Review Article

HBV cccDNA and Its Potential as a Therapeutic Target

Anjing Zhu, Xinzhong Liao, Shuang Li, Hang Zhao, Limin Chen, Min Xu* and Xiaoqiong Duan*
Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China

Abstract Integration of the HBV DNA into the host genome begins
immediately after infection of hepatocytes.11 After transcrip-
Chronic hepatitis B virus infection continues to be a major tion and translation, pgRNA is packaged and assembled in the
health burden worldwide. It can cause various degrees of liver nucleocapsid, where it is transcribed to relaxed circular DNA.
damage and is strongly associated with the development of Finally, the nucleocapsid is enveloped and the virion is
liver cirrhosis and hepatocellular carcinoma. Covalently secreted from the hepatocyte.12,13
closed circular DNA in the nucleus of infected cells cannot The current standard therapy for HBV infection includes
be disabled by present therapies which may lead to HBV PEGylated interferon a and nucleotide analogues (NAs). For
persistence and relapse. In this review, we summarized the example, a recent study showed that interferon a induces a
current knowledge on hepatitis B virus covalently closed long-term and sustainable suppression of cccDNA transcrip-
circular DNA and its potential role as a therapeutic target. tion, possibly by altering epigenetic modification of cccDNA
Citation of this article: Zhu A, Liao X, Li S, Zhao H, Chen L, minichromosomes.13 The inhibition of HBV replication by NAs
Xu M, et al. HBV cccDNA and its potential as a therapeutic is mediated by targeting the viral RNA-dependent DNA poly-
target. J Clin Transl Hepatol 2019;7(3):258–262. doi: merase which catalyzes the reverse transcription of pgRNA to
10.14218/JCTH.2018.00054. mature viral DNA. Five NAs have been approved for clinical
use since 1998, including lamivudine, entecavir, telbivudine,
adefovir dipivoxil and tenofovir.14 Although NAs have been
considered as first-line therapy for the treatment of chronic
Hepatitis B virus (HBV) life cycle and therapies HBV infection due to their high efficiency,13 they have failed to
cure HBV in most cases because they cannot clear cccDNA.15
HBV was discovered by Blumberg and colleagues1 in 1965. As such, virus rebound occurs following the termination of
Today, there are about 257 million people chronically infected NAs treatment. Obviously, disabling the cccDNA is key in
with HBV, which causes 650000 deaths worldwide every year. terms of curing HBV infection.16
According to World Health Organization reports, HBV caused
887000 deaths, mostly from complex diseases (including cir-
HBV cccDNA: Formation and modification
rhosis and hepatocellular carcinoma) in 2015. HBV infection
brings a huge burden to people and even society.
cccDNA is stable and acts as a virus transcription template.
The HBV genome is a circular, partially double-stranded
There are 3–50 copies of cccDNA per infected cell, and the
DNA, which is enveloped by an outer lipoprotein with an inner
number of copies decreases when the host cell divides. Thus,
nucleocapsid core. HBV DNA is only 3.2kb in length and
recycling of the new relaxed circular form to the nucleus
contains four overlapping open reading frames, known as S,
occurs in order to maintain the relatively stable cccDNA
C, P and X. The four open reading frames code for 7 viral
copy number.17 There are three main steps in conversion of
proteins: pre-S1, pre-S2, S, C, pre-C, X protein (commonly
the relaxed circular form to cccDNA: (1) unlocking of the
known as HBX), and HBV polymerase.2–4 Once infected, HBV
protein, which is covalently linked to the 5′ end of the
enters host hepatocytes by binding to the sodium taurocho-
(–)-DNA; (2) removal of the 5′ end of the (+)-strand consist-
late cotransporting polypeptide NTCP and being endocy-
ing of an RNA oligonucleotide; and (3) covalently ligation
tosed.5 After entry, the virus releases its DNA-containing
of both strands.18,19 Recent studies have shown that the
nucleocapsid into the cytoplasm, which is then transported
host DNA damage response is involved in the formation of
to the nucleus. In the nucleus, the viral DNA is converted
cccDNA, but the exact mechanism remains to be clarified.20
from its relaxed circular form to closed covalent circular
Epigenetic modifications, like histone acetylation and DNA
DNA (cccDNA).6–9 The cccDNA is transcribed into HBV prege-
methylation, play an important role in the transcriptional
nomic RNA (pgRNA) and several subgenomic RNAs.6,10
activity of cccDNA.21–24 Six CpG islands have been reported
in the HBV genome. The CpG islands distribute differently in
Keywords: Hepatitis B virus (HBV); Covalently closed circular DNA (cccDNA);
Therapeutic target.
HBV genotypes, overlapping some functional genes. Three
Abbreviations: cccDNA, covalently closed circular DNA; CRISPR/Cas, clustered conventional CpG islands (I, II, III) are potential targets for
regularly interspaced short palindromic repeats/CRISPR associated; HBV, chronic HBV DNA methylation (Fig. 1).25
hepatitis B virus; NA, nucleotide analogue; pgRNA, pregenomic RNA; TALEN, tran-
scription activator-like effector nuclease; ZFN, zinc finger nuclease.
Received: 26 September 2018; Revised: 2 April 2019; Accepted: 10 July 2019 Novel strategies for eradicating HBV cccDNA
*Correspondence to: Min Xu and Xiaoqiong Duan, Institute of Blood Transfusion,
Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu
610052, China. Tel: +86-135-4080-7307, E-mail: xumin@[Link] (MX) HBV cure depends on disabling of the HBV cccDNA, which is
or xiaoqiongduan@[Link] (XD) very difficult because cccDNA resides in the nucleus as an

258 Journal of Clinical and Translational Hepatology 2019 vol. 7 | 258–262

Copyright: © 2019 Authors. This article has been published under the terms of Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0), which
permits noncommercial unrestricted use, distribution, and reproduction in any medium, provided that the following statement is provided. “This article has been published
in Journal of Clinical and Translational Hepatology at DOI: 10.14218/JCTH.2018.00054 and can also be viewed on the Journal’s website at [Link]
Zhu A. et al: HBV cccDNA as a therapeutic target

gene editing can be used in combination with gene-silencing

technologies for antiHBV therapy. Using TALEN-mediated
homology directed recombination to introduce artificial
primary miRNAs into HBV genome could boost the antiviral
efficacy of TALENs.35
The CRISPR/Cas system is the adaptive immunity of
bacteria and archaea, and acts against invading foreign DNA
via RNA-guided DNA cleavage.36–40 The CRISPR/Cas9 system
is a newly developed programmable genome-editing tool and
allows for sequence-specific cleavage of DNA. Compared to
other genome-editing tools, the advantage of the CRIPSR/
Cas9 system lies in its simplicity and flexibility in design.
Several studies have demonstrated that the CRISPR/Cas9
system can efficiently destroy HBV cccDNA.41–45 Using the
CRISPR/Cas9 system, scientists have completely excised a
full-length 3175bp integrated HBV DNA from the host
genome and disrupted the HBV cccDNA in a stable HBV cell
line.44,45 Therefore, gene editing seems highly promising for
disabling HBV cccDNA.29,34
These three genome-editing technologies work similarly
by targeting and modifying DNA sequences using engineered
nucleases, thereby inducing a targeted DNA double-strand
break that stimulates the cellular DNA repair mechanisms.
However, the engineered nucleases, recognition area, and the
molecular mechanism are different.46 Engineered ZFNs and
TALENs are composed of a DNA binding domain and a Fok I
nuclease motif. The CRISPR-Cas9 genome editing system
Fig. 1. Distribution diagram of CpG islands. Island I overlaps the start site of
the S gene; island II contains enhance I and the X gene promoter, close to the core
consists of two components: a “guide” RNA and a nonspecific
gene promoter and enhance II; island III overlaps the Sp1 promoter and the start CRISPR-associated endonuclease (the Cas9). The recognition
codon of the P gene. areas of the ZFNs, TALENs, CRISPR/Cas system are zinc-
finger proteins, NLS, and sgRNA, respectively. ZFNs and
TALENs use the Fok I enzyme to cleave target DNA, while
episomal plasmid-like molecule to produce progeny CRISPR/Cas uses the Cas protein. The target sizes among
virus.15,26 Several gene therapy strategies have been pro- the three genome-editing tools are also different, that is
posed to disable HBV cccDNA. (9-12bp)*2 for ZFNs and TALENs, 20bp+ NGG for CRISPR/
Cas.29,46,47
Silencing cccDNA expression by gene editing There are some limitations for these technologies. First,
techniques specificity can be altered by context-dependent effects
caused by interactions among neighboring zinc fingers of
Several genomic editing technologies, including zinc finger the DNA binding domain, Second, the large size of TALENs
nucleases (ZFNs), transcription activator-like effector makes it difficult to deliver. Finally, the CRISPR/Cas9 system
nucleases (TALENs) and the clustered regularly interspaced faces several challenges like in vivo delivery efficiency and
short palindromic repeats/CRISPR associated (CRISPR/Cas) off-target cleavage.48,49 Cutting integrated HBV genomes by
system, have been used to disrupt HBV cccDNA. CRISPR/Cas9 also raises serious concerns, because this
ZFNs, a custom DNA endonuclease, are used to create a manipulation can cause genome instability. Compared to
DNA double-strand break in a specific target site and repair by ZFNs and TALENs, the two advantages of the CRISPR/Cas9
creating sequence alterations at the cleavage sites.27–29 system have contributed to the advancement of the new tech-
Through a proof-of-concept experiment, scientists found nology and generated widespread interest, according to its
that anti-HBV ZFNs (cognate 6L and 6R ZFN pair) disrupted simplicity and flexibility. Thus, we believe that the CRISPR/
36% of plasmid-derived viral sequences in a cell culture Cas9 system is very promising for curing chronic HBV once
model. Weber et al.30 designed three ZFNs targeting HBV several challenges are solved.50
polymerase, gene X and core, and delivered them into the
HepAD38 cells by self-complementary adeno-associated Silencing cccDNA transcription by epigenetic
viral vectors, respectively. They found these HBV-targeted modifications
ZFNs produced a sustained suppression of HBV levels over
the course of the experiment.31 HBV cccDNA exists in the nucleus as multiple copies of
TALENs are similar to ZFNs. However, the DNA-binding nucleosome-decorated minichromosomes, which indicates
domain of TALENs is highly repeated and derived from tran- that epigenetic modifications may influence HBV replication
scription activator-like III effectors, which are proteins and persistent infection.51 It has been shown that DNA meth-
secreted by Xanthomonas bacteria.32,33 The efficiency of ylation and histone acetylation are required for cccDNA
TALENs in reducing HBV productions in cell culture was first formation.22 Therefore, regulation of DNA methylation or
described by Bloom et al.34 in 2013. The investigators found histone acetylation is a potential method to reduce
that targeting the S and C regions of cccDNA led to 35% cccDNA.21,52–55 Interferon a treatment has been adopted to
decrease in cccDNA molecules in HepG2 cell lines. Moreover, silence cccDNA through epigenetic modification. It has been

Journal of Clinical and Translational Hepatology 2019 vol. 7 | 258–262 259

 Zhu A. et al: HBV cccDNA as a therapeutic target

found that interferon a inhibits HBV replication by regulating problem and finding ways to deliver in vivo. What is more, for
the epigenetic modification of HBV cccDNA. Interferon a a long time, lack of robust, reliable and quantifiable HBV
represses the transcription of HBV cccDNA through recruit- cccDNA models has delayed the development of cccDNA
ment of the histone deacetylases HDAC1 and hSirt1 and therapies. Recently, Yuan et al.68 established a cell line
decreasing the acetylation of cccDNA-bound histones.56 through integrating 2–60 copies of the monomeric HBV
Hyun et al.24 found that short hairpin RNA induced HBV genome into HepG2-derived cell lines, where the cccDNA
cccDNA methylation to inhibit its transcription in human hep- could be produced and detected with specific primers. The
atoma cells. HBV cccDNA transcription was regulated by CpG establishment of a cell line will provide a proper model for
methylation during chronic HBV infection.21 CpG island II evaluation of drugs or therapies targeting on cccDNA.
methylation has been shown to significantly decrease Though there are challenges to overcome for both gene
cccDNA transcription and subsequent viral core DNA replica- editing- and epigenetic modification-based strategies and it
tion, while CpG island III methylation has been shown to be there remains a long road from basic research to clinical appli-
associated with low serum HBsAg titers (Fig. 1).21,53,54,57–59 cation, it is very likely that we will conquer HBV similar to the
Interestingly, HBx can reduce chromatin-mediated transcrip- hepatitis C virus cure in the future.
tional repression of HBV cccDNA caused by SETDB1 histone
methyltransferase and therefore allow the establishment of
Conflict of interest
active chromatin.60 Recently, it has also been reported that
HB core carboxyl-terminal domain arginine residues reduced
The authors have no conflict of interests related to this
acetylation of cccDNA-bound histones and thus reduced the
publication.
interaction of HBc with cccDNA.61 Two enzymes have been
found to regulate the methylation.62,63 Protein arginine meth-
yltransferase 5 can regulate symmetric dimethylation of argi- Author contributions
nine 3 on histone 4 of cccDNA.62 The silent mating type
information regulation 2 homolog 3 SIRT3 restricts HBV tran- Collected the data and wrote the manuscript (AZ, XD),
scription and replication via epigenetic regulation of cccDNA, revised the manuscript and answered the reviewersquestions
involving SUV39H1 and SETD1A histone methyltransferases.63 (AZ, MX), directed and wrote the manuscript with comments
HBV replication is also regulated by the acetylation status (LC), helped to edit the manuscript (XL, SL, HZ). All authors
of HBV cccDNA-bound histone 3 and histone 4.22 The Np95/ have seen and approved the content of this manuscript.
ICBP90-like RING finger protein NIRF, a novel E3 ubiquitin
ligase, has been found to inhibit HBV DNA replication and
References
hepatitis B e antigen secretion in HepG2 cells through reduc-
ing HBV cccDNA-bound histone 3.64 Curcumin can inhibit HBV [1] Blumberg BS, Gerstley BJ, Hungerford DA, London WT, Sutnick AI. A serum
replication via down-regulation of cccDNA-bound histone ace- antigen (Australia antigen) in Down’s syndrome, leukemia, and hepatitis.
tylation.65 Retinoid X receptor a was also reported to be able Ann Intern Med 1967;66:924–931. doi: 10.7326/0003-4819-66-5-924.
to regulate the replication of HBV and modulate the HBV [2] Tiollais P, Pourcel C, Dejean A. The hepatitis B virus. Nature 1985;317:489–
495. doi: 10.1038/317489a0.
cccDNA epigenetically.66 In addition, basal core promoter [3] Arzumanyan A, Reis HM, Feitelson MA. Pathogenic mechanisms in HBV- and
mutations were found to inhibit viral replication through mod- HCV-associated hepatocellular carcinoma. Nat Rev Cancer 2013;13:123–
ulating the acetylation and deacetylation of cccDNA-bound 135. doi: 10.1038/nrc3449.
histones, while preCore mutations have been shown to have [4] Doo EC, Ghany MG. Hepatitis B virology for clinicians. Clin Liver Dis 2010;14:
397–408. doi: 10.1016/[Link].2010.05.001.
no effect on viral replication.67 These collective findings indi- [5] Li W. The hepatitis B virus receptor. Annu Rev Cell Dev Biol 2015;31:125–
cate that regulation of the DNA methylation and histone ace- 147. doi: 10.1146/annurev-cellbio-100814-125241.
tylation may inactivate cccDNA transcription and thus inhibit [6] Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev 2000;
HBV replication. 64:51–68. doi: 10.1128/mmbr.64.1.51-68.2000.
[7] Bock CT, Schranz P, Schröder CH, Zentgraf H. Hepatitis B virus genome is
Disabling cccDNA is the key to curing hepatitis B. Studies organized into nucleosomes in the nucleus of the infected cell. Virus Genes
have shown the great potential of gene editing technologies 1994;8:215–229. doi: 10.1007/bf01703079.
for disrupting the cccDNA in cultured cells and in hydro- [8] Newbold JE, Xin H, Tencza M, Sherman G, Dean J, Bowden S, et al. The
dynamically-injected mice. However, in the case of HBV covalently closed duplex form of the hepadnavirus genome exists in situ as
a heterogeneous population of viral minichromosomes. J Virol 1995;69:
infection, there are some disadvantages of using gene 3350–3357.
editing technologies. First, HBV DNA often integrates into [9] Zoulim F. New insight on hepatitis B virus persistence from the study of intra-
the host genome and undesirable gene cleavage may occur. hepatic viral cccDNA. J Hepatol 2005;42:302–308. doi: 10.1016/[Link].
Second, off-target effects has been observed in some studies. 2004.12.015.
[10] Block TM, Guo H, Guo JT. Molecular virology of hepatitis B virus for clinicians.
Finally, the means by how to deliver in vivo remains a chal- Clin Liver Dis 2007;11:685–706. doi: 10.1016/[Link].2007.08.002.
lenge for therapeutic application of these technologies. The [11] Tu T, Budzinska MA, Vondran FWR, Shackel NA, Urban S. Hepatitis B Virus
epigenetic modifications mentioned above are related to the DNA Integration Occurs Early in the Viral Life Cycle in an In Vitro Infection
replication and transcription of HBV cccDNA. While they can Model via Sodium Taurocholate Cotransporting Polypeptide-Dependent
Uptake of Enveloped Virus Particles. J Virol 2018;92:e02007–17. doi: 10.
regulate the HBV cccDNA, they cannot eliminate the HBV 1128/JVI.02007-17.
cccDNA. [12] Huovila AP, Eder AM, Fuller SD. Hepatitis B surface antigen assembles in a
post-ER, pre-Golgi compartment. J Cell Biol 1992;118:1305–1320. doi: 10.
1083/jcb.118.6.1305.
Conclusions [13] Tang LSY, Covert E, Wilson E, Kottilil S. Chronic hepatitis B infection: A
review. JAMA 2018;319:1802–1813. doi: 10.1001/jama.2018.3795.
In conclusion, disabling or complete inactivation of cccDNA is [14] Liaw YF, Sung JJ, Chow WC, Farrell G, Lee CZ, Yuen H, et al. Lamivudine for
patients with chronic hepatitis B and advanced liver disease. N Engl J Med
the desired end-point of HBV treatment. However, the most 2004;351:1521–1531. doi: 10.1056/NEJMoa033364.
urgent issues should be addressed before using gene editing [15] Zhou T, Guo H, Guo JT, Cuconati A, Mehta A, Block TM. Hepatitis B virus e
technologies for HBV treatment include solving the off-target antigen production is dependent upon covalently closed circular (ccc) DNA in

260 Journal of Clinical and Translational Hepatology 2019 vol. 7 | 258–262

Zhu A. et al: HBV cccDNA as a therapeutic target

HepAD38 cell cultures and may serve as a cccDNA surrogate in antiviral [39] Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, et al.
screening assays. Antiviral Res 2006;72:116–124. doi: 10.1016/[Link]. The CRISPR/Cas bacterial immune system cleaves bacteriophage and
2006.05.006. plasmid DNA. Nature 2010;468:67–71. doi: 10.1038/nature09523.
[16] Seeger C, Mason WS. Molecular biology of hepatitis B virus infection. Virol- [40] Manica A, Zebec Z, Teichmann D, Schleper C. In vivo activity of CRISPR-
ogy 2015;479–480:672–686. doi: 10.1016/[Link].2015.02.031. mediated virus defence in a hyperthermophilic archaeon. Mol Microbiol
[17] Ji M, Hu K. Recent advances in the study of hepatitis B virus covalently closed 2011;80:481–491. doi: 10.1111/j.1365-2958.2011.07586.x.
circular DNA. Virol Sin 2017;32:454–464. doi: 10.1007/s12250-017-4009-4. [41] Seeger C, Sohn JA. Complete Spectrum of CRISPR/Cas9-induced Mutations
[18] Qi Y, Gao Z, Xu G, Peng B, Liu C, Yan H, et al. DNA polymerase k is a key on HBV cccDNA. Mol Ther 2016;24:1258–1266. doi: 10.1038/mt.2016.94.
cellular factor for the formation of covalently closed circular DNA of hepatitis [42] Ramanan V, Shlomai A, Cox DB, Schwartz RE, Michailidis E, Bhatta A, et al.
B virus. PLoS Pathog 2016;12:e1005893. doi: 10.1371/[Link]. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus.
1005893. Sci Rep 2015;5:10833. doi: 10.1038/srep10833.
[19] Königer C, Wingert I, Marsmann M, Rösler C, Beck J, Nassal M. Involvement [43] Karimova M, Beschorner N, Dammermann W, Chemnitz J, Indenbirken D,
of the host DNA-repair enzyme TDP2 in formation of the covalently closed Bockmann JH, et al. CRISPR/Cas9 nickase-mediated disruption of hepatitis
circular DNA persistence reservoir of hepatitis B viruses. Proc Natl Acad Sci B virus open reading frame S and X. Sci Rep 2015;5:13734. doi: 10.
U S A 2014;111:E4244–E4253. doi: 10.1073/pnas.1409986111. 1038/srep13734.
[20] Schreiner S, Nassal M. A role for the host DNA damage response in hepatitis [44] Liu X, Hao R, Chen S, Guo D, Chen Y. Inhibition of hepatitis B virus by the
B virus cccDNA formation-and beyond? Viruses 2017;9:E125. doi: 10. CRISPR/Cas9 system via targeting the conserved regions of the viral
3390/v9050125. genome. J Gen Virol 2015;96:2252–2261. doi: 10.1099/vir.0.000159.
[21] Kim JW, Lee SH, Park YS, Hwang JH, Jeong SH, Kim N, et al. Replicative [45] Li H, Sheng C, Wang S, Yang L, Liang Y, Huang Y, et al. Removal of integrated
activity of hepatitis B virus is negatively associated with methylation of cova- hepatitis B virus DNA using CRISPR-Cas9. Front Cell Infect Microbiol 2017;7:
lently closed circular DNA in advanced hepatitis B virus infection. Intervirol- 91. doi: 10.3389/fcimb.2017.00091.
ogy 2011;54:316–325. doi: 10.1159/000321450. [46] Gaj T, Gersbach CA, Barbas CF 3rd. ZFN, TALEN, and CRISPR/Cas-based
[22] Pollicino T, Belloni L, Raffa G, Pediconi N, Squadrito G, Raimondo G, et al. methods for genome engineering. Trends Biotechnol 2013;31:397–405.
Hepatitis B virus replication is regulated by the acetylation status of hepatitis doi: 10.1016/[Link].2013.04.004.
B virus cccDNA-bound H3 and H4 histones. Gastroenterology 2006;130: [47] Holkers M, Maggio I, Liu J, Janssen JM, Miselli F, Mussolino C, et al. Differ-
823–837. doi: 10.1053/[Link].2006.01.001. ential integrity of TALE nuclease genes following adenoviral and lentiviral
[23] Liu F, Campagna M, Qi Y, Zhao X, Guo F, Xu C, et al. Alpha-interferon sup- vector gene transfer into human cells. Nucleic Acids Res 2013;41:e63.
presses hepadnavirus transcription by altering epigenetic modification of doi: 10.1093/nar/gks1446.
cccDNA minichromosomes. PLoS Pathog 2013;9:e1003613. doi: 10. [48] Mussolino C, Morbitzer R, Lütge F, Dannemann N, Lahaye T, Cathomen T. A
1371/[Link].1003613. novel TALE nuclease scaffold enables high genome editing activity in combi-
[24] Park HK, Min BY, Kim NY, Jang ES, Shin CM, Park YS, et al. Short hairpin RNA nation with low toxicity. Nucleic Acids Res 2011;39:9283–9293. doi: 10.
induces methylation of hepatitis B virus covalently closed circular DNA in 1093/nar/gkr597.
human hepatoma cells. Biochem Biophys Res Commun 2013;436:152– [49] Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, et al. GUIDE-
155. doi: 10.1016/[Link].2013.04.108. seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas
[25] Zhang Y, Li C, Zhang Y, Zhu H, Kang Y, Liu H, et al. Comparative analysis of nucleases. Nat Biotechnol 2015;33:187–197. doi: 10.1038/nbt.3117.
CpG islands among HBV genotypes. PLoS One 2013;8:e56711. doi: 10. [50] Yang HC, Chen PJ. The potential and challenges of CRISPR-Cas in eradication
1371/[Link].0056711. of hepatitis B virus covalently closed circular DNA. Virus Res 2018;244:304–
[26] Werle-Lapostolle B, Bowden S, Locarnini S, Wursthorn K, Petersen J, Lau G, 310. doi: 10.1016/[Link].2017.06.010.
et al. Persistence of cccDNA during the natural history of chronic hepatitis B [51] Levrero M, Pollicino T, Petersen J, Belloni L, Raimondo G, Dandri M. Control of
and decline during adefovir dipivoxil therapy. Gastroenterology 2004;126: cccDNA function in hepatitis B virus infection. J Hepatol 2009;51:581–592.
1750–1758. doi: 10.1053/[Link].2004.03.018. doi: 10.1016/[Link].2009.05.022.
[27] Cathomen T, Keith Joung J. Zinc-finger nucleases: The next generation [52] Miller RH, Robinson WS. Integrated hepatitis B virus DNA sequences specify-
emerges. Mol Ther 2008;16:1200–1207. doi: 10.1038/mt.2008.114. ing the major viral core polypeptide are methylated in PLC/PRF/5 cells. Proc
[28] Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger Natl Acad Sci U S A 1983;80:2534–2538. doi: 10.1073/pnas.80.9.2534.
fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 1996;93:1156– [53] Guo Y, Li Y, Mu S, Zhang J, Yan Z. Evidence that methylation of hepatitis B
1160. doi: 10.1073/pnas.93.3.1156. virus covalently closed circular DNA in liver tissues of patients with chronic
[29] Cradick TJ, Keck K, Bradshaw S, Jamieson AC, McCaffrey AP. Zinc-finger hepatitis B modulates HBV replication. J Med Virol 2009;81:1177–1183.
nucleases as a novel therapeutic strategy for targeting hepatitis B virus doi: 10.1002/jmv.21525.
DNAs. Mol Ther 2010;18:947–954. doi: 10.1038/mt.2010.20. [54] Vivekanandan P, Thomas D, Torbenson M. Methylation regulates hepatitis B
[30] Weber ND, Stone D, Sedlak RH, De Silva Feelixge HS, Roychoudhury P, viral protein expression. J Infect Dis 2009;199:1286–1291. doi: 10.
Schiffer JT, et al. AAV-mediated delivery of zinc finger nucleases targeting 1086/597614.
hepatitis B virus inhibits active replication. PLoS One 2014;9:e97579. doi: [55] Curradi M, Izzo A, Badaracco G, Landsberger N. Molecular mechanisms of
10.1371/[Link].0097579. gene silencing mediated by DNA methylation. Mol Cell Biol 2002;22:3157–
[31] Bloom K, Maepa MB, Ely A, Arbuthnot P. Gene therapy for chronic 3173. doi: 10.1128/mcb.22.9.3157-3173.2002.
HBV-Can we eliminate cccDNA? Genes (Basel) 2018;9:E207. doi: 10. [56] Belloni L, Allweiss L, Guerrieri F, Pediconi N, Volz T, Pollicino T, et al. IFN-a
3390/genes9040207. inhibits HBV transcription and replication in cell culture and in humanized
[32] Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eich- mice by targeting the epigenetic regulation of the nuclear cccDNA minichro-
tinger M, et al. Rapid “open-source” engineering of customized zinc-finger mosome. J Clin Invest 2012;122:529–537. doi: 10.1172/JCI58847.
nucleases for highly efficient gene modification. Mol Cell 2008;31:294–301. [57] Zhang Y, Mao R, Yan R, Cai D, Zhang Y, Zhu H, et al. Transcription of hepatitis
doi: 10.1016/[Link].2008.06.016. B virus covalently closed circular DNA is regulated by CpG methylation during
[33] Boch J, Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery chronic infection. PLoS One 2014;9:e110442. doi: 10.1371/[Link].
and function. Annu Rev Phytopathol 2010;48:419–436. doi: 10. 0110442.
1146/annurev-phyto-080508-081936. [58] Vivekanandan P, Thomas D, Torbenson M. Hepatitis B viral DNA is methylated
[34] Bloom K, Ely A, Mussolino C, Cathomen T, Arbuthnot P. Inactivation of hep- in liver tissues. J Viral Hepat 2008;15:103–107. doi: 10.1111/j.1365-2893.
atitis B virus replication in cultured cells and in vivo with engineered tran- 2007.00905.x.
scription activator-like effector nucleases. Mol Ther 2013;21:1889–1897. [59] Vivekanandan P, Kannangai R, Ray SC, Thomas DL, Torbenson M. Compre-
doi: 10.1038/mt.2013.170. hensive genetic and epigenetic analysis of occult hepatitis B from liver tissue
[35] Dreyer T, Nicholson S, Ely A, Arbuthnot P, Bloom K. Improved antiviral effi- samples. Clin Infect Dis 2008;46:1227–1236. doi: 10.1086/529437.
cacy using TALEN-mediated homology directed recombination to introduce [60] Rivière L, Gerossier L, Ducroux A, Dion S, Deng Q, Michel ML, et al. HBx
artificial primary miRNAs into DNA of hepatitis B virus. Biochem Biophys Res relieves chromatin-mediated transcriptional repression of hepatitis B viral
Commun 2016;478:1563–1568. doi: 10.1016/[Link].2016.08.152. cccDNA involving SETDB1 histone methyltransferase. J Hepatol 2015;63:
[36] Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing 1093–1102. doi: 10.1016/[Link].2015.06.023.
systems in bacteria and archaea. Nature 2012;482:331–338. doi: 10. [61] Chong CK, Cheng CYS, Tsoi SYJ, Huang FY, Liu F, Seto WK, et al. Role of
1038/nature10886. hepatitis B core protein in HBV transcription and recruitment of histone ace-
[37] Shah SA, Garrett RA. CRISPR/Cas and Cmr modules, mobility and evolution tyltransferases to cccDNA minichromosome. Antiviral Res 2017;144:1–7.
of adaptive immune systems. Res Microbiol 2011;162:27–38. doi: 10. doi: 10.1016/[Link].2017.05.003.
1016/[Link].2010.09.001. [62] Zhang W, Chen J, Wu M, Zhang X, Zhang M, Yue L, et al. PRMT5 restricts
[38] Jore MM, Brouns SJ, van der Oost J. RNA in defense: CRISPRs protect pro- hepatitis B virus replication through epigenetic repression of covalently
karyotes against mobile genetic elements. Cold Spring Harb Perspect Biol closed circular DNA transcription and interference with pregenomic RNA
2012;4:a003657. doi: 10.1101/cshperspect.a003657. encapsidation. Hepatology 2017;66:398–415. doi: 10.1002/hep.29133.

Journal of Clinical and Translational Hepatology 2019 vol. 7 | 258–262 261

 Zhu A. et al: HBV cccDNA as a therapeutic target

[63] Ren JH, Hu JL, Cheng ST, Yu HB, Wong VKW, Law BYK, et al. SIRT3 restricts [66] Song M, Sun Y, Tian J, He W, Xu G, Jing Z, et al. Silencing retinoid X receptor
hepatitis B virus transcription and replication through epigenetic regulation alpha expression enhances early-stage hepatitis B virus infection in cell cul-
of covalently closed circular DNA involving suppressor of variegation 3-9 tures. J Virol 2018;92:e01771–17. doi: 10.1128/JVI.01771-17.
homolog 1 and SET domain containing 1A histone methyltransferases. Hep- [67] Koumbi L, Pollicino T, Raimondo G, Stampoulis D, Khakoo S, Karayiannis P.
atology 2018;68:1260–1276. doi: 10.1002/hep.29912. Hepatitis B virus basal core promoter mutations show lower replication
[64] Qian G, Hu B, Zhou D, Xuan Y, Bai L, Duan C. NIRF, a novel ubiquitin ligase, fitness associated with cccDNA acetylation status. Virus Res 2016;220:
inhibits hepatitis B virus replication through effect on HBV core protein and 150–160. doi: 10.1016/[Link].2016.04.022.
H3 histones. DNA Cell Biol 2015;34:327–332. doi: 10.1089/dna.2014.2714. [68] Wu M, Li J, Yue L, Bai L, Li Y, Chen J, et al. Establishment of Cre-mediated
[65] Wei ZQ, Zhang YH, Ke CZ, Chen HX, Ren P, He YL, et al. Curcumin inhibits HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral
hepatitis B virus infection by down-regulating cccDNA-bound histone acetylation. screening assays. Antiviral Res 2018;152:45–52. doi: 10.1016/[Link].
World J Gastroenterol 2017;23:6252–6260. doi: 10.3748/wjg.v23.i34.6252. 2018.02.007.

262 Journal of Clinical and Translational Hepatology 2019 vol. 7 | 258–262