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. 1987 Mar;6(3):675–680. doi: 10.1002/j.1460-2075.1987.tb04807.x

Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line.

C M Chang, K S Jeng, C P Hu, S J Lo, T S Su, L P Ting, C K Chou, S H Han, E Pfaff, J Salfeld, et al.
PMCID: PMC553450  PMID: 3034605

Abstract

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core-related antigens (HBc/HBeAg) if well-differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH-7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH-7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus-associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS--PAGE after labeling with [35S]methionine. Antisera specific for X-gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV-related gene function and HBV replication.

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Selected References

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

  1. Aden D. P., Fogel A., Plotkin S., Damjanov I., Knowles B. B. Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature. 1979 Dec 6;282(5739):615–616. doi: 10.1038/282615a0. [DOI] [PubMed] [Google Scholar]
  2. Asselbergs F. A., Will H., Wingfield P., Hirschi M. A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein. J Mol Biol. 1986 Jun 5;189(3):401–411. doi: 10.1016/0022-2836(86)90312-8. [DOI] [PubMed] [Google Scholar]
  3. Cattaneo R., Will H., Darai G., Pfaff E., Schaller H. Detection of an element of the SV40 late promoter in vectors used for expression studies in COS cells. EMBO J. 1983;2(4):511–514. doi: 10.1002/j.1460-2075.1983.tb01455.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cattaneo R., Will H., Schaller H. Hepatitis B virus transcription in the infected liver. EMBO J. 1984 Sep;3(9):2191–2196. doi: 10.1002/j.1460-2075.1984.tb02113.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang C., Lin Y., O-Lee T. W., Chou C. K., Lee T. S., Liu T. J., P'eng F. K., Chen T. Y., Hu C. P. Induction of plasma protein secretion in a newly established human hepatoma cell line. Mol Cell Biol. 1983 Jun;3(6):1133–1137. doi: 10.1128/mcb.3.6.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang C., Simmons D. T., Martin M. A., Mora P. T. Identification and partial characterization of new antigens from simian virus 40-transformed mouse cells. J Virol. 1979 Aug;31(2):463–471. doi: 10.1128/jvi.31.2.463-471.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Charnay P., Pourcel C., Louise A., Fritsch A., Tiollais P. Cloning in Escherichia coli and physical structure of hepatitis B virion DNA. Proc Natl Acad Sci U S A. 1979 May;76(5):2222–2226. doi: 10.1073/pnas.76.5.2222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Doi I. Establishment of a cell line and its clonal sublines from a patient with hepatoblastoma. Gan. 1976 Feb;67(1):1–10. [PubMed] [Google Scholar]
  10. Fogh J., Fogh J. M., Orfeo T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst. 1977 Jul;59(1):221–226. doi: 10.1093/jnci/59.1.221. [DOI] [PubMed] [Google Scholar]
  11. Gerin J. L., Purcell R. H., Hoggan M. D., Holland P. V., Chanock R. M. Biophysical properties of Australia antigen. J Virol. 1969 Nov;4(5):763–768. doi: 10.1128/jvi.4.5.763-768.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gough N. M., Murray K. Expression of the hepatitis B virus surface, core and E antigen genes by stable rat and mouse cell lines. J Mol Biol. 1982 Nov 25;162(1):43–67. doi: 10.1016/0022-2836(82)90161-9. [DOI] [PubMed] [Google Scholar]
  13. Heermann K. H., Goldmann U., Schwartz W., Seyffarth T., Baumgarten H., Gerlich W. H. Large surface proteins of hepatitis B virus containing the pre-s sequence. J Virol. 1984 Nov;52(2):396–402. doi: 10.1128/jvi.52.2.396-402.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hirschman S. Z., Price P., Garfinkel E., Christman J., Acs G. Expression of cloned hepatitis B virus DNA in human cell cultures. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5507–5511. doi: 10.1073/pnas.77.9.5507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Howard C. R., Zuckerman A. J. Core antigen and circulating anti-core antibody in hepatitis B infection. J Immunol Methods. 1977;14(3-4):291–301. doi: 10.1016/0022-1759(77)90140-5. [DOI] [PubMed] [Google Scholar]
  16. Kaplan P. M., Greenman R. L., Gerin J. L., Purcell R. H., Robinson W. S. DNA polymerase associated with human hepatitis B antigen. J Virol. 1973 Nov;12(5):995–1005. doi: 10.1128/jvi.12.5.995-1005.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Nakabayashi H., Taketa K., Miyano K., Yamane T., Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 1982 Sep;42(9):3858–3863. [PubMed] [Google Scholar]
  20. Pfaff E., Klinkert M. Q., Theilmann L., Schaller H. Characterization of large surface proteins of hepatitis B virus by antibodies to preS-S encoded amino acids. Virology. 1986 Jan 15;148(1):15–22. doi: 10.1016/0042-6822(86)90399-5. [DOI] [PubMed] [Google Scholar]
  21. Pourcel C., Louise A., Gervais M., Chenciner N., Dubois M. F., Tiollais P. Transcription of the hepatitis B surface antigen gene in mouse cells transformed with cloned viral DNA. J Virol. 1982 Apr;42(1):100–105. doi: 10.1128/jvi.42.1.100-105.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Seeger C., Ganem D., Varmus H. E. Biochemical and genetic evidence for the hepatitis B virus replication strategy. Science. 1986 Apr 25;232(4749):477–484. doi: 10.1126/science.3961490. [DOI] [PubMed] [Google Scholar]
  23. Seeger C., Ganem D., Varmus H. E. Nucleotide sequence of an infectious molecularly cloned genome of ground squirrel hepatitis virus. J Virol. 1984 Aug;51(2):367–375. doi: 10.1128/jvi.51.2.367-375.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sprengel R., Kuhn C., Manso C., Will H. Cloned duck hepatitis B virus DNA is infectious in Pekin ducks. J Virol. 1984 Dec;52(3):932–937. doi: 10.1128/jvi.52.3.932-937.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Summers J., Mason W. S. Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell. 1982 Jun;29(2):403–415. doi: 10.1016/0092-8674(82)90157-x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Tiollais P., Pourcel C., Dejean A. The hepatitis B virus. Nature. 1985 Oct 10;317(6037):489–495. doi: 10.1038/317489a0. [DOI] [PubMed] [Google Scholar]
  28. Tuttleman J. S., Pourcel C., Summers J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell. 1986 Nov 7;47(3):451–460. doi: 10.1016/0092-8674(86)90602-1. [DOI] [PubMed] [Google Scholar]
  29. Tuttleman J. S., Pugh J. C., Summers J. W. In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus. J Virol. 1986 Apr;58(1):17–25. doi: 10.1128/jvi.58.1.17-25.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wang Y., Schäfer-Ridder M., Stratowa C., Wong T. K., Hofschneider P. H. Expression of hepatitis B surface antigen in unselected cell culture transfected with recircularized HBV DNA. EMBO J. 1982;1(10):1213–1216. doi: 10.1002/j.1460-2075.1982.tb00015.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Will H., Cattaneo R., Darai G., Deinhardt F., Schellekens H., Schaller H. Infectious hepatitis B virus from cloned DNA of known nucleotide sequence. Proc Natl Acad Sci U S A. 1985 Feb;82(3):891–895. doi: 10.1073/pnas.82.3.891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Will H., Cattaneo R., Koch H. G., Darai G., Schaller H., Schellekens H., van Eerd P. M., Deinhardt F. Cloned HBV DNA causes hepatitis in chimpanzees. Nature. 1982 Oct 21;299(5885):740–742. doi: 10.1038/299740a0. [DOI] [PubMed] [Google Scholar]
  33. Will H., Cattaneo R., Pfaff E., Kuhn C., Roggendorf M., Schaller H. Expression of hepatitis B antigens with a simian virus 40 vector. J Virol. 1984 May;50(2):335–342. doi: 10.1128/jvi.50.2.335-342.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Will H., Salfeld J., Pfaff E., Manso C., Theilmann L., Schaler H. Putative reverse transcriptase intermediates of human hepatitis B virus in primary liver carcinomas. Science. 1986 Feb 7;231(4738):594–596. doi: 10.1126/science.2418501. [DOI] [PubMed] [Google Scholar]

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