Eliminating cccDNA to cure hepatitis B virus infection

Discovery of a first-in-class orally available HBV cccDNA inhibitorJournal of HepatologyVol. 78Issue 4PreviewThe persistence of covalently closed circular DNA (cccDNA) in infected hepatocytes is the major barrier preventing viral eradication with existing therapies in patients with chronic hepatitis B. Therapeutic agents that can eliminate cccDNA are urgently needed to achieve viral eradication and thus HBV cure. Full-Text PDF See Article, pages 742–753 See Article, pages 742–753 Current treatment of chronic hepatitis B can suppress HBV replication efficiently. However, a functional cure of infection defined by hepatitis B surface antigen (HBsAg) loss rarely occurs,[1]Jeng W.J. Lok A.S. Should treatment indications for chronic hepatitis B Be expanded?.Clin Gastroenterol Hepatol. 2021; 19: 2006-2014Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar and thus long-term nucleos(t)ide analogue (NA) therapy is needed in the majority of patients. Because of the persistence of the viral minichromosome in the infected liver, i.e. covalently closed circular (ccc) DNA,2Martinez M.G. Boyd A. Combe E. Testoni B. Zoulim F. Covalently closed circular DNA: the ultimate therapeutic target for curing HBV infections.J Hepatol. 2021; 75: 706-717Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 3Boyd A. Lacombe K. Lavocat F. Maylin S. Miailhes P. Lascoux-Combe C. et al.Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.J Hepatol. 2016; 65: 683-691Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 4Villeret F. Lebosse F. Radenne S. Samuel D. Roche B. Mabrut J.-Y. et al.Early recurrence of Hepatitis B Virus infection on the liver graft: results of a prospective cohort study in transplanted patients.JHEP Rep. 2023; (in press)Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar viral relapse is almost universal when treatment is stopped prior to HBsAg loss. The major barriers to HBV cure include the persistence of a reservoir of HBV-infected cells harbouring cccDNA and/or integrated viral sequences, and the impaired innate and adaptive immune responses against HBV.[2]Martinez M.G. Boyd A. Combe E. Testoni B. Zoulim F. Covalently closed circular DNA: the ultimate therapeutic target for curing HBV infections.J Hepatol. 2021; 75: 706-717Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar,5Fanning G.C. Zoulim F. Hou J. Bertoletti A. Therapeutic strategies for hepatitis B virus infection: towards a cure.Nat Rev Drug Discov. 2019; 18: 827-844Crossref PubMed Scopus (280) Google Scholar, 6Maini M.K. Burton A.R. Restoring, releasing or replacing adaptive immunity in chronic hepatitis B.Nat Rev Gastroenterol Hepatol. 2019; 16: 662-675Crossref PubMed Scopus (76) Google Scholar, 7Meier M.A. Calabrese D. Suslov A. Terracciano L.M. Heim M.H. Wieland S. Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load.J Hepatol. 2021; 75: 840-847Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Wang et al. make an important contribution to the HBV cure field and report on their attempts to identify small molecules to reduce cccDNA in chronically infected hepatocytes.[8]Wang L. Zhu Q. Zhang J.D. Zhang Y. Ni X. Xiang K. et al.Discovery of a first-in-class orally available HBV cccDNA inhibitor.J Hepatol. 2023; 78: 742-753Abstract Full Text Full Text PDF Scopus (5) Google Scholar The episomal HBV cccDNA is located in the hepatocyte nucleus and serves as a transcriptional template for all HBV RNAs including pregenomic RNA (pgRNA) which is reverse transcribed into HBV DNA, and four messenger RNAs which are translated into viral proteins.[2]Martinez M.G. Boyd A. Combe E. Testoni B. Zoulim F. Covalently closed circular DNA: the ultimate therapeutic target for curing HBV infections.J Hepatol. 2021; 75: 706-717Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar cccDNA is derived not only from incoming virions but also from intracellular recycling of nucleocapsids. The dual source of cccDNA and its long half-life explain why cccDNA levels are minimally decreased even after many years of NA therapy.[3]Boyd A. Lacombe K. Lavocat F. Maylin S. Miailhes P. Lascoux-Combe C. et al.Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.J Hepatol. 2016; 65: 683-691Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar,[9]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.Gastroenterology. 2004; 126: 1750-1758Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar Permanent cccDNA elimination or silencing is the key objective to prevent any risk of viral reactivation, and thus cccDNA is considered as the ultimate target for HBV cure.[2]Martinez M.G. Boyd A. Combe E. Testoni B. Zoulim F. Covalently closed circular DNA: the ultimate therapeutic target for curing HBV infections.J Hepatol. 2021; 75: 706-717Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar In that respect, efforts have been made to standardize the technologies for cccDNA quantification to monitor the effect of new strategies aimed at HBV cure.[10]Allweiss L. Testoni B. Yu M. Lucifora J. Ko C. Qu B. et al.Quantification of the Hepatitis B Virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure.Gut. 2023 Jan 27; (gutjnl-2022-328380)https://doi.org/10.1136/gutjnl-2022-328380Crossref PubMed Scopus (5) Google Scholar After viral internalization via the cellular receptor sodium-taurocholate cotransporting polypeptide,[11]Yan H. Zhong G. Xu G. He W. Jing Z. Gao Z. et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife. 2012; 1e00049Crossref PubMed Scopus (1475) Google Scholar HBV delivers its 3.2 kb relaxed cicurlar (rc) DNA genome into the nuclei of the host cell. rcDNA is then repaired into a fully double stranded cccDNA, through a multistep process that involves several nuclear enzymes12Wei L. Ploss A. Hepatitis B virus cccDNA is formed through distinct repair processes of each strand.Nat Commun. 2021; 12: 1591Crossref PubMed Scopus (41) Google Scholar, 13Wei L. Ploss A. Mechanism of hepatitis B virus cccDNA formation.Viruses. 2021; 13Crossref Scopus (29) Google Scholar, 14Wei L. Ploss A. Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation.Nat Microbiol. 2020; 5: 715-726Crossref PubMed Scopus (62) Google Scholar (Fig. 1). Since NAs do not target the rcDNA-to-cccDNA conversion, and do not induce a complete suppression of viral DNA synthesis, a low level replenishment of cccDNA through new rounds of infection and/or intracellular recycling may occur even during effective NA therapy.[3]Boyd A. Lacombe K. Lavocat F. Maylin S. Miailhes P. Lascoux-Combe C. et al.Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.J Hepatol. 2016; 65: 683-691Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar,[15]Burdette D.L. Lazerwith S. Yang J. Chan H.L.Y. Delaney Iv W.E. Fletcher S.P. et al.Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.PLoS One. 2022; 17e0262516Crossref Scopus (6) Google Scholar It was also shown that the dynamics of cccDNA are affected by infected cell death and the resulting hepatocyte turnover which may lead to cccDNA clearance, provided that new virions are not produced or are neutralized by antibodies.[16]Summers J. Mason W.S. Residual integrated viral DNA after hepadnavirus clearance by nucleoside analog therapy.Proc Natl Acad Sci USA. 2004; 101: 638-640Crossref PubMed Scopus (76) Google Scholar cccDNA is wrapped around nucleosomes containing core histones and is associated with viral capsid and HBx proteins and host transcription factors, forming a highly stable minichromosome[2]Martinez M.G. Boyd A. Combe E. Testoni B. Zoulim F. Covalently closed circular DNA: the ultimate therapeutic target for curing HBV infections.J Hepatol. 2021; 75: 706-717Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar,[17]Tropberger P. Mercier A. Robinson M. Zhong W. Ganem D.E. Holdorf M. Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation.Proc Natl Acad Sci U S A. 2015; 112: E5715-E5724Crossref PubMed Scopus (174) Google Scholar (Fig. 1). The mechanism and kinetics of cccDNA chromatin configuration, including histone deposition and recycling, and epigenetic regulation are not well defined. It was shown that rcDNA repair into cccDNA and histone deposition are coordinated, as is the case for host chromosomes.[18]Locatelli M. Quivy J.P. Chapus F. Michelet M. Fresquet J. Maadadi S. et al.HIRA supports hepatitis B virus minichromosome establishment and transcriptional activity in infected hepatocytes.Cell Mol Gastroenterol Hepatol. 2022; 14: 527-551Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar Like cellular genes, cccDNA is subjected to the histone code thanks to the dynamic exchange between histone modifiers, chromatin remodelers and transcription factors.[19]Hong X. Kim E.S. Guo H. Epigenetic regulation of hepatitis B virus covalently closed circular DNA: implications for epigenetic therapy against chronic hepatitis B.Hepatology. 2017; 66: 2066-2077Crossref PubMed Scopus (138) Google Scholar Beside the elimination of the reservoir of infected cells, for instance by specific immune-mediated killing of infected hepatocytes, direct targeting of cccDNA remains a challenge.[5]Fanning G.C. Zoulim F. Hou J. Bertoletti A. Therapeutic strategies for hepatitis B virus infection: towards a cure.Nat Rev Drug Discov. 2019; 18: 827-844Crossref PubMed Scopus (280) Google Scholar Understanding the factors and mechanisms involved in cccDNA formation has revealed novel targets for inhibiting cccDNA biogenesis. It has been shown that cccDNA can be reduced by small molecule inhibitors that target various repair factors including: aphidicolin, a peptide derived from the cyclin-dependent kinase inhibitor p21, FEN-1 endonuclease inhibitor PTPD, topoisomerase inhibitors, DNA ligase inhibitors, and inhibitors of DNA checkpoint kinase ATR and CHK1.[13]Wei L. Ploss A. Mechanism of hepatitis B virus cccDNA formation.Viruses. 2021; 13Crossref Scopus (29) Google Scholar,[20]Xia Y. Guo H. Hepatitis B virus cccDNA: formation, regulation and therapeutic potential.Antivir Res. 2020; 180104824Crossref Scopus (69) Google Scholar,[21]Cai D. Mills C. Yu W. Yan R. Aldrich C.E. Saputelli J.R. et al.Identification of the disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation.Antimicrob Agents Chemother. 2012; 56: 4277-4288Crossref PubMed Scopus (187) Google Scholar Whether an approach targeting the host DNA repair pathway will be feasible to treat chronic HBV infections with a sufficient safety margin will require specific investigations. Moreover, it remains to be seen if blocking rcDNA to cccDNA conversion would lead to a significant reduction of the already established pool of cccDNA. Targeting the pool of already established cccDNA for its degradation or silencing of its transcription/expression has thus been the subject of several investigations. It was shown that interferon-alpha (IFN-α) and a lymphotoxin beta receptor agonist led to the non-cytolytic clearance of cccDNA via the upregulation of apolipoprotein B mRNA-editing enzyme 3 (APOBEC3) in HBV-infected HepaRG cells and primary human hepatocytes.[22]Lucifora J. Xia Y. Reisinger F. Zhang K. Stadler D. Cheng X. et al.Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.Science. 2014; 343: 1221-1228Crossref PubMed Scopus (701) Google Scholar Indeed, APOBEC3 members recognize and deaminate foreign DNA, leading to destruction of HBV DNA, and potentially to cccDNA deamination. However, other studies showed that APOBECs preferentially target single-stranded DNA and rcDNA[23]Nair S. Zlotnick A. Asymmetric modification of hepatitis B virus (HBV) genomes by an endogenous cytidine deaminase inside HBV cores informs a model of reverse transcription.J Virol. 2018; 92Crossref PubMed Google Scholar. Studies in in vitro-infected hepatocytes showed that IFN-α treatment did not lead to cccDNA deamination by APOBECs, and instead suggested that G-A hypermutations in virions occurred independently of IFN-α.[24]Seeger C. Sohn J.A. Complete spectrum of CRISPR/Cas9-induced mutations on HBV cccDNA.Mol Ther. 2016; 24: 1258-1266Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Thus, while HBV suppression was clearly demonstrated, direct cccDNA targeting by APOBECs remains controversial. Advances in the gene editing field have given rise to a wide range of tools available for various applications including the targeting of the HBV genome and cccDNA.[25]Martinez M.G. Smekalova E. Combe E. Gregoire F. Zoulim F. Testoni B. Gene editing technologies to target HBV cccDNA.Viruses. 2022; 14Crossref Scopus (6) Google Scholar Gene-editing approaches using meganucleases, Zinc-finger nucleases and TALENs (transcriptional activator-like effector nucleases) can disrupt the viral genome in a permanent manner.[26]Cradick T.J. Keck K. Bradshaw S. Jamieson A.C. McCaffrey A.P. Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs.Mol Ther. 2010; 18: 947-954Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar,[27]Bloom K. Ely A. Mussolino C. Cathomen T. Arbuthnot P. Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.Mol Ther. 2013; 21: 1889-1897Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar CRISPR-Cas9 can be redirected to DNA sequences by redesigning the guide RNAs so that they are complementary to the desired target.[28]Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (10186) Google Scholar It was shown that CRISPR-Cas9 can target HBV DNA and cccDNA in HBV-infected hepatocytes.[2]Martinez M.G. Boyd A. Combe E. Testoni B. Zoulim F. Covalently closed circular DNA: the ultimate therapeutic target for curing HBV infections.J Hepatol. 2021; 75: 706-717Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar,[25]Martinez M.G. Smekalova E. Combe E. Gregoire F. Zoulim F. Testoni B. Gene editing technologies to target HBV cccDNA.Viruses. 2022; 14Crossref Scopus (6) Google Scholar,[29]Martinez M.G. Combe E. Inchauspe A. Mangeot P.E. Delberghe E. Chapus F. et al.CRISPR-Cas9 targeting of hepatitis B virus covalently closed circular DNA generates transcriptionally active episomal variants.mBio. 2022; 13e0288821Crossref Scopus (9) Google Scholar Several challenges remain to be addressed for the development of CRISPR-Cas9 technologies to treat chronic HBV infections, including potential off target effects, double stranded breaks leading to chromosomal recombination, and efficient delivery of the ribonucleoprotein complex to the infected liver.[25]Martinez M.G. Smekalova E. Combe E. Gregoire F. Zoulim F. Testoni B. Gene editing technologies to target HBV cccDNA.Viruses. 2022; 14Crossref Scopus (6) Google Scholar The development of new base and prime editing technologies has enabled DNA rewriting without cleavage, allowing for more precise DNA sequence modification and reducing the risk of host genome rearrangements. Yang et al. recently showed that cytosine base editors targeting the S open reading frame led to a reduction of HBV replication in cultured hepatocytes.[30]Yang Y.C. Chen Y.H. Kao J.H. Ching C. Liu I.J. Wang C.C. et al.Permanent inactivation of HBV genomes by CRISPR/Cas9-Mediated non-cleavage base editing.Mol Ther Nucleic Acids. 2020; 20: 480-490Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Further studies evaluating the direct effect of base editors on cccDNA and its fate after editing are awaited.[31]Martinez M.G. Smekalova E. Combe E. Packer M.S. Barrera L.A. Dejene S. et al.CCCDNA inactivation using cytosine base editors.Hepatology. 2021; 74 (504A-A)PubMed Google Scholar Epigenetic silencing of cccDNA to shut down its transcription is another potential approach to functional cure. cccDNA transcription is regulated by the host cell epigenetic machinery. It was shown that IFN-α treatment leads to changes in post-translational modifications of cccDNA-associated histones and recruitment of transcriptional repressors, which could lead to a reduction in its transcriptional activity. Pro-inflammatory cytokines such as IL-6 and IL-1β also have a direct effect on the transcriptional activity of cccDNA, without affecting its levels.[32]Isorce N. Testoni B. Locatelli M. Fresquet J. Rivoire M. Luangsay S. et al.Antiviral activity of various interferons and pro-inflammatory cytokines in non-transformed cultured hepatocytes infected with hepatitis B virus.Antivir Res. 2016; 130: 36-45Crossref PubMed Scopus (57) Google Scholar cccDNA-associated histones can be directly targeted to silence cccDNA transcription. Drugs interfering with histone acetylation or methylation were shown to suppress cccDNA transcription (reviewed in19Hong X. Kim E.S. Guo H. Epigenetic regulation of hepatitis B virus covalently closed circular DNA: implications for epigenetic therapy against chronic hepatitis B.Hepatology. 2017; 66: 2066-2077Crossref PubMed Scopus (138) Google Scholar, 20Xia Y. Guo H. Hepatitis B virus cccDNA: formation, regulation and therapeutic potential.Antivir Res. 2020; 180104824Crossref Scopus (69) Google Scholar). Though epigenetic modifiers led to a decrease in viral parameters, a major caveat is that they could also interfere with host gene regulation. The discovery of the role of HBx in cccDNA transcription and the initiation of infection has opened new perspectives on the search for virus-specific targets to silence cccDNA.[33]Decorsiere A. Mueller H. van Breugel P.C. Abdul F. Gerossier L. Beran R.K. et al.Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.Nature. 2016; 531: 386-389Crossref PubMed Scopus (348) Google Scholar Small-interfering RNAs (siRNAs) targeting HBx mRNA transiently reduced HBx levels, leading to Smc5/6 complex re-appearance.[34]Kornyeyev D. Ramakrishnan D. Voitenleitner C. Livingston C.M. Xing W. Hung M. et al.Spatiotemporal analysis of hepatitis B virus X protein in primary human hepatocytes.J Virol. 2019; 93Crossref PubMed Scopus (29) Google Scholar Moreover, it was shown that silencing all viral transcripts using a combination of siRNAs and peg-IFNα substantially decreased HBx protein levels, leading to Smc6 rebound in vivo.[35]Allweiss L. Giersch K. Pirosu A. Volz T. Muench R.C. Beran R.K. et al.Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo.Gut. 2022; 71: 372-381Crossref PubMed Scopus (28) Google Scholar Small molecules targeting the HBx-DDB1 interaction, nitazoxanide and pevonedistat (MLN4924), have been tested in cultured hepatocytes and were shown to induce a rebound of Smc5/6 associated with a decrease in both cccDNA transcriptional activity and viral parameters.[36]Sekiba K. Otsuka M. Ohno M. Yamagami M. Kishikawa T. Suzuki T. et al.Inhibition of HBV transcription from cccDNA with nitazoxanide by targeting the HBx-DDB1 interaction.Cel Mol Gastroenterol Hepatol. 2019; 7: 297-312Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar,[37]Sekiba K. Otsuka M. Ohno M. Yamagami M. Kishikawa T. Seimiya T. et al.Pevonedistat, a neuronal precursor cell-expressed developmentally down-regulated protein 8-activating enzyme inhibitor, is a potent inhibitor of hepatitis B virus.Hepatology. 2019; 69: 1903-1915Crossref PubMed Scopus (38) Google Scholar The in vivo confirmation of these findings is warranted. Given the complexity of cccDNA biology and its key role in viral persistence, the discovery of orally available antiviral agents that could deplete the pool of intrahepatic cccDNA would change the HBV cure paradigm. Wang et al.[8]Wang L. Zhu Q. Zhang J.D. Zhang Y. Ni X. Xiang K. et al.Discovery of a first-in-class orally available HBV cccDNA inhibitor.J Hepatol. 2023; 78: 742-753Abstract Full Text Full Text PDF Scopus (5) Google Scholar report the discovery of a small molecule, ccc_R08, that decreases the HBV cccDNA reservoir in highly relevant study models, including a high-throughput screening in HBV-infected primary human hepatocytes and HepDES19 cells, and experimental validation in the HBV circle mouse model and the uPA-SCID humanized liver mouse model. The compound ccc_R08 demonstrated its ability to reduce HBsAg, HBeAg, HBV DNA, and pgRNA in the blood circulation and to decrease the cccDNA reservoir in the liver. It is the first time that a small molecule has been shown to act on the already established cccDNA pool. Although the authors called their compound a cccDNA 'inhibitor', the exact mode of action of the compound remains to be determined as it may be ascribed to cellular processes involved in cccDNA degradation or instability (Fig. 1). Interestingly, their findings showed that ccc_R08 administration was associated with a strong reduction in viral transcripts, i.e. pre-C/pregenomic and X transcripts, as well as an impact on host gene expression. In their study models, where integration is thought to be negligeable, HBsAg is mainly produced by transcriptionally active cccDNA. Therefore, the observation that cccDNA levels were reduced to a lesser extent than HBsAg levels suggests that the compound might preferentially destabilize the transcriptionally active cccDNA species. Thus, it will be interesting to study the chromatin configuration of residual cccDNA to determine if compacted cccDNA species would be less sensitive to ccc_R08. The integrated analysis of host transcriptomics and viral RNA expression and replication suggested that the anti-HBV effect of ccc_R08 is most likely mediated by the regulation of a host gene regulatory network. To overcome the difficulties encountered with knockdown and knockout approaches, the authors used a clever forward and reverse pharmacology approach and identified two gene lists that are likely involved in the pharmacological effect of ccc_R08. This enabled them to identify already reported putative targets of ccc_R08 including CHEK1, CHEK2, TOP2A, and ATM,[13]Wei L. Ploss A. Mechanism of hepatitis B virus cccDNA formation.Viruses. 2021; 13Crossref Scopus (29) Google Scholar but also novel candidates to be explored by future studies, such as transcription factors, druggable proteins, and signal transduction proteins. Further functional investigations of these candidates may reveal new avenues for the identification of molecular targets involved in cccDNA depletion. Altogether, this elegant study by Wang et al.,[8]Wang L. Zhu Q. Zhang J.D. Zhang Y. Ni X. Xiang K. et al.Discovery of a first-in-class orally available HBV cccDNA inhibitor.J Hepatol. 2023; 78: 742-753Abstract Full Text Full Text PDF Scopus (5) Google Scholar not only provides new hope in the search for drugs to eliminate cccDNA, but also provides the ground to generate new knowledge on the cellular networks involved in the persistence and regulation of the HBV minichromosome. FZ and BT have received public grants overseen by the French National Research Agency (ANR) as part of the second "Investissements d'Avenir" program (reference: ANR-17-RHUS-0003) and by the European Union (grant EU H2020-847939-IP-cure-B). FZ received consulting fees from Aicuris, Aligos, Antios, Assembly, Blue Jay, Evotec, Gilead, GSK, Zhimeng. FZ and BT received research grants from Assembly, Beam, Janssen, Viravaxx. Please refer to the accompanying ICMJE disclosure forms for further details. FZ wrote the manuscript, BT prepared the figure and revised the manuscript. 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