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Epigenetic Regulation of Hepatitis B Virus Replication

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In Young Moon and Jin-Wook Kim

Submitted: October 17th, 2017 Reviewed: September 27th, 2018 Published: November 15th, 2018

DOI: 10.5772/intechopen.81711

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Abstract

Hepatitis B virus (HBV) is the most important cause of chronic viral hepatitis worldwide. The genome of HBV is 3.2 kb partially double-stranded DNA, which is translocated to the nuclei of infected hepatocytes and converted to complete double-stranded DNA, aka covalently closed circular DNA (cccDNA). Typical course of chronic HBV infection results in inactive carrier state with clearance of viral particles in the bloodstream. However, the cccDNA can be detected in the hepatocytes from inactive carriers by sensitive methods. It has been increasingly known that epigenetic mechanisms contribute to the control of HBV replication in the inactive stage of HBV infection. Histone modification and DNA methylation have been identified in the HBV cccDNA, leading to modification of transcriptional activity. The understanding of epigenetic control of transcription will shed light on the development of new therapeutic strategy against HBV cccDNA.

Keywords

  • hepatitis B virus
  • covalently closed circular DNA
  • histone modification
  • inactive carrier

1. Introduction

Hepatitis B virus (HBV) is the most important cause of chronic viral hepatitis worldwide. About 240–350 million people are infected with HBV globally [1]. Development of potent nucleos(t)ide analogs (Nas) has revolutionized the treatment of HBV, but current treatment cannot eradicate the DNA genome of HBV, i.e., covalently closed circular DNA (cccDNA) from the nuclei of infected hepatocytes. Since there have been no innate clearing mechanisms identified for foreign double-stranded DNA in mammalian cells, theoretically HBV cccDNA in the liver stem cells will dilute out but persist indefinitely in some portion of the hepatocytes [2]. Therefore, prolonged use of current NA therapy is recommended without interruption, which is very costly.

It has been known that transcriptional activity of HBV cccDNA varies according to the stage of natural history of chronic hepatitis B (CHB) [3, 4]. Interestingly, many patients with chronic hepatitis B are free from circulating HBV during the natural course despite the presence of HBV cccDNA in the infected nuclei [5]. These findings raise the possibility that replication of HBV is regulated at the transcriptional level. Genetic changes, i.e., DNA mutation, are an attractive explanation for the variable transcriptional activity since the reverse transcriptase activity of HBV is error-prone. However, no universal mutations have been identified associated with transcriptional suppression [6]. Consequently, epigenetic control has been proposed as the mechanism of these variable transcriptional activities in CHB patients [7], and this article covers the current knowledge of epigenetic mechanisms contributing to the transcriptional control of HBV replication.

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2. Organization of HBV cccDNA and its transcriptional control

Hepatitis B virus (HBV) is a partially double-stranded circular DNA virus [8]. The viral DNA goes into nuclei of infected hepatocytes where it is converted to cccDNA [8]. The cccDNA is a viral minichromosome, which takes the form of “beads-on-a-string” conformation of nucleosomal packaging [9], analogous to DNA packaging by mammalian nucleosome. HBV core protein and X protein along with histone H3 and H4 are components of HBV minichromosome [9, 10, 11]. A variety of cellular transcription factors bind HBV cccDNA, which in turn control transcriptional activity of HBV promoters: the preC/pregenomic, S1, S2, and X promoters [12]. The core promoter initiates transcription of preC and pregenomic RNA, the template for the viral genome by reverse transcription. Ubiquitous transcription factors such as specificity protein 1 (SP1), nuclear factor kappa B (NF-κβ), activator protein 1 (AP-1), and liver-enriched transcription factors such as hepatocyte nuclear factor 3 (HNF3), CAAT enhancer-binding protein (C/EBP), and several nuclear receptors such as hepatocyte nuclear factor 4 (HNF4), peroxisome proliferator-activated receptors (PPAR) and retinoid X receptors (RXRα), farnesoid acid receptor (FXR), small heterodimer partner (SHP), and testicular orphan receptor 4 (TR4) can bind core promoter [13, 14].

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3. Epigenetic control of HBV transcription: histone modification

As described above, ultrastructure of HBV cccDNA simulates that of mammalian nucleosome, suggesting the possibility of histone molecules as a main factor for transcriptional control [9]. Indeed, Pollicino et al. demonstrated the feasibility of HBV-associated histone modification as a major transcriptional regulator of HBV [11]. Genome-wise search for posttranslational modification (PTM) of HBV-infected liver cell lines has revealed that active marks of transcription such as H3K4me3, H3K27ac, and H3K122ac are abundant in active chromatin, especially in the core promoter region [19]. Interestingly, however, the repressive marks of transcription, i.e., H3K27me3 and H3K9me2, are depleted in the HBV cccDNA, suggesting that modified histones regulate HBV transcription mainly in favor of active replication. Pol2 enrichment is co-localized at the H3K27me-enriched transcription start site of precore/pregenomic area, and treatment with interferon alpha reduces the active PTMs, also suggesting that active marks of histone modification contribute to the transcriptional activity of HBV.

Much is unknown regarding the effects of histone modification on the binding of these transcription factors. Hepatitis X protein (HBx) is the most studied modulator of HBV-bound histone [20, 21]. HBx is bound to the cccDNA and enhance transcription by increasing histone acetylation and recruiting cellular coactivators p300, CBP, and PCAF [22], by inhibiting protein arginine methyltransferase 1 and reducing H4 methylation [23]. HBx also increases histone acetylation and H3K4me3 and decreases HP1 binding and H3K9me3 on the cccDNA [24]. Other host transcription factors, mainly suppressors, that act via epigenetic control of HBV include SIRT3 [25], zinc finger and homeoboxes 2 (ZHX2) [26], KDM2B [27], protein arginine methyltransferase 5 (PRMT5) [28], and SETDB1 [24]. Interestingly, mutations in the basal core promoter are also reported to be associated with histone modification [29]. The effect of histone modification on HBV replication is summarized in Table 1.

Histone changeModifierHBV response and mechanismsReferences
H3K4me3MLL3Activation(H3K4me modification of the NTCP promoter by MLL3 may facilitate HBV infection in vivo.)[19, 30]
Zinc finger and homeoboxes (ZHX2)Repression (ZHX2 inhibited trimethylation of H3K4. Overexpression of ZHX2 also decreases the acetylation levels of H3K27 and H3K122.)[26]
H3K27acEZH2Activation(Knockdown of EZH2 resulted in upregulation of HBeAg and ABsAg, indicating a repressive function of EZH2 on HBV gene expression.)[28, 31]
H3K122acP300/CBPActivation[19, 32, 33]
AcH3/AcH4 H3K4me3HBxActivation[20, 21, 22, 24]
H4R3me2sPRMT5Repression(PRMT5-mediated histone H4 dimethyl Arg3 symmetric (H4R3me2s) represses cccDNA transcription.)[28]
H3K9me3SETDB1Repression(upon HBV infection, cellular mechanisms involving SETDB1-mediated H3K9me3 and HP1 induce silencing of HBV cccDNA transcription through modulation of chromatin structure.)[24]
SIRT3Repression(SIRT3 is a novel host factor epigenetically restricting HBV cccDNA transcription by acting cooperatively with histone methyltransferase.)[25]
H3K27me3Suz12Repression(downregulation of Suz12 and Znf198 enhances HBV replication.)[34]
H3K79meKDM2BRepression(KDM2B as an H3K79 demethylase and link its function to transcriptional repression via SIRT1-mediated chromatin silencing.)[27]
etcBCP mutationRepression(BCP mutations decrease viral replication capacity possibly by modulating the acetylation and deacetylation of cccDNA-bound histones.)[29]
IFNαRepression[19, 35, 36]

Table 1.

Histone modification affecting HBV transcription.

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4. Epigenetic control of HBV transcription: cccDNA methylation

Isolation and testing of HBV cccDNA shows methylation not only of HBV DNA integrated in host chromosome [39, 40] but also of HBV cccDNA. Methylation is speculated to affect replicative activity of HBV [38], and this hypothesis was confirmed in the hepatoma cell lines [41] and human liver tissue [15, 16]. HBV cccDNA has three CpG islands which harbor methylation in human liver and hepatoma cell lines [37, 38]. Methylation of HBV in the CpG islands of cccDNA is associated with suppressed transcriptional activity of HBV [15, 16] (for recent reviews, see [17, 18]). Especially, methylation of CpG island II is associated with reduced HBV replicability [42].

Although DNA methyltransferases, i.e., DNMT1, DNMT3a, and DNMT3b, are expressed in normal tissues [45], the level of expression is higher in HCC [46], which may explain the increased levels of methylation in hepatocellular carcinoma (HCC) compared to noncancerous tissues [43, 44]. In addition, since the hepatic expression of DNMT3a and DNMT3b, the de novo methylators, increases with age [47], methylation of HBV may also increase with age [42], which might explain suppressed replicative activity of HBV in the later stage of natural history. Degree of methylation also depends on HBeAg positivity [15, 42] and degree of hepatic fibrosis [42].

The mechanism of HBV DNA methylation is still unknown (Table 2). From the specific patterns of methylation in the HBV genome [42], it can be speculated that some kind of molecular chaperon (s) may guide the de novo methylation enzymes to the specific target sequence. HBx may play a role, because it recruits DNMT3A to the regulatory promoters [48]. Small RNAs may be a plausible candidate, as suggested by our in vitro study in which short hairpin RNA induced methylation of the target site in HBV [49].

HBV areaModifierHBV replicationReferences
CpG2HBcIncreased(The relative abundances of HBc binding to CpG island 2 were associated with the binding of CREB binding protein (CBP) and with hypomethylation in CpG island 2 of HBV cccDNA minichromosomes.)[50]
CpG3PEG-IFNDecreased(PEG-IFN treatment significantly increased methylation of HBV cccDNA in CpG island III.)[51]

Table 2.

Mechanisms of methylation in HBV cccDNA.

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5. Therapeutic implications and future perspectives of epigenetics in chronic hepatitis B

NAs, the most commonly used modality of CHB therapy, is costly without definite duration. Interferon induces sustained virologic response with finite duration, but the response rate is suboptimal. The realistic goal of CHB therapy is to render the patients to the clinical situation similar to inactive carrier stage, i.e., normal alanine aminotransferase levels with low or negative serum HBV DNA levels. Since epigenetic silencing may contribute to the suppressive HBV replication status of inactive carrier stage, it would be theoretically feasible and clinically useful to induce epigenetic suppression of HBV replication simulating natural inactive stage of disease. Further studies will be needed to elucidate the mechanisms and long-term consequences of epigenetic suppression of HBV replication.

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6. Conclusions

Epigenetic modification is an important mechanism of host-viral interaction in the transcriptional control of HBV. Current treatment strategy focuses on the inactivation/elimination of HBV cccDNA [17, 52], and knowledge on the epigenetic control is prerequisite for the novel development of HBV cure in the foreseeable future.

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Acknowledgments

This work is supported by a National Research Foundation of Korea (NRF) grant to J-W Kim that was funded by the Korean Government (2017R1D1A1B03031483).

References

  1. 1. MacLachlan JH, Cowie BC. Hepatitis B virus epidemiology. Cold Spring Harbor Perspectives in Medicine. 2015;5:a021410
  2. 2. Li M, Sohn JA, Seeger C. Distribution of hepatitis B virus nuclear DNA. Journal of Virology. 2018;92:e01391-e01317
  3. 3. Laras A, Koskinas J, Dimou E, Kostamena A, Hadziyannis SJ. Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients. Hepatology. 2006;44:694-702
  4. 4. Volz T, Lutgehetmann M, Wachtler P, Jacob A, Quaas A, Murray JM, et al. Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients. Gastroenterology. 2007;133:843-852
  5. 5. 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;126:1750-1758
  6. 6. Jammeh S, Tavner F, Watson R, Thomas HC, Karayiannis P. Effect of basal core promoter and pre-core mutations on hepatitis B virus replication. The Journal of General Virology. 2008;89:901-909
  7. 7. Levrero M, Pollicino T, Petersen J, Belloni L, Raimondo G, Dandri M. Control of cccDNA function in hepatitis B virus infection. Journal of Hepatology. 2009;51:581-592
  8. 8. Seeger C, Mason WS. Molecular biology of hepatitis B virus infection. Virology. 2015;479-480:672-686
  9. 9. Bock CT, Schwinn S, Locarnini S, Fyfe J, Manns MP, Trautwein C, et al. Structural organization of the hepatitis B virus minichromosome. Journal of Molecular Biology. 2001;307:183-196
  10. 10. Lucifora J, Arzberger S, Durantel D, Belloni L, Strubin M, Levrero M, et al. Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection. Journal of Hepatology. 2011;55:996-1003
  11. 11. 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. Gastroenterology. 2006;130:823-837
  12. 12. Moolla N, Kew M, Arbuthnot P. Regulatory elements of hepatitis B virus transcription. Journal of Viral Hepatitis. 2002;9:323-331
  13. 13. Quasdorff M, Protzer U. Control of hepatitis B virus at the level of transcription. Journal of Viral Hepatitis. 2010;17:527-536
  14. 14. Hensel KO, Rendon JC, Navas MC, Rots MG, Postberg J. Virus-host interplay in hepatitis B virus infection and epigenetic treatment strategies. The FEBS Journal. 2017;284:3550-3572
  15. 15. Guo Y, Li Y, Mu S, Zhang J, Yan Z. Evidence that methylation of hepatitis B virus covalently closed circular DNA in liver tissues of patients with chronic hepatitis B modulates HBV replication. Journal of Medical Virology. 2009;81:1177-1183
  16. 16. Kim JW, Lee SH, Park YS, Hwang JH, Jeong SH, Kim N, et al. Replicative activity of hepatitis B virus is negatively associated with methylation of covalently closed circular DNA in advanced hepatitis B virus infection. Intervirology. 2011;54:316-325
  17. 17. Hong X, Kim ES, Guo H. Epigenetic regulation of hepatitis B virus covalently closed circular DNA: Implications for epigenetic therapy against chronic hepatitis B. Hepatology. 2017;66:2066-2077
  18. 18. Koumbi L, Karayiannis P. The epigenetic control of hepatitis B virus modulates the outcome of infection. Frontiers in Microbiology. 2015;6:1491
  19. 19. Tropberger P, Mercier A, Robinson M, Zhong W, Ganem DE, Holdorf M. Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation. Proceedings of the National Academy of Sciences of the United States of America. 2015;112:E5715-E5724
  20. 20. Luo L, Chen S, Gong Q, Luo N, Lei Y, Guo J, et al. Hepatitis B virus X protein modulates remodelling of minichromosomes related to hepatitis B virus replication in HepG2 cells. International Journal of Molecular Medicine. 2013;31:197-204
  21. 21. Protzer U. Hepatitis: Epigenetic control of HBV by HBx protein—Releasing the break? Nature Reviews Gastroenterology & Hepatology. 2015;12:558-559
  22. 22. Belloni L, Pollicino T, De Nicola F, Guerrieri F, Raffa G, Fanciulli M, et al. Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:19975-19979
  23. 23. Benhenda S, Ducroux A, Riviere L, Sobhian B, Ward MD, Dion S, et al. Methyltransferase PRMT1 is a binding partner of HBx and a negative regulator of hepatitis B virus transcription. Journal of Virology. 2013;87:4360-4371
  24. 24. Riviere L, Gerossier L, Ducroux A, Dion S, Deng Q, Michel ML, et al. HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase. Journal of Hepatology. 2015;63:1093-1102
  25. 25. Ren JH, Hu JL, Cheng ST, Yu HB, Wong VKW, Law BYK, et al. SIRT3 restricts HBV transcription and replication via epigenetic regulation of cccDNA involving SUV39H1 and SETD1A histone methyltransferases. Hepatology. 2018;68(4):1260-1276
  26. 26. Xu L, Wu Z, Tan S, Wang Z, Lin Q, Li X, et al. Tumor suppressor ZHX2 restricts hepatitis B virus replication via epigenetic and non-epigenetic manners. Antiviral Research. 2018;153:114-123
  27. 27. Kang JY, Kim JY, Kim KB, Park JW, Cho H, Hahm JY, et al. KDM2B is a histone H3K79 demethylase and induces transcriptional repression via sirtuin-1-mediated chromatin silencing. The FASEB Journal. 2018;32(10):5737-5750, fj201800242R
  28. 28. Zhang W, Chen J, Wu M, Zhang X, Zhang M, Yue L, et al. PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation. Hepatology. 2017;66:398-415
  29. 29. Koumbi L, Pollicino T, Raimondo G, Stampoulis D, Khakoo S, Karayiannis P. Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status. Virus Research. 2016;220:150-160
  30. 30. Ananthanarayanan M, Li Y, Surapureddi S, Balasubramaniyan N, Ahn J, Goldstein JA, et al. Histone H3K4 trimethylation by MLL3 as part of ASCOM complex is critical for NR activation of bile acid transporter genes and is downregulated in cholestasis. American Journal of Physiology. Gastrointestinal and Liver Physiology. 2011;300:G771-G781
  31. 31. Allis CD, Jenuwein T. The molecular hallmarks of epigenetic control. Nature Reviews. Genetics. 2016;17:487-500
  32. 32. Tropberger P, Pott S, Keller C, Kamieniarz-Gdula K, Caron M, Richter F, et al. Regulation of transcription through acetylation of H3K122 on the lateral surface of the histone octamer. Cell. 2013;152:859-872
  33. 33. Pradeepa MM, Grimes GR, Kumar Y, Olley G, Taylor GC, Schneider R, et al. Histone H3 globular domain acetylation identifies a new class of enhancers. Nature Genetics. 2016;48:681-686
  34. 34. Andrisani OM. Deregulation of epigenetic mechanisms by the hepatitis B virus X protein in hepatocarcinogenesis. Viruses. 2013;5:858-872
  35. 35. Belloni L, Allweiss L, Guerrieri F, Pediconi N, Volz T, Pollicino T, et al. IFN-alpha inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome. The Journal of Clinical Investigation. 2012;122:529-537
  36. 36. Liu F, Campagna M, Qi Y, Zhao X, Guo F, Xu C, et al. Alpha-interferon suppresses hepadnavirus transcription by altering epigenetic modification of cccDNA minichromosomes. PLoS Pathogens. 2013;9:e1003613
  37. 37. Vivekanandan P, Thomas D, Torbenson M. Hepatitis B viral DNA is methylated in liver tissues. Journal of Viral Hepatitis. 2008;15:103-107
  38. 38. Vivekanandan P, Kannangai R, Ray SC, Thomas DL, Torbenson M. Comprehensive genetic and epigenetic analysis of occult hepatitis B from liver tissue samples. Clinical Infectious Diseases. 2008;46:1227-1236
  39. 39. Yamamura K, Tsurimoto T, Ebihara T, Kamino K, Fujiyama A, Ochiya T, et al. Methylation of hepatitis B virus DNA and liver-specific suppression of RNA production in transgenic mouse. Japanese Journal of Cancer Research. 1987;78:681-688
  40. 40. Watanabe Y, Yamamoto H, Oikawa R, Toyota M, Yamamoto M, Kokudo N, et al. DNA methylation at hepatitis B viral integrants is associated with methylation at flanking human genomic sequences. Genome Research. 2015;25:328-337
  41. 41. Vivekanandan P, Thomas D, Torbenson M. Methylation regulates hepatitis B viral protein expression. The Journal of Infectious Diseases. 2009;199:1286-1291
  42. 42. Zhang Y, Mao R, Yan R, Cai D, Zhang Y, Zhu H, et al. Transcription of hepatitis B virus covalently closed circular DNA is regulated by CpG methylation during chronic infection. PLoS One. 2014;9:e110442
  43. 43. Jain S, Chang TT, Chen S, Boldbaatar B, Clemens A, Lin SY, et al. Comprehensive DNA methylation analysis of hepatitis B virus genome in infected liver tissues. Scientific Reports. 2015;5:10478
  44. 44. Kaur P, Paliwal A, Durantel D, Hainaut P, Scoazec JY, Zoulim F, et al. DNA methylation of hepatitis B virus (HBV) genome associated with the development of hepatocellular carcinoma and occult HBV infection. The Journal of Infectious Diseases. 2010;202:700-704
  45. 45. Robertson KD, Uzvolgyi E, Liang G, Talmadge C, Sumegi J, Gonzales FA, et al. The human DNA methyltransferases (DNMTs) 1, 3a and 3b: Coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Research. 1999;27:2291-2298
  46. 46. Oh BK, Kim H, Park HJ, Shim YH, Choi J, Park C, et al. DNA methyltransferase expression and DNA methylation in human hepatocellular carcinoma and their clinicopathological correlation. International Journal of Molecular Medicine. 2007;20:65-73
  47. 47. Xiao Y, Word B, Starlard-Davenport A, Haefele A, Lyn-Cook BD, Hammons G. Age and gender affect DNMT3a and DNMT3b expression in human liver. Cell Biology and Toxicology. 2008;24:265-272
  48. 48. Zheng DL, Zhang L, Cheng N, Xu X, Deng Q, Teng XM, et al. Epigenetic modification induced by hepatitis B virus X protein via interaction with de novo DNA methyltransferase DNMT3A. Journal of Hepatology. 2009;50:377-387
  49. 49. Park HK, Min BY, Kim NY, Jang ES, Shin CM, Park YS, et al. Short hairpin RNA induces methylation of hepatitis B virus covalently closed circular DNA in human hepatoma cells. Biochemical and Biophysical Research Communications. 2013;436:152-155
  50. 50. Guo YH, Li YN, Zhao JR, Zhang J, Yan Z. HBc binds to the CpG islands of HBV cccDNA and promotes an epigenetic permissive state. Epigenetics. 2011;6:720-726
  51. 51. Uchida T, Imamura M, Hayes CN, Hiraga N, Kan H, Tsuge M, et al. Persistent loss of hepatitis B virus markers in serum without cellular immunity by combination of peginterferon and entecavir therapy in humanized mice. Antimicrobial Agents and Chemotherapy. 2017;61:e00725-e00717
  52. 52. Seeger C. Control of viral transcripts as a concept for future HBV therapies. Current Opinion in Virology. 2018;30:18-23

Written By

In Young Moon and Jin-Wook Kim

Submitted: October 17th, 2017 Reviewed: September 27th, 2018 Published: November 15th, 2018