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HBV culture and infectious systems

Hepatology International volume 10, pages 559–566 (2016)Cite this article

Abstract

While an effective vaccine against hepatitis B virus (HBV) has long been available, chronic HBV infection remains a severe global public health concern. Current treatment options have limited effectiveness, and long-term therapy is required to suppress HBV replication; however, complete elimination of the virus is rare. The lack of suitable animal models and infection systems has hindered efforts to unravel the HBV life cycle, particularly the early events in HBV entry, which appear to be highly species- and tissue-specific. Human primary hepatocytes remain the gold standard for HBV replication studies but are limited by availability and variability. While the HepaRG cell line is permissive for HBV replication, other hepatoma cell lines such as HepG2 do not support HBV replication. The recent discovery of sodium taurocholate transporting peptide (NTCP) as a primary receptor for HBV binding has led to the development of replication-competent cell lines such as HepG2–NTCP. Human hepatocytes grown in chimeric mice have provided another approach that allows primary human hepatocytes to be used while overcoming many of their limitations. Although the difficulty in developing HBV infection systems has hindered development of effective treatments, the variability and limited replication efficiency among cell lines point to additional liver-specific factors involved in HBV infection. It is hoped that HBV infection studies will lead to novel drug targets and therapeutic options for the treatment of chronic HBV infection.

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Fig. 1
Fig. 2

Abbreviations

cccDNA:

Covalently closed circular DNA

DMSO:

Dimethyl sulfoxide

HBV:

Hepatitis B virus

NTCP:

Sodium taurocholate transporting peptide

References

  1. Urban S, Bartenschlager R, Kubitz R, Zoulim F. Strategies to inhibit entry of HBV and HDV into hepatocytes. Gastroenterology 2014;147(1):48–64. doi:10.1053/j.gastro.2014.04.030

  2. Wieland S, Thimme R, Purcell RH, Chisari FV. Genomic analysis of the host response to hepatitis B virus infection. Proc Natl Acad Sci USA 2004;101(17):6669–6674. doi:10.1073/pnas.0401771101

  3. Gripon P, Diot C, Theze N, Fourel I, Loreal O, Brechot C, et al. Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide. J Virol 1988;62(11):4136–4143

  4. Walter E, Keist R, Niederost B, Pult I, Blum HE. Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo. Hepatology 1996;24(1):1–5. doi:10.1002/hep.510240101

  5. Shimizu Y, Nambu S, Kojima T, Sasaki H. Replication of hepatitis B virus in culture systems with adult human hepatocytes. J Med Virol 1986;20(4):313–327

  6. Gripon P, Diot C, Theze N, Fourel I, Loreal O, Brechot C, et al. Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide. J Virol 1988;62(11):4136–4143

  7. Ochiya T, Tsurimoto T, Ueda K, Okubo K, Shiozawa M, Matsubara K. An in vitro system for infection with hepatitis B virus that uses primary human fetal hepatocytes. Proc Natl Acad Sci USA 1989;86(6):1875–1879

  8. Gripon P, Diot C, Guguen-Guillouzo C. Reproducible high level infection of cultured adult human hepatocytes by hepatitis B virus: effect of polyethylene glycol on adsorption and penetration. Virology 1993;192(2):534–540. doi:10.1006/viro.1993.1069

  9. Galle PR, Hagelstein J, Kommerell B, Volkmann M, Schranz P, Zentgraf H. In vitro experimental infection of primary human hepatocytes with hepatitis B virus. Gastroenterology 1994;106(3):664–673

  10. Wilkening S, Stahl F, Bader A. Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties. Drug Metab Dispos 2003;31(8):1035–1042. doi:10.1124/dmd.31.8.1035

  11. Guillouzo A, Corlu A, Aninat C, Glaise D, Morel F, Guguen-Guillouzo C. The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem Biol Interact 2007;168(1):66–73. doi:10.1016/j.cbi.2006.12.003

  12. Wilkening S, Bader A. Influence of culture time on the expression of drug-metabolizing enzymes in primary human hepatocytes and hepatoma cell line HepG2. J Biochem Mol Toxicol 2003;17(4):207–213. doi:10.1002/jbt.10085

  13. Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 1980;209(4455):497–499

  14. Sells MA, Chen ML, Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci USA 1987;84(4):1005–1009

  15. Ladner SK, Otto MJ, Barker CS, Zaifert K, Wang GH, Guo JT, et al. Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother 1997;41(8):1715–1720

  16. Neurath AR, Kent SB, Strick N, Parker K. Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus. Cell 1986;46(3):429–436

  17. Petit MA, Dubanchet S, Capel F, Voet P, Dauguet C, Hauser P. HepG2 cell binding activities of different hepatitis B virus isolates: inhibitory effect of anti-HBs and anti-preS1(21-47). Virology 1991;180(2):483–491

  18. Qiao M, Macnaughton TB, Gowans EJ. Adsorption and penetration of hepatitis B virus in a nonpermissive cell line. Virology 1994;201(2):356–363. doi:10.1006/viro.1994.1301

  19. Glebe D, Urban S. Viral and cellular determinants involved in hepadnaviral entry. World J Gastroenterol 2007;13(1):22–38

  20. Bchini R, Capel F, Dauguet C, Dubanchet S, Petit MA. In vitro infection of human hepatoma (HepG2) cells with hepatitis B virus. J Virol 1990;64(6):3025–3032

  21. Mabit H, Dubanchet S, Capel F, Dauguet C, Petit MA. In vitro infection of human hepatoma cells (HepG2) with hepatitis B virus (HBV): spontaneous selection of a stable HBV surface antigen-producing HepG2 cell line containing integrated HBV DNA sequences. J Gen Virol 1994;75(Pt 10):2681–2689. doi:10.1099/0022-1317-75-10-2681

  22. Paran N, Geiger B, Shaul Y. HBV infection of cell culture: evidence for multivalent and cooperative attachment. Embo J 2001;20(16):4443–4453. doi:10.1093/emboj/20.16.4443

  23. Natarajan AT, Darroudi F. Use of human hepatoma cells for in vitro metabolic activation of chemical mutagens/carcinogens. Mutagenesis 1991;6(5):399–403

  24. Rodriguez-Antona C, Donato MT, Boobis A, Edwards RJ, Watts PS, Castell JV, et al. Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica 2002;32(6):505–520. doi:10.1080/00498250210128675

  25. Jover R, Bort R, Gomez-Lechon MJ, Castell JV. Cytochrome P450 regulation by hepatocyte nuclear factor 4 in human hepatocytes: a study using adenovirus-mediated antisense targeting. Hepatology 2001;33(3):668–675. doi:10.1053/jhep.2001.22176

  26. Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I, et al. Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci USA 2002;99(24):15655–15660. doi:10.1073/pnas.232137699

  27. Hantz O, Parent R, Durantel D, Gripon P, Guguen-Guillouzo C, Zoulim F. Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells. J Gen Virol 2009;90(Pt 1):127–135. doi:10.1099/vir.0.004861-0

  28. Lucifora J, Durantel D, Testoni B, Hantz O, Levrero M, Zoulim F. Control of hepatitis B virus replication by innate response of HepaRG cells. Hepatology 2010;51(1):63–72. doi:10.1002/hep.23230

  29. Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I, et al. Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci USA 2002;99(24):15655–15660. doi:10.1073/pnas.232137699

  30. Kanebratt KP, Andersson TB. Evaluation of HepaRG cells as an in vitro model for human drug metabolism studies. Drug Metab Dispos 2008;36(7):1444–1452. doi:10.1124/dmd.107.020016

  31. Kanebratt KP, Andersson TB. HepaRG cells as an in vitro model for evaluation of cytochrome P450 induction in humans. Drug Metab Dispos 2008;36(1):137–145. doi:10.1124/dmd.107.017418

  32. Hao Z, Zheng L, Kluwe L, Huang W. Ferritin light chain and squamous cell carcinoma antigen 1 are coreceptors for cellular attachment and entry of hepatitis B virus. Int J Nanomedicine 2012;7:827–834. doi:10.2147/IJN.S27803

  33. 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;1:e00049. doi:10.7554/eLife.00049

  34. Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Falth M, et al. Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Gastroenterology 2014;146(4):1070–1083. doi:10.1053/j.gastro.2013.12.024

  35. Elinger S. HBV: stowaway of NTCP. Clin Res Hepatol Gastroenterol 2014;38(6):661–663. doi:10.1016/j.clinre.2014.07.009

  36. Watashi K, Urban S, Li W, Wakita T. NTCP and beyond: opening the door to unveil hepatitis B virus entry. Int J Mol Sci 2014;15(2):2892–2905. doi:10.3390/ijms15022892

  37. Chen ZJ, Ye J. Getting to grips with hepatitis. Elife 2012;1:e00301. doi:10.7554/eLife.00301

  38. Schulze A, Mills K, Weiss TS, Urban S. Hepatocyte polarization is essential for the productive entry of the hepatitis B virus. Hepatology 2012;55(2):373–383. doi:10.1002/hep.24707

  39. Hagenbuch B, Meier PJ. Molecular cloning, chromosomal localization, and functional characterization of a human liver Na+/bile acid cotransporter. J Clin Investig 1994;93(3):1326–1331. doi:10.1172/JCI117091

  40. Stieger B. The role of the sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation. Handb Exp Pharmacol. 2011(201):205–259. doi:10.1007/978-3-642-14541-4_5

  41. Yan H, Liu Y, Sui J, Li W. NTCP opens the door for hepatitis B virus infection. Antivir Res 2015;121:24–30. doi:10.1016/j.antiviral.2015.06.002

  42. Iwamoto M, Watashi K, Tsukuda S, Aly HH, Fukasawa M, Fujimoto A, et al. Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP. Biochem Biophys Res Commun 2014;443(3):808–813. doi:10.1016/j.bbrc.2013.12.052

  43. Oehler N, Volz T, Bhadra OD, Kah J, Allweiss L, Giersch K, et al. Binding of hepatitis B virus to its cellular receptor alters the expression profile of genes of bile acid metabolism. Hepatology 2014;60(5):1483–1493. doi:10.1002/hep.27159

  44. Jiang Y, Wang AH, Shao LH, Wang G, Yao YY, Sai LT, et al. A new cell culture system for infection with hepatitis B virus that fuses HepG2 cells with primary human hepatocytes. J Int Med Res 2009;37(3):650–661

  45. Yan R, Zhang Y, Cai D, Liu Y, Cuconati A, Guo H. Spinoculation enhances HBV infection in NTCP-reconstituted hepatocytes. PLoS ONE 2015;10(6):e0129889. doi:10.1371/journal.pone.0129889

  46. Iwamoto M, Watashi K, Tsukuda S, Aly HH, Fukasawa M, Fujimoto A, et al. Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP. Biochem Biophys Res Commun 2013. doi:10.1016/j.bbrc.2013.12.052

  47. Verrier ER, Colpitts CC, Bach C, Heydmann L, Weiss A, Renaud M, et al. A targeted functional RNA interference screen uncovers glypican 5 as an entry factor for hepatitis B and D viruses. Hepatology 2016;63(1):35–48. doi:10.1002/hep.28013

  48. Ishida Y, Yamasaki C, Yanagi A, Yoshizane Y, Fujikawa K, Watashi K, et al. Novel robust in vitro hepatitis B virus infection model using fresh human hepatocytes isolated from humanized mice. Am J Pathol 2015;185(5):1275–1285. doi:10.1016/j.ajpath.2015.01.028

  49. Tateno C, Yoshizane Y, Saito N, Kataoka M, Utoh R, Yamasaki C, et al. Near completely humanized liver in mice shows human-type metabolic responses to drugs. Am J Pathol 2004;165(3):901–912. doi:10.1016/S0002-9440(10)63352-4

  50. Nishimura M, Yokoi T, Tateno C, Kataoka M, Takahashi E, Horie T, et al. Induction of human CYP1A2 and CYP3A4 in primary culture of hepatocytes from chimeric mice with humanized liver. Drug Metab Pharmacokinet 2005;20(2):121–126

  51. Yoshitsugu H, Nishimura M, Tateno C, Kataoka M, Takahashi E, Soeno Y, et al. Evaluation of human CYP1A2 and CYP3A4 mRNA expression in hepatocytes from chimeric mice with humanized liver. Drug Metab Pharmacokinet 2006;21(6):465–474

  52. Yamasaki C, Kataoka M, Kato Y, Kakuni M, Usuda S, Ohzone Y, et al. In vitro evaluation of cytochrome P450 and glucuronidation activities in hepatocytes isolated from liver-humanized mice. Drug Metab Pharmacokinet 2010;25(6):539–550

  53. Tsuge M, Hiraga N, Takaishi H, Noguchi C, Oga H, Imamura M, et al. Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis B virus. Hepatology 2005;42(5):1046–1054. doi:10.1002/Hep.20892

  54. Tsuge M, Hiraga N, Akiyama R, Tanaka S, Matsushita M, Mitsui F, et al. HBx protein is indispensable for development of viraemia in human hepatocyte chimeric mice. J Gen Virol 2010;91(Pt 7):1854–1864. doi:10.1099/vir.0.019224-0

  55. Meuleman P, Libbrecht L, Wieland S, De Vos R, Habib N, Kramvis A, et al. Immune suppression uncovers endogenous cytopathic effects of the hepatitis B virus. J Virol 2006;80(6):2797–2807. doi:10.1128/JVI.80.6.2797-2807.2006

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Acknowledgements

This work was supported in part by Grants-in-Aid for scientific research and development from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labor and Welfare, Government of Japan.

Compliance with ethical requirements

This article does not contain any studies with human or animal subjects.

Conflict of interest

C.N. Hayes and K. Chayama declare that they have nothing to disclose regarding funding or conflict of interest with respect to this manuscript.

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Affiliations

  1. Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan

    C. Nelson Hayes & Kazuaki Chayama

  2. Liver Research Project Center, Hiroshima University, Hiroshima, 734-8551, Japan

    C. Nelson Hayes & Kazuaki Chayama

  3. Laboratory for Digestive Diseases, Center for Genomic Medicine, RIKEN, Hiroshima, 734-8551, Japan

    Kazuaki Chayama

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  1. C. Nelson Hayes
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Correspondence to Kazuaki Chayama.

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Hayes, C.N., Chayama, K. HBV culture and infectious systems. Hepatol Int 10, 559–566 (2016). https://doi.org/10.1007/s12072-016-9712-y

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Keywords

  • Hepatitis B virus
  • HepaRG
  • HepG2
  • NTCP
  • Human hepatocyte chimeric mice