Skip to main content
NCBI home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1997 Aug;41(8):1715–1720. doi: 10.1128/aac.41.8.1715

Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.

S K Ladner 1, M J Otto 1, C S Barker 1, K Zaifert 1, G H Wang 1, J T Guo 1, C Seeger 1, R W King 1
PMCID: PMC163991  PMID: 9257747

Abstract

We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Acs G., Sells M. A., Purcell R. H., Price P., Engle R., Shapiro M., Popper H. Hepatitis B virus produced by transfected Hep G2 cells causes hepatitis in chimpanzees. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4641–4644. doi: 10.1073/pnas.84.13.4641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Christman J. K., Gerber M., Price P. M., Flordellis C., Edelman J., Acs G. Amplification of expression of hepatitis B surface antigen in 3T3 cells cotransfected with a dominant-acting gene and cloned viral DNA. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1815–1819. doi: 10.1073/pnas.79.6.1815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cullen B. R. Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism. Cell. 1986 Sep 26;46(7):973–982. doi: 10.1016/0092-8674(86)90696-3. [DOI] [PubMed] [Google Scholar]
  4. Davis M. G., Jansen R. W. Inhibition of hepatitis B virus in tissue culture by alpha interferon. Antimicrob Agents Chemother. 1994 Dec;38(12):2921–2924. doi: 10.1128/aac.38.12.2921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dienstag J. L., Perrillo R. P., Schiff E. R., Bartholomew M., Vicary C., Rubin M. A preliminary trial of lamivudine for chronic hepatitis B infection. N Engl J Med. 1995 Dec 21;333(25):1657–1661. doi: 10.1056/NEJM199512213332501. [DOI] [PubMed] [Google Scholar]
  6. Fallows D. A., Goff S. P. Mutations in the epsilon sequences of human hepatitis B virus affect both RNA encapsidation and reverse transcription. J Virol. 1995 May;69(5):3067–3073. doi: 10.1128/jvi.69.5.3067-3073.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fattovich G., Brollo L., Alberti A., Pontisso P., Giustina G., Realdi G. Long-term follow-up of anti-HBe-positive chronic active hepatitis B. Hepatology. 1988 Nov-Dec;8(6):1651–1654. doi: 10.1002/hep.1840080630. [DOI] [PubMed] [Google Scholar]
  8. Ganem D., Varmus H. E. The molecular biology of the hepatitis B viruses. Annu Rev Biochem. 1987;56:651–693. doi: 10.1146/annurev.bi.56.070187.003251. [DOI] [PubMed] [Google Scholar]
  9. Gish R. G., Lau J. Y., Brooks L., Fang J. W., Steady S. L., Imperial J. C., Garcia-Kennedy R., Esquivel C. O., Keeffe E. B. Ganciclovir treatment of hepatitis B virus infection in liver transplant recipients. Hepatology. 1996 Jan;23(1):1–7. doi: 10.1002/hep.510230101. [DOI] [PubMed] [Google Scholar]
  10. Gossen M., Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547–5551. doi: 10.1073/pnas.89.12.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Honkoop P., de Man R. A., Heijtink R. A., Schalm S. W. Hepatitis B reactivation after lamivudine. Lancet. 1995 Oct 28;346(8983):1156–1157. doi: 10.1016/s0140-6736(95)91829-9. [DOI] [PubMed] [Google Scholar]
  12. Knowles B. B., Howe C. C., Aden D. P. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980 Jul 25;209(4455):497–499. doi: 10.1126/science.6248960. [DOI] [PubMed] [Google Scholar]
  13. Korba B. E., Boyd M. R. Penciclovir is a selective inhibitor of hepatitis B virus replication in cultured human hepatoblastoma cells. Antimicrob Agents Chemother. 1996 May;40(5):1282–1284. doi: 10.1128/aac.40.5.1282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Korba B. E., Gerin J. L. Use of a standardized cell culture assay to assess activities of nucleoside analogs against hepatitis B virus replication. Antiviral Res. 1992 Jul 1;19(1):55–70. doi: 10.1016/0166-3542(92)90056-b. [DOI] [PubMed] [Google Scholar]
  15. Korba B. E., Milman G. A cell culture assay for compounds which inhibit hepatitis B virus replication. Antiviral Res. 1991 Mar-Apr;15(3):217–228. doi: 10.1016/0166-3542(91)90068-3. [DOI] [PubMed] [Google Scholar]
  16. Lau J. Y., Wright T. L. Molecular virology and pathogenesis of hepatitis B. Lancet. 1993 Nov 27;342(8883):1335–1340. doi: 10.1016/0140-6736(93)92249-s. [DOI] [PubMed] [Google Scholar]
  17. Lin T. S., Luo M. Z., Liu M. C., Pai S. B., Dutschman G. E., Cheng Y. C. Synthesis and biological evaluation of 2',3'-dideoxy-L-pyrimidine nucleosides as potential antiviral agents against human immunodeficiency virus (HIV) and hepatitis B virus (HBV). J Med Chem. 1994 Mar 18;37(6):798–803. doi: 10.1021/jm00032a013. [DOI] [PubMed] [Google Scholar]
  18. Nair V., Jahnke T. S. Antiviral activities of isometric dideoxynucleosides of D- and L-related stereochemistry. Antimicrob Agents Chemother. 1995 May;39(5):1017–1029. doi: 10.1128/aac.39.5.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Popper H., Shafritz D. A., Hoofnagle J. H. Relation of the hepatitis B virus carrier state to hepatocellular carcinoma. Hepatology. 1987 Jul-Aug;7(4):764–772. doi: 10.1002/hep.1840070425. [DOI] [PubMed] [Google Scholar]
  20. Price P. M., Banerjee R., Acs G. Inhibition of the replication of hepatitis B virus by the carbocyclic analogue of 2'-deoxyguanosine. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8541–8544. doi: 10.1073/pnas.86.21.8541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Scaglioni P. P., Melegari M., Wands J. R. Posttranscriptional regulation of hepatitis B virus replication by the precore protein. J Virol. 1997 Jan;71(1):345–353. doi: 10.1128/jvi.71.1.345-353.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schinazi R. F., Gosselin G., Faraj A., Korba B. E., Liotta D. C., Chu C. K., Mathé C., Imbach J. L., Sommadossi J. P. Pure nucleoside enantiomers of beta-2',3'-dideoxycytidine analogs are selective inhibitors of hepatitis B virus in vitro. Antimicrob Agents Chemother. 1994 Sep;38(9):2172–2174. doi: 10.1128/aac.38.9.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sells M. A., Chen M. L., Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A. 1987 Feb;84(4):1005–1009. doi: 10.1073/pnas.84.4.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sells M. A., Zelent A. Z., Shvartsman M., Acs G. Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions. J Virol. 1988 Aug;62(8):2836–2844. doi: 10.1128/jvi.62.8.2836-2844.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shih C. H., Li L. S., Roychoudhury S., Ho M. H. In vitro propagation of human hepatitis B virus in a rat hepatoma cell line. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6323–6327. doi: 10.1073/pnas.86.16.6323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sureau C., Eichberg J. W., Hubbard G. B., Romet-Lemonne J. L., Essex M. A molecularly cloned hepatitis B virus produced in vitro is infectious in a chimpanzee. J Virol. 1988 Aug;62(8):3064–3067. doi: 10.1128/jvi.62.8.3064-3067.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sureau C., Romet-Lemonne J. L., Mullins J. I., Essex M. Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Cell. 1986 Oct 10;47(1):37–47. doi: 10.1016/0092-8674(86)90364-8. [DOI] [PubMed] [Google Scholar]
  28. Tagieva N. E., Gizatullin R. Z., Zakharyev V. M., Kisselev L. L. A genome-integrated hepatitis B virus DNA in human neuroblastoma. Gene. 1995 Jan 23;152(2):277–278. doi: 10.1016/0378-1119(94)00665-f. [DOI] [PubMed] [Google Scholar]
  29. Tsurimoto T., Fujiyama A., Matsubara K. Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA. Proc Natl Acad Sci U S A. 1987 Jan;84(2):444–448. doi: 10.1073/pnas.84.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Weiss L., Kekulè A. S., Jakubowski U., Bürgelt E., Hofschneider P. H. The HBV-producing cell line HepG2-4A5: a new in vitro system for studying the regulation of HBV replication and for screening anti-hepatitis B virus drugs. Virology. 1996 Feb 1;216(1):214–218. doi: 10.1006/viro.1996.0049. [DOI] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES