of the pregenomic RNA (pgRNA) [
      • Mason W.S.
      • Aldrich C.
      • Summers J.
      • Taylor J.M.
      Asymmetric replication of duck hepatitis B virus DNA in liver cells: free minus-strand DNA.
      ]. Of note, experiments performed using the duck model revealed that the major source of cccDNA in infected hepatocytes comes from newly synthesized nucleocapsids, which are not enveloped and secreted into the blood, but are transported into the nucleus to ensure accumulation, and later maintenance of the cccDNA pool [
      • Wu T.T.
      • Coates L.
      • Aldrich C.E.
      • Summers J.
      • Mason W.S.
      In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway.
      ,
      • Zoulim F.
      New insight on hepatitis B virus persistence from the study of intrahepatic viral cccDNA.
      ]. Thus, these studies indicate that multiple rounds of infection are not needed to establish a stable cccDNA pool in infected cells. Evidence from animal models indicated that 1 to 50 cccDNA molecules accumulate per cell [
      • Newbold J.E.
      • Xin H.
      • Tencza M.
      • Sherman G.
      • Dean J.
      • Bowden S.
      • et al.
      The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes.
      ,
      • Dandri M.
      • Burda M.R.
      • Will H.
      • Petersen J.
      Increased hepatocyte turnover and inhibition of woodchuck hepatitis B virus replication by adefovir in vitro do not lead to reduction of the closed circular DNA.
      ,
      • Moraleda G.
      • Saputelli J.
      • Aldrich C.E.
      • Averett D.
      • Condreay L.
      • Mason W.S.
      Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus.
      ,
      • Wong D.K.
      • Yuen M.F.
      • Yuan H.
      • Sum S.S.
      • Hui C.K.
      • Hall J.
      • et al.
      Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients.
      ] and analysis of cccDNA distribution at the single cell level revealed that cccDNA amount varied significantly from cell to cell in the course of chronic infection in the duck model, where a fraction of cells was found to contain only one cccDNA copy [
      • Zhang Y.Y.
      • Zhang B.H.
      • Theele D.
      • Litwin S.
      • Toll E.
      • Summers J.
      Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver.
      ]. However, the viral and host factors that control cccDNA formation and cccDNA pool size are yet poorly defined. A negative-feedback mechanism might involve the viral large surface (LS) protein of DHBV suppressing cccDNA amplification [
      • Summers J.
      • Smith P.M.
      • Horwich A.L.
      Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification.
      ,
      • Lenhoff R.J.
      • Summers J.
      Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus.
      ]: when nuclear cccDNA levels reach 10–50 copies sufficient LS protein is made to shut off efficiently the cccDNA amplification pathway and redirect mature nucleocapsids (containing RC DNA) to envelopment and extracellular secretion.

      2. Control of cccDNA pool in hepadnavirus infection

      cccDNA is very stable in quiescent hepatocytes and the size of cccDNA pool, once established, may be reduced through different mechanisms that entail cell killing, dilution by cell proliferation and cell “cure” mediated by inflammatory cytokines. More recently, the possibility that the functionality of persisting cccDNA might be controlled by a reduction of its transcriptional activity has been proposed. Most of our knowledge on cccDNA pool size control in infected livers comes from transient and chronic hepatitis B infection in the woodchuck model [
      • Mason W.S.
      • Litwin S.
      • Xu C.
      • Jilbert A.R.
      Hepatocyte turnover in transient and chronic hepadnavirus infections.
      ]. It has to be emphasized, however, that differences may exist in both cccDNA dynamics and the efficiency and mechanisms of viral DNA integration into the host genome between HBV and WHV. In general, cccDNA can persist throughout the life span of the quiescent hepatocytes without affecting their viability. On the contrary, cell division may favour dilution/loss of cccDNA during mitosis, so that selection of cccDNA-free cells may occur when cells are forced to divide to compensate the loss of infected cells killed by the T cell response. In the acute phase hepadnavirus infection can spread to the entire hepatocyte population in the liver and, the uninfected hepatocytes present in the recovered liver have been shown to derive from previously infected hepatocytes [
      • Mason W.S.
      • Litwin S.
      • Xu C.
      • Jilbert A.R.
      Hepatocyte turnover in transient and chronic hepadnavirus infections.
      ,
      • Summers J.
      • Jilbert A.R.
      • Yang W.
      • Aldrich C.E.
      • Saputelli J.
      • Litwin S.
      • et al.
      Hepatocyte turnover during resolution of a transient hepadnaviral infection.
      ]. During virus clearance antiviral cytokines are thought to block virus replication and the formation of new cccDNA. Clearance of infected hepatocytes from both replicative intermediates and cccDNA would occur through three modalities. In the first scenario, cccDNA is primarily eliminated by cytokines and cell death or cell proliferation do not contribute significantly [the “cure” model]. In the second, cytokines suppress cytoplasmic viral replication and new cccDNA formation but do not act on pre-existing cccDNA that is eliminated by both cell death and mitotic loss and would need a turnover of approximately 0.7 of the total liver mass [the “death and compensatory proliferation” model]. In the third, cccDNA survives hepatocytes mitosis, is distributed in a binomial fashion to the progeny hepatocytes and it is eliminated only by the elimination of infected cells, requiring an estimated turnover exceeding 2.5 times the total liver mass [the “cell death” model] (reviewed in [
      • Mason W.S.
      • Litwin S.
      • Xu C.
      • Jilbert A.R.
      Hepatocyte turnover in transient and chronic hepadnavirus infections.
      ]). An elegant computer modelling study based on the analysis of hepatocyte turnover during the resolution of untreated or entecavir-treated transiently infected woodchucks, indicated that cccDNA clearance requires, in addition to cell death, a combination of cytokine suppression and mitotic loss [
      • Mason W.S.
      • Xu C.
      • Low H.C.
      • Saputelli J.
      • Aldrich C.E.
      • Scougall C.
      • et al.
      The amount of hepatocyte turnover that occurred during resolution of transient hepadnavirus infections was lower when virus replication was inhibited with entecavir.
      ]. In chronically infected woodchuck livers, the identification of uninfected cell clones, which have lost cccDNA, but still contain “traces” of the infection in form of viral integrations has been explained as a cccDNA clearance without cell destruction [
      • Summers J.
      • Mason W.S.
      Residual integrated viral DNA after hepadnavirus clearance by nucleoside analog therapy.
      ,
      • Mason W.S.
      • Jilbert A.R.
      • Summers J.
      Clonal expansion of hepatocytes during chronic woodchuck hepatitis virus infection.
      ]. Since liver cell death and regeneration usually occur in chronically infected woodchuck livers, an alternative hypothesis might be that surviving hepatocytes harbouring both viral integrations and cccDNA may divide, cccDNA would be lost through cell division while the integrated DNA would persist in daughter cells [
      • Summers J.
      • Mason W.S.
      Residual integrated viral DNA after hepadnavirus clearance by nucleoside analog therapy.
      ,
      • Mason W.S.
      • Jilbert A.R.
      • Summers J.
      Clonal expansion of hepatocytes during chronic woodchuck hepatitis virus infection.
      ]. Thus, in chronic infection, killing of hepatocytes may be instrumental not only to eliminate infected cells but also to induce hepatocyte proliferation which, in turn, may favour cccDNA loss. On the other hand, studies have shown that very low levels of cccDNA can persist indefinitely, possibly explaining life-long immune responses to HBV despite clinical resolution of HBV infection [
      • Penna A.
      • Artini M.
      • Cavalli A.
      • Levrero M.
      • Bertoletti A.
      • Pilli M.
      • et al.
      Longlasting memory T cell responses following self-limited acute hepatitis B.
      ,
      • Rehermann B.
      • Ferrari C.
      • Pasquinelli C.
      • Chisari F.V.
      The hepatitis B virus persists for decades after patients’ recovery from acute viral hepatitis despite active maintenance of a cytotoxic T-lymphocyte response.
      ].
      As HBV polymerase inhibitors do not directly affect the cccDNA, a decrease in cccDNA levels is supposed to derive from the lack of sufficient recycling of viral nucleocapsids to the nucleus, due to the strong inhibition of viral DNA synthesis in the cytoplasm, and less incoming viruses from the blood. Following this scenario, cccDNA depletion will require many years of nucleos(t)ide drug administration and it is expected to lead to the selection of resistant variants [
      • Zoulim F.
      Assessment of treatment efficacy in HBV infection and disease.
      ]. Importantly, currently available nucleos(t)ide analogs do not prevent cccDNA formation when cells are exposed to a new round of viral infection [
      • Kock J.
      • Baumert T.F.
      • Delaney WEt
      • Blum H.E.
      • von Weizsacker F.
      Inhibitory effect of adefovir and lamivudine on the initiation of hepatitis B virus infection in primary tupaia hepatocytes.
      ,
      • Delmas J.
      • Schorr O.
      • Jamard C.
      • Gibbs C.
      • Trepo C.
      • Hantz O.
      • et al.
      Inhibitory effect of adefovir on viral DNA synthesis and covalently closed circular DNA formation in duck hepatitis B virus-infected hepatocytes in vivo and in vitro.
      ,
      • Le Guerhier F.
      • Pichoud C.
      • Guerret S.
      • Chevallier M.
      • Jamard C.
      • Hantz O.
      • et al.
      Characterization of the antiviral effect of 2′,3′-dideoxy-2′, 3′-didehydro-beta-Lfluorocytidine in the duck hepatitis B virus infection model.
      ], which in turn has important clinical implications in terms of treatment duration as persisting viremia during the antiviral treatment might be responsible for new rounds of infection. Simultaneous treatment with antiviral agents acting through different mechanisms and, possibly, limiting hepatocytes reinfection [
      • Sung J.J.
      • Wong M.L.
      • Bowden S.
      • Liew C.T.
      • Hui A.Y.
      • Wong V.W.
      • et al.
      Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy.
      ,
      • Wursthorn K.
      • Lutgehetmann M.
      • Dandri M.
      • Volz T.
      • Buggisch P.
      • Zollner B.
      • et al.
      Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B.
      ,
      • Lutgehetmann M.
      • Volzt T.
      • Quaas A.
      • Zankel M.
      • Fischer C.
      • Dandri M.
      • et al.
      Sequential combination therapy leads to biochemical and histological improvement despite low ongoing intrahepatic hepatitis B virus replication.
      ,
      • Petersen J.
      • Dandri M.
      • Mier W.
      • Lütgehetmann M.
      • Volz T.
      • von Weizsäcker F.
      • et al.
      Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein.
      ], as well as the development of novel therapeutic strategies aimed at eradicating cccDNA from the liver may be necessary to eventually achieve clearance of infection.

      3. Structure of the cccDNA minichromosome

      Nuclear hepadnaviral cccDNA molecules are organized into a chromatin-like structure as a viral minichromosome that displays the typical beads-on-a string arrangement at electron microscopy and reveals the nucleosomal packaging of transcriptionally inactive chromatin [
      • Newbold J.E.
      • Xin H.
      • Tencza M.
      • Sherman G.
      • Dean J.
      • Bowden S.
      • et al.
      The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes.
      ,
      • Bock C.T.
      • Schranz P.
      • Schroder C.H.
      • Zentgraf H.
      Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell.
      ]. Organization of viral DNAs into a nucleosomal structure has been described for the polyoma viruses [
      • Prieto-Soto A.
      • Gourlie B.
      • Miwa M.
      • Pigiet V.
      • Sugimura T.
      • Malik N.
      • et al.
      Polyoma virus minichromosomes: poly ADP-ribosylation of associated chromatin proteins.
      ], simian virus 40 (SV40) [
      • Griffith J.D.
      Chromatin structure: deduced from a minichromosome.
      ], human (HPVs) [
      • Favre M.
      • Breitburd F.
      • Croissant O.
      • Orth G.
      Chromatin-like structures obtained after alkaline disruption of bovine and human papillomaviruses.
      ] and bovine papilloma viruses (BPV) [
      • Rosl F.
      • Waldeck W.
      • Zentgraf H.
      • Sauer G.
      Properties of intracellular bovine papillomavirus chromatin.
      ], adenovirus [
      • Tate V.E.
      • Philipson L.
      Parental adenovirus DNA accumulates in nucleosome-like structures in infected cells.
      ], latent EBV [
      • Shaw J.E.
      • Levinger L.F.
      • Carter Jr, C.W.
      Nucleosomal structure of Epstein–Barr virus DNA in transformed cell lines.
      ] and Herpes simplex 1 [
      • Deshmane S.L.
      • Fraser N.W.
      During latency, herpes simplex virus type 1 DNA is associated with nucleosomes in a chromatin structure.
      ] viruses. These viruses, with the possible exception of Herpes simplex, all produce covalently closed double-stranded DNAs (cccDNAs) that are found in the nuclei of infected cells. Two populations of Hepadnaviral minichromosomes, corresponding to a full or half complement of nucleosomes, can be detected possibly reflecting dynamic changes related to transcriptional regulation [
      • Newbold J.E.
      • Xin H.
      • Tencza M.
      • Sherman G.
      • Dean J.
      • Bowden S.
      • et al.
      The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes.
      ]. HBV minichromosome has been shown to consist of both histone and non-histone proteins [
      • Bock C.T.
      • Schwinn S.
      • Locarnini S.
      • Fyfe J.
      • Manns M.P.
      • Trautwein C.
      • et al.
      Structural organization of the hepatitis B virus minichromosome.
      ]. Histone proteins H3 and H2B were the most prominent species but lower levels of histone proteins H4, H2A, and H1 were also detectable by immunoblotting on purified nuclear nucleocapsid complexes [
      • Bock C.T.
      • Schwinn S.
      • Locarnini S.
      • Fyfe J.
      • Manns M.P.
      • Trautwein C.
      • et al.
      Structural organization of the hepatitis B virus minichromosome.
      ]. The virally encoded core protein is a structural component of the HBV minichromosome, binds preferentially to HBV double-stranded DNA and its binding results in a reduction of the nucleosomal spacing of the HBV nucleoprotein complexes by 10% from 200 bp to 180 bp [
      • Bock C.T.
      • Schwinn S.
      • Locarnini S.
      • Fyfe J.
      • Manns M.P.
      • Trautwein C.
      • et al.
      Structural organization of the hepatitis B virus minichromosome.
      ]. A combination of in vitro approaches, including classical oligonucleotide gel retardation (EMSA) and DNAse protection assays, have shown that a number of transcription factors bind in vitro to the HBV enhancers 1 and 2 and regulate transcription driven by the same HBV regulatory elements [
      • Moolla N.
      • Kew M.
      • Arbuthnot P.
      Regulatory elements of hepatitis B virus transcription.
      ] but their association with the viral minicromosome has not been proven in vivo. The chromatin immuno-precipitation (ChIP) method is a powerful technique that allows the identification in vivo, at a resolution of a few hundred base pairs, of the DNA binding sites of virtually any chromosome component [
      • Orlando V.
      Mapping chromosomal proteins in vivo by formaldehyde-crosslinked chromatin immunoprecipitation.
      ,
      • Kuo M.H.
      • Allis C.D.
      In vivo cross-linking and immunoprecipitation for studying dynamic protein: DNA associations in a chromatin environment.
      ]. The principle relies upon the fixation of protein–DNA and protein–protein interactions in vivo by the cross-linking agent formaldehyde. Chromatin is then disrupted by sonication, and the cross-linked protein of interest immuno-precipitated with specific antibody directed against histones, covalently modified (acetylated, methylated, SUMOlated etc.) histones, transcription factors, transcriptional co-activators and co-repressors or their post-translationally modified versions. After the cross-links are released by heating the immuno-precipitated DNA is used as a template in classic or real-time PCRs using oligonucleotides that amplify the region presumably bound by the protein of interest. ChIP-based protocols have evolved over time to work on as low as 1000 cultured cells as well as on a variety of surgical and biopsy tissue samples. We have recently developed a ChIP-based quantitative technique to study the recruitment in vivo of cellular and viral proteins onto the HBV minichromosome [
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ] (Fig. 2). The HBV cccDNA ChIP assay combines a cccDNA ChIP step with a sensitive and specific real-time PCR protocol for cccDNA quantification [
      • Werle-Lapostolle B.
      • Bowden S.
      • Locarnini S.
      • Wursthorn K.
      • Petersen J.
      • Lau G.
      • et al.
      Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.
      ].
      Figure thumbnail gr2
      Fig. 2The cccDNA chromatin immuno-precipitation assay.
      The assay has been first used to immunoprecipitate and analyse the cccDNA in a cellular model of HBV replication based upon the transient transfection of linear full-length HBV monomers into HuH7 and HepG2 hepatoma cells [
      • Gunther S.
      • Li B.C.
      • Miska S.
      • Kruger D.H.
      • Meisel H.
      • Will H.
      A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients.
      ] and subsequently adapted to study the cccDNA in baculovirus-HBV-infected cells [
      • Lucifora J.
      • Durantel D.
      • Belloni L.
      • Barraud L.
      • Villet S.
      • Vincent I.E.
      • et al.
      Initiation of hepatitis B virus genome replication and production of infectious virus following delivery in HepG2 cells by novel recombinant baculovirus vector.
      ], hepatocytes derived from the livers of humanized chimera mice infected in vivo with HBV (Dandri and Belloni, unpublished observations) and liver biopsy samples from chronic hepatitis B patients [
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ]. Using the cccDNA-ChIP assay we and others have confirmed the recruitment in vivo of H3 and H4 histones [
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ,

      Cougot D, Wu Y, Buendia MA, Neuveut C. Role of HBx and its cellular partners in the regulation of viral transcription. In: 2007 international meeting the molecular biology of hepatitis B viruses, Rome, Italy, September 16–20, 2007.

      ] and of HBV core proteins on the cccDNA minichromosome [
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ]. Using the same approach several cellular transcription factors, including CREB, ATF, STAT1 and STAT2, and chromatin modifying enzymes have been shown to bind to the cccDNA in cells replicating HBV [
      • Belloni L.
      • Pollicino T.
      • Cimino L.
      • Raffa G.
      • Raimondo G.
      • Levrero M.
      HBX binds in vivo on the HBV minichromosome, modifies the epigenetic regulation of ccc-DNA function and potentiates HBV replication.
      ]. The demonstration that the cellular histone acetyltransferases CBP (a coactivator of both CREB and ATF transcription factors), p300 and PCAF/GCN5 and the histone deacetylases HDAC1 and hSirtl are recruited with different kinetics onto the cccDNA in cells replicating HBV imply that cccDNA bound histones may undergo regulatory post-translational modifications.

      4. cccDNA quantification and activity in HBV chronic carriers

      The development of highly selective real-time polymerase chain reaction (PCR) assays has provided new sensitive tools to investigate the replicative activity [
      • Laras A.
      • Koskinas J.
      • Dimou E.
      • Kostamena A.
      • Hadziyannis S.J.
      Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients.
      ,
      • Volz T.
      • Lutgehetmann M.
      • Wachtler P.
      • Jacob A.
      • Quaas A.
      • Murray J.M.
      • et al.
      Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.
      ] and the effectiveness of antiviral therapy [
      • Sung J.J.
      • Wong M.L.
      • Bowden S.
      • Liew C.T.
      • Hui A.Y.
      • Wong V.W.
      • et al.
      Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy.
      ,
      • Lutgehetmann M.
      • Volzt T.
      • Quaas A.
      • Zankel M.
      • Fischer C.
      • Dandri M.
      • et al.
      Sequential combination therapy leads to biochemical and histological improvement despite low ongoing intrahepatic hepatitis B virus replication.
      ,
      • Werle-Lapostolle B.
      • Bowden S.
      • Locarnini S.
      • Wursthorn K.
      • Petersen J.
      • Lau G.
      • et al.
      Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.
      ] in infected patients by enabling accurate determination of serological and intrahepatic HBV-DNA levels, including specific detection and quantification of the cccDNA in human liver biopsies. Studies have shown that cccDNA loads vary significantly in the different phases of CH-B infection and that patients who underwent HBeAg seroconversion were more likely to have lower cccDNA levels at the beginning of therapy [
      • Werle-Lapostolle B.
      • Bowden S.
      • Locarnini S.
      • Wursthorn K.
      • Petersen J.
      • Lau G.
      • et al.
      Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.
      ]. The lower viremia generally observed in HBeAg-negative patients appeared to be due not only to the lower cccDNA content (>1 log) but also to reduced replication efficiency, which was defined as the amount of relaxed circular HBV DNA (rcDNA) produced per cccDNA molecule [
      • Volz T.
      • Lutgehetmann M.
      • Wachtler P.
      • Jacob A.
      • Quaas A.
      • Murray J.M.
      • et al.
      Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.
      ]. Quantitative measurements of HBV RNA transcripts also indicated that lower steady-state levels of pgRNA found in HBeAg-negative patients accounted for the reduced virion productivity observed at the DNA level.
      The good correlation observed between pgRNA and rcDNA copy number indicated that impairment of replicative activity in HBeAg-negative patients was not due to differences in reverse transcription efficiency [
      • Laras A.
      • Koskinas J.
      • Dimou E.
      • Kostamena A.
      • Hadziyannis S.J.
      Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients.
      ,
      • Volz T.
      • Lutgehetmann M.
      • Wachtler P.
      • Jacob A.
      • Quaas A.
      • Murray J.M.
      • et al.
      Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.
      ]. Surprisingly, impairment in virion productivity was not linked to the presence of common PC/BCP variants, since HBV replicative activity was mostly reduced in HBeAg-negative patients without detectable PC/BCP mutations [
      • Volz T.
      • Lutgehetmann M.
      • Wachtler P.
      • Jacob A.
      • Quaas A.
      • Murray J.M.
      • et al.
      Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.
      ]. Although viral titres correlated with the amount of cccDNA present in the liver in HBeAg-positive patients, there was poor correlation between viremia and intrahepatic cccDNA amounts detected in HBeAg-negative patients, implying that viral productivity can be specifically impaired in the low replicative HBeAg-negative phase of chronic hepatitis B, and strengthen the notion that viremia measurements in HBeAg-negative patients are not good predictive markers of infection in the liver [
      • Sung J.J.
      • Wong M.L.
      • Bowden S.
      • Liew C.T.
      • Hui A.Y.
      • Wong V.W.
      • et al.
      Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy.
      ,
      • Volz T.
      • Lutgehetmann M.
      • Wachtler P.
      • Jacob A.
      • Quaas A.
      • Murray J.M.
      • et al.
      Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.
      ]. The finding that levels of preS/S RNA transcripts and HBsAg concentrations per cccDNA molecule did not differ significantly between the two HBeAg groups, implied that only the replicative pathway was specifically impaired in those HBeAg-negative patients and hence, that the production of viral and subviral particles may be differently regulated in the course of HBV-chronic infection [
      • Volz T.
      • Lutgehetmann M.
      • Wachtler P.
      • Jacob A.
      • Quaas A.
      • Murray J.M.
      • et al.
      Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.
      ].
      Nevertheless, the molecular mechanisms responsible for the lower HBV replicative activity determined in treatment naı¨ve HBeAg-negative patients remain to be elucidated.

      5. Factors regulating cccDNA activity

      Due to the narrow host range of HBV and limited animal models available [
      • Dandri M.
      • Lutgehetmann M.
      • Volz T.
      • Petersen J.
      Small animal model systems for studying hepatitis B virus replication and pathogenesis.
      ], knowledge of the factors regulating the transcriptional activity of the cccDNA in vivo is still very limited. These factors need to be elucidated since they will affect the size and ultimately the stability of the cccDNA pool, and therefore, will influence the clinical course of ongoing infection.

      5.1 Immune-mediated factors

      Experiments with HBV-replicating transgenic mice and chimpanzees have shown that inflammatory cytokines, such as type 1 interferon and TNF alpha, can efficiently suppress viral replication through non-cytolytic, immune-mediated mechanisms, which also contribute to diminish the cccDNA reservoirs from infected cells [
      • Guidotti L.G.
      • Rochford R.
      • Chung J.
      • Shapiro M.
      • Purcell R.
      • Chisari F.V.
      Viral clearance without destruction of infected cells during acute HBV infection.
      ]. However, in the chronic phase these processes may be ineffective, and in the absence of cell division, the long half-life of the hepatocytes will guarantee cccDNA survival in infected hepatocytes. Kupffer cells and dendritic cells play a major role in mediating both innate and adaptive immune responses and the molecular mechanisms by which the innate and adaptive immune response can affect viral replication are only starting to emerge [
      • Guidotti L.G.
      • Rochford R.
      • Chung J.
      • Shapiro M.
      • Purcell R.
      • Chisari F.V.
      Viral clearance without destruction of infected cells during acute HBV infection.
      ,
      • Boni C.
      • Fisicaro P.
      • Valdatta C.
      • Amadei B.
      • Di Vincenzo P.
      • Giuberti T.
      • et al.
      Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection.
      ,
      • Puro R.
      • Schneider R.J.
      Tumor necrosis factor activates a conserved innate antiviral response to hepatitis B virus that destabilizes nucleocapsids and reduces nuclear viral DNA.
      ,
      • Visvanathan K.
      • Skinner N.A.
      • Thompson A.J.
      • Riordan S.M.
      • Sozzi V.
      • Edwards R.
      • et al.
      Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.
      ]. A growing body of evidence indicates that several viruses evolved mechanisms to directly suppress the host defence by altering different pathways of the innate immune response [
      • Schwabe R.F.
      • Brenner D.A.
      Mechanisms of liver injury. I. TNF-alpha-induced liver injury: role of IKK, JNK, and ROS pathways.
      ,
      • Wu J.
      • Lu M.
      • Meng Z.
      • Trippler M.
      • Broering R.
      • Szczeponek A.
      • et al.
      Toll-like receptor-mediated control of HBV replication by nonparenchymal liver cells in mice.
      ]. Notably, chronic hepatitis B infection has been associated with a significant reduction of dendritic cell functions and impairment of the innate immune response [
      • van der Molen R.G.
      • Sprengers D.
      • Binda R.S.
      • de Jong E.C.
      • Niesters H.G.
      • Kusters J.G.
      • et al.
      Functional impairment of myeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B.
      ]. Down-regulation of Toll-like receptor signaling was recently demonstrated in the liver and blood of HBeAg-positive chronic carriers, revealing the occurrence of potentially important interactions between virions, HBeAg and the innate immune response [
      • Visvanathan K.
      • Skinner N.A.
      • Thompson A.J.
      • Riordan S.M.
      • Sozzi V.
      • Edwards R.
      • et al.
      Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.
      ,
      • Chen W.
      • Zhang Z.
      • Shi M.
      • Chen L.
      • Fu J.
      • Shi F.
      • et al.
      Activated plasmacytoid dendritic cells act synergistically with hepatitis B core antigen-pulsed monocyte-derived dendritic cells in the induction of hepatitis B virus-specific CD8 T-cell response.
      ].

      5.2 Virological factors

      The complex interplay between virus and host factors occurring in the various phases of chronic infection is likely to promote the selection of certain HBV variants. A recent in vivo virological study [
      • Lim S.G.
      • Cheng Y.
      • Guindon S.
      • Seet B.L.
      • Lee L.Y.
      • Hu P.
      • et al.
      Viral quasi-species evolution during hepatitis Be antigen seroconversion.
      ] demonstrated ongoing significant accumulation of viral genetic diversity in HBeAg seroconverters due to selective pressure operated by the immune response. Transcription of pregenomic RNA is under the control of the basal core promoter (BCP) and mutations in this region and in the precore (PC) region are common in HBeAg-negative individuals and have been shown to affect viral replication in vitro[
      • Gunther S.
      • Sommer G.
      • Von Breunig F.
      • Iwanska A.
      • Kalinina T.
      • Sterneck M.
      • et al.
      Amplification of full-length hepatitis B virus genomes from samples from patients with low levels of viremia: frequency and functional consequences of PCR-introduced mutations.
      ,
      • Parekh S.
      • Zoulim F.
      • Ahn S.H.
      • Tsai A.
      • Li J.
      • Kawai S.
      • et al.
      Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promoter mutants.
      ,
      • Jammeh S.
      • Tavner F.
      • Watson R.
      • Thomas H.C.
      • Karayiannis P.
      Effect of basal core promoter and pre-core mutations on hepatitis B virus replication.
      ]. Occurrence of mutations within the regulatory regions of the virus may also affect HBV replication activity in vivo. However, current knowledge of the transcriptional state of different HBV isolates is mostly based on measurements performed in vitro after transfection of the genomic HBV-DNA inserted on a plasmid backbone. In these systems, structure and organization of the cccDNA cannot be recapitulated, and hence, the factors influencing the transcriptional activity of the cccDNA need to be assessed in vivo.
      Recent studies have shown that in a minority of HBeAg-negative patients only cccDNA molecules harbouring PC/BCP mutations were detected. These individuals displayed significantly higher levels of replicative activity (median 150 vs. 6 rcDNA/cccDNA) and higher mutation frequency (0.46% vs. 0.18%; p = 0.001) compared to HBeAg-negative patients without detectable PC/BCP mutations [
      • Koepke A.
      • Volz T.
      • Lutgehetmann M.
      • Lohse A.W.
      • Dandri M.
      • Petersen J.
      Genetic variability of the cccDNA regulatory region changes significantly in the different phases of chronic HBV infection.
      ]. Of note, the selection of BCP mutations was associated with higher replication levels and worsening of clinical manifestations [
      • Volz T.
      • Lutgehetmann M.
      • Wachtler P.
      • Jacob A.
      • Quaas A.
      • Murray J.M.
      • et al.
      Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.
      ].

      5.3 Epigenetic factors

      Transcriptional regulation of gene expression rely on nuclear enzymatic activities that constantly modify chromatin to make it competent or refractory to gene activation. The amino-terminal tails of the four core histones on the nucleosome surface are subjected to a variety of enzyme-catalyzed, posttranslational modifications of selected amino acids, including lysine acetylation, lysine and arginine methylation, serine phosphorylation, and attachment of the small peptides ubiquitin and SUMO, that form a “histone code” specifying patterns of gene expression. Histone acetylation has long been associated with transcription initiation, whereas reduced acetylation of histones H3 and H4 N-terminal tails is a key signal for the subsequent recruitment of histone methylases and DNA methylation enzymes that results in chromatin based repression of gene expression and hetero-chromatinization [
      • Sterner D.E.
      • Berger S.L.
      Acetylation of histones and transcription related factors.
      ,
      • Strahl B.D.
      • Allis C.D.
      The language of covalent histone modifications.
      ,
      • Turner B.M.
      Cellular memory and the histone code.
      ]. The principal histone tails and DNA modifications and their functional impact on transcription are outlined in Fig. 3. In all eukaryotes protein coding genes transcribed by the Pol II are under the control of short discrete DNA elements arranged in promoters and enhancers. These sequences, and their complex embricate juxtapositions, dictate the binding of sequence-specific transcription factors (TFs) to activate, or repress, transcription in a gene-specific way. A common set of “general transcription farctors” (GTFs) are involved in the activation of essentially all genes. Among these, of primary importance is TBP and the associated TAFs, forming the TFIID complex. The accessibility of TFs to their target DNA sequences is regulated by the activity of a number of non-DNA binding coactivators, such as p300, CBP, PCAF/GCN5, CARM1, that (a) almost invariably harbor enzymatic activities to modify the histone tails in the nucleosomes, (b) form large >1 MDa-complexes with 10 or more subunits (SAGA, TFTC, Ccr4-Not) and (c) mediate the interplay between the gene specific factors and the general transcriptional machinery [
      • Sterner D.E.
      • Berger S.L.
      Acetylation of histones and transcription related factors.
      ,
      • Strahl B.D.
      • Allis C.D.
      The language of covalent histone modifications.
      ,
      • Turner B.M.
      Cellular memory and the histone code.
      ]. Since HBV cccDNA has a nucleosomal organization in infected cells and it is the template for cellular polymerases to transcribe all viral mRNAs and the RNA pregenome acetylation and deacetylation of cccDNA-bound histones might regulate transcription of viral chromatin and, by inference, viral replication. Using an antiacetylated-H3 or -H4 cccDNA ChIP assay we found that HBV replication is indeed regulated, both in cell based replication systems [
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ,
      • Lucifora J.
      • Durantel D.
      • Belloni L.
      • Barraud L.
      • Villet S.
      • Vincent I.E.
      • et al.
      Initiation of hepatitis B virus genome replication and production of infectious virus following delivery in HepG2 cells by novel recombinant baculovirus vector.
      ] and in the liver of HBV chronically infected patients [
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ] by the acetylation status of H3/H4 histones bound to the viral cccDNA in the nuclei of HBV-infected cells. The co-recruitment histone acetyltransferases (PCAF and p300/CBP) parallels viral replication in vitro[
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ,

      Cougot D, Wu Y, Buendia MA, Neuveut C. Role of HBx and its cellular partners in the regulation of viral transcription. In: 2007 international meeting the molecular biology of hepatitis B viruses, Rome, Italy, September 16–20, 2007.

      ] whereas histone deacetylase 1 (HDAC1) recruitment onto the cccDNA correlate with low HBV replication in vitro and with low viremia in vivo[
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ] (Fig. 4). The importance of cccDNA histone epigenetic modifications in the regulation of the viral transcription/replication cycle is further confirmed by the observation that abrogation of PCAF expression by specific siRNAs reduces HBV replication and acetylation of cccDNA bound histones whereas the class I and class III histone deacetylases inhibitors trichostatin (TSA), valproate (VPA) and nicotinamide (NAM) induce an evident increase of both cccDNA-bound acetylated H4 and HBV replication [
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ,
      • Belloni L.
      • Pollicino T.
      • Cimino L.
      • Raffa G.
      • Raimondo G.
      • Levrero M.
      HBX binds in vivo on the HBV minichromosome, modifies the epigenetic regulation of ccc-DNA function and potentiates HBV replication.
      ]. Interestingly, valproate, that is used clinically as an anticonvulsant and more recently in the treatment of bipolar syndromes, has been shown to also increase BPV, CMV and HIV viral replication in vitro, to reactivate lytic replication of latent HHV8 in primary effusion lymphoma (PEL) cells [
      • Klass C.M.
      • Krug L.T.
      • Pozharskaya V.P.
      • Offermann M.K.
      The targeting of primary effusion lymphoma cells for apoptosis by inducing lytic replication of human herpesvirus 8 while blocking virus production.
      ] and FDA has recently raised a warning for the use of the drug in CMV or HIV infected patients. Although VPA has been frequently associated with hepatoxicity and severe acute hepatis this has been mainly attributed to a direct mitochondrial toxicity and the possible interaction with a co-existing HBV infection has not been assessed. By coupling cccDNA quantification and the cccDNA ChIP assay we also found that HBV genotype (A vs. D) has no effect on the accumulation of cccDNA whereas the YMDD mutant displays a reduced cccDNA accumulation [
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ]. Interestingly, the acetylation of cccDNA-bound H4 histone is not affected either by the HBV genotype or by the presence of the YMDD mutation, suggesting that the YMDD mutant has a defect in cccDNA molecules accumulation but the cccDNA itself is then properly assembled as a minichromosome [
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ]. In addition, we confirmed that anti-polymerase drugs, notably lamivudine and adefovir, do not have a noticeable effect on the accumulation of cccDNA (cccDNA molecules per cell) and on the acetylation of cccDNA-bound histone acetylation [
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ]. On the contrary, in response to IFNα HDAC1 is actively recruited onto the HBV minichromosone and cccDNA-bound histone acetylation sharply decreases, without observing, in these short-term cell culture-based experiments, significant changes in the levels of cccDNA [
      • Belloni L.
      • Testoni B.
      • Scisciani C.
      • Pollicino T.
      • Raimondo G.
      • Levrero M.
      Modulation of the epigenetic regulation of the cccDNA function contributes to IFNα inhibition of HBV replication.
      ]. These observations suggest that the ability of class I interferon to inhibit HBV replication may be mediated, in addition to the known post-translational mechanisms and degradation of HBV transcripts [
      • Guidotti L.G.
      • Borrow P.
      • Brown A.
      • McClary H.
      • Koch R.
      • Chisari F.V.
      Noncytopathic clearance of lymphocytic choriomeningitis virus from the hepatocyte.
      ,
      • Rang A.
      • Gunther S.
      • Will H.
      Effect of interferon alpha on hepatitis B virus replication and gene expression in transiently transfected human hepatoma cells.
      ], also by the epigenetic modulation of the cccDNA function. We have also shown that the HBx regulatory protein produced in HBV replicating cells is recruited onto the cccDNA minichromosome. HBV mutants that do not express HBx are impaired in their replication and that exogenously expressed HBx trans-complements the replication defects [
      • Belloni L.
      • Pollicino T.
      • Cimino L.
      • Raffa G.
      • Raimondo G.
      • Levrero M.
      HBX binds in vivo on the HBV minichromosome, modifies the epigenetic regulation of ccc-DNA function and potentiates HBV replication.
      ,
      • Keasler V.V.
      • Hodgson A.J.
      • Madden C.R.
      • Slagle B.L.
      Enhancement of hepatitis B virus replication by the regulatory X protein in vitro and in vivo.
      ]. The kinetics of HBx recruitment on the cccDNA parallels HBV replication and is similar to that of the PCAF/GCN5 acetyltransferase [
      • Belloni L.
      • Pollicino T.
      • Cimino L.
      • Raffa G.
      • Raimondo G.
      • Levrero M.
      HBX binds in vivo on the HBV minichromosome, modifies the epigenetic regulation of ccc-DNA function and potentiates HBV replication.
      ]. Despite this observation and the physical interaction between the two proteins, we could not find any significant change in the recruitment of PCAF on the cccDNA in cells replicating the HBx defective virus. Instead, we found that cccDNA-bound histones are more rapidly hypo-acetylated in cells replicating the HBx mutant and the recruitment of the p300 acetyltransferase is severely impaired whereas the recruitment of the histone deacetylases hSirtl and HDAC1 is increased (Fig. 4) and occurs earlier. Accordingly, we show that in cells replicating the HBx mutant the pool of cccDNA is not reduced but the HBx mutant cccDNA transcribes significantly less pgRNA [
      • Belloni L.
      • Pollicino T.
      • Cimino L.
      • Raffa G.
      • Raimondo G.
      • Levrero M.
      HBX binds in vivo on the HBV minichromosome, modifies the epigenetic regulation of ccc-DNA function and potentiates HBV replication.
      ].
      Figure thumbnail gr3
      Fig. 3Chromatin modifications and transcription. (Upper left panel) Schematic representation of nucleosomes. (Upper right panel) Type, site and function of known chromatin marks. Numbers within brackets identify the aminoacid residues involved in specific modifications. (Lower panel) Schematic representation of a nucleosome with the more frequent histone modifications.
      Figure thumbnail gr4
      Fig. 4Schematic representation of cccDNA-bound histones acetylation status and the recruitment of chromatin modifying enzymes onto the viral minichromosome in relation to viral replication and infection phase [
      • Bock C.T.
      • Schwinn S.
      • Locarnini S.
      • Fyfe J.
      • Manns M.P.
      • Trautwein C.
      • et al.
      Structural organization of the hepatitis B virus minichromosome.
      ,
      • Pollicino T.
      • Belloni L.
      • Raffa G.
      • Pediconi N.
      • Squadrito G.
      • Raimondo G.
      • et al.
      Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
      ,
      • Belloni L.
      • Pollicino T.
      • Cimino L.
      • Raffa G.
      • Raimondo G.
      • Levrero M.
      HBX binds in vivo on the HBV minichromosome, modifies the epigenetic regulation of ccc-DNA function and potentiates HBV replication.
      ,
      • Belloni L.
      • Testoni B.
      • Scisciani C.
      • Pollicino T.
      • Raimondo G.
      • Levrero M.
      Modulation of the epigenetic regulation of the cccDNA function contributes to IFNα inhibition of HBV replication.
      ].
      In addition to post-translational modifications of histones DNA methylation of CpG islands also contribute to regulate gene expression. In silico analysis of HBV genotype A identifies two CpG islands in proximity to the HBV surface gene start codon (island 1) and to the enhancer 1/X gene promoter region (island 2) [
      • Vivekanandan P.
      • Thomas D.
      • Torbenson M.
      Hepatitis B viral DNA is methylated in liver tissues.
      ]. The HBV-DNA extracted from circulating virions does not display significant methylation whereas the HBV CpG islands are methylated in transfected HepG2 cells and in about 50% of HBV-infected liver tissues [
      • Vivekanandan P.
      • Thomas D.
      • Torbenson M.
      Hepatitis B viral DNA is methylated in liver tissues.
      ]. Although this study does not specifically analyze the HBV cccDNA methylation status it shows that HBV-DNA can be methylated in human tissues and thus DNA methylation may also play a role in the regulation of HBV gene expression.

      6. cccDNA is “suppressed” in occult hepatitis B

      Stability and long-lasting persistence of HBV cccDNA in the nuclei of hepatocytes represent the molecular basis of occult hepatitis B infection (OBI), a form of HBV infection characterized by the persistence of HBV-DNA in the liver (and in some cases also in the serum) of individuals who test negative for the HBV surface antigen (HBsAg) [
      • Vivekanandan P.
      • Thomas D.
      • Torbenson M.
      Hepatitis B viral DNA is methylated in liver tissues.
      ]. On the basis of the HBV antibody profile, OBI has been distinguished in seropositive-occult hepatitis B (when anti-HBc and/or anti-HBs are present) and seronegative OBI (when anti-HBc and anti-HBs are absent) [
      • Raimondo G.
      • Allain J.P.
      • Brunetto M.R.
      • Buendia M.A.
      • Chen D.S.
      • Colombo M.
      • et al.
      Statements from the Taormina expert meeting on occult hepatitis B virus infection.
      ]. In a small number of these individuals, failure to detect HBsAg and occult infection are due to HBV variants either producing an antigenically modified HBsAg that is not recognized by commercially available detection assays, or carrying mutations in the HBV regulatory regions or in the Pol gene responsible for defective protein synthesis and/or replication activity [
      • Brechot C.
      • Thiers V.
      • Kremsdorf D.
      • Nalpas B.
      • Pol S.
      • Paterlini-Brechot P.
      Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely occult?.
      ,
      • Hu K.Q.
      Occult hepatitis B virus infection and its clinical implications.
      ,
      • Torbenson M.
      • Thomas D.L.
      Occult hepatitis B.
      ,
      • Raimondo G.
      • Pollicino T.
      • Cacciola I.
      • Squadrito G.
      Occult hepatitis B virus infection.
      ]. Extensive molecular analyses of entire viral genomes have revealed that occult HBV populations have a large intra-individual genetic heterogeneity that does not differ from what is found in HBsAg-positive subjects [
      • Chaudhuri V.
      • Tayal R.
      • Nayak B.
      • Acharya S.K.
      • Panda S.K.
      Occult hepatitis B virus infection in chronic liver disease: full-length genome and analysis of mutant surface promoter.
      ,
      • Pollicino T.
      • Raffa G.
      • Costantino L.
      • Lisa A.
      • Campello C.
      • Squadrito G.
      • et al.
      Molecular and functional analysis of occult hepatitis B virus isolates from patients with hepatocellular carcinoma.
      ,
      • Vivekanandan P.
      • Kannangai R.
      • Ray S.C.
      • Thomas D.L.
      • Torbenson M.
      Comprehensive genetic and epigenetic analysis of occult hepatitis B from liver tissue samples.
      ]. In most cases viral genomic variability does not appear to play a fundamental role in inducing the occult HBV status and the majority of occult HBV cases are infected with replication-competent HBVs displaying a strong suppression of replication activity and gene expression with extremely low levels of virion production [
      • Chaudhuri V.
      • Tayal R.
      • Nayak B.
      • Acharya S.K.
      • Panda S.K.
      Occult hepatitis B virus infection in chronic liver disease: full-length genome and analysis of mutant surface promoter.
      ,
      • Pollicino T.
      • Raffa G.
      • Costantino L.
      • Lisa A.
      • Campello C.
      • Squadrito G.
      • et al.
      Molecular and functional analysis of occult hepatitis B virus isolates from patients with hepatocellular carcinoma.
      ]. In fact, in vitro functional analysis showed that occult viral isolates “re-acquire” normal capacities of replication, transcription and protein synthesis once extrapolated from the host’s liver microenvironment [
      • Pollicino T.
      • Raffa G.
      • Costantino L.
      • Lisa A.
      • Campello C.
      • Squadrito G.
      • et al.
      Molecular and functional analysis of occult hepatitis B virus isolates from patients with hepatocellular carcinoma.
      ]. In addition, several molecular virology studies have demonstrated the presence of both HBV cccDNA molecules and viral transcripts (including the viral pregenome) in the liver of occult infected individuals who may have or may not have HBV-DNA in the serum [
      • Pollicino T.
      • Raffa G.
      • Costantino L.
      • Lisa A.
      • Campello C.
      • Squadrito G.
      • et al.
      Molecular and functional analysis of occult hepatitis B virus isolates from patients with hepatocellular carcinoma.
      ,
      • Mason A.L.
      • Xu L.
      • Guo L.
      • Kuhns M.
      • Perrillo R.P.
      Molecular basis for persistent hepatitis B virus infection in the liver after clearance of serum hepatitis B surface antigen.
      ,
      • Marusawa H.
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      ,
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      Hepatitis B virus maintains its pro-oncogenic properties in the case of occult HBV infection.
      ]. Real-time PCR analysis has shown median intrahepatic HBV cccDNA levels about 1 log lower than those observed in HBeAg-negative chronic HBV carriers (0.002 copies/cell vs. 0.01 copies/cell) [
      • Werle-Lapostolle B.
      • Bowden S.
      • Locarnini S.
      • Wursthorn K.
      • Petersen J.
      • Lau G.
      • et al.
      Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy.
      ]. The persistence of such minute amounts of cccDNA is consistent with the hypothesis that the clinical resolution of HBV infection does not occur through a complete eradication of the virus from the liver and rather viral replication is controlled by the host’s immune system. The presence of a complete HBV replication cycle in HBsAg-negative cases is confirmed both by the demonstration of HBV-specific T-cell responses in patients with occult HBV infection, even when serum HBV markers were completely negative [
      • Zerbini A.
      • Pilli M.
      • Boni C.
      • Fisicaro P.
      • Penna A.
      • Di Vincenzo P.
      • et al.
      The characteristics of the cell-mediated immune response identify different profiles of occult hepatitis B virus infection.
      ] and by the observations that occult HBV can be transmitted through blood transfusion or organ transplantation and occult HBV carriers are at risk of HBV reactivation when exposed to immune suppression (i.e. hematologic malignancies, HIV infection, cancer chemotherapy, etc.) [
      • Lalazar G.
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      Screening, prevention and treatment of viral hepatitis B reactivation in patients with haematological malignancies.
      ,
      • Marzano A.
      • Angelucci E.
      • Andreone P.
      • Brunetto M.
      • Bruno R.
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      • et al.
      Prophylaxis and treatment of hepatitis B in immunocompromised patients.
      ]. The molecular mechanisms that keep viral replication under control in seronegative occult infections have not been fully defined. Innate immune response and/or interference by coinfecting agents (i.e. hepatitis C virus) may contribute as additional factors negatively influencing HBV replication and gene expression. However, according to our findings on the important role played by chromatin remodelling enzymes and the epigenetic modulation of cccDNA function we have found that the profound suppression of viral replication and viral transcription in OBI livers gene associates with the recruitment of several cellular several histone deacetylases and histone methylases onto the HBV minichromosome (Pollicino, personal observations) (Fig. 4) and with a specific pattern of HBV-DNA methylation in occult infected patients [
      • Vivekanandan P.
      • Kannangai R.
      • Ray S.C.
      • Thomas D.L.
      • Torbenson M.
      Comprehensive genetic and epigenetic analysis of occult hepatitis B from liver tissue samples.
      ].

      7. Conclusions

      The availability of sensitive molecular assays for direct cccDNA quantitation in liver tissues and the development of new techniques based upon the analysis of chromatin dynamics to study the regulation of cccDNA function both in cellular models of HBV replication and in vivo has significantly advanced our knowledge of the molecular mechanisms regulating HBV replication and persistence. One major conceptual advance has been the recognition of the existence of a complex network of epigenetic events that influence both cccDNA function and HBV replication. Although more work is needed to expand our knowledge, results suggest that inactive carriers, anti-HBe positive patients during low replication phases and patients with occult HBV infection may share, although to a different degree, common mechanisms of active cccDNA suppression mediated by epigenetic modifications of cccDNA and cccDNA-bound histones. The feasibility to modulate in a cell culture model cccDNA transcription using zinc finger proteins (ZFPs) engineered to specifically bind and repress the activity of the DHBV enhancer has been recently reported [
      • Zimmerman K.A.
      • Fischer K.P.
      • Joyce M.A.
      • Tyrrell D.L.
      Zinc finger proteins designed to specifically target duck hepatitis B virus covalently closed circular DNA inhibit viral transcription in tissue culture.
      ]. Although attractive, in principle this approach might be limited by the need for ZFPs to be delivered using viral vectors or included in liposomes or nanoparticles designed in order to target the infected liver. The identification of epigenetic marks of active and inactive cccDNA and the precise definition of the cellular enzymatic activities modulating transcription of the cccDNA minichromosome, as well as the cellular signaling pathways that modulate their activity, opens new perspectives for the identification of relevant therapeutic targets to permanently suppress HBV transcription/replication. On the other hand, we must continue our efforts to understand the molecular basis of cccDNA stability. Relevant issues would include: (a) the identification of the cellular proteins that are involved in the conversion of RC DNA into the cccDNA; (b) the identification of proteins and pathways affecting the size of the nuclear HBV cccDNA pool in different categories of HBV patients; (c) the comparison of naked vs. chromatinized cccDNA half-life to elucidate whether open/transcribed cccDNA would be less stable compared to packed/inactive cccDNA. This knowledge will hopefully allow us, in the long term, to move a step forward from the control of HBV replication and disease to the cure of HBV infection.

      Acknowledgements

      M.L. and G.R. are supported by grants from Associazione Italiana per la Ricerca sul Cancro and PRIN-MIUR (Progetti di Ricerca di Interesse Nazionale del Ministero dell’Istruzione, dell’Università e della Ricerca) J.P. and M.D. are supported by the Deutsche Forschungsgemeinschaft (DFG) Pe608/2-5. M.L. and J.P. were also supported by the Vigilance Network for the management of antiviral drug resistance (Virgil).

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