Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Apr;87(8):2970–2974. doi: 10.1073/pnas.87.8.2970

A trans-activator function is generated by integration of hepatitis B virus preS/S sequences in human hepatocellular carcinoma DNA.

W H Caselmann 1, M Meyer 1, A S Kekulé 1, U Lauer 1, P H Hofschneider 1, R Koshy 1
PMCID: PMC53815  PMID: 2158099

Abstract

The X gene of wild-type hepatitis B virus or integrated DNA has recently been shown to stimulate transcription of a variety of enhancers and promoters. To further delineate the viral sequences responsible for trans-activation in hepatomas, we cloned the single hepatitis B virus insert from human hepatocellular carcinoma DNA M1. The plasmid pM1 contains 2004 base pairs of hepatitis B virus DNA subtype adr, including truncated preS/S sequences and the enhancer element. The X promoter and 422 nucleotides of the X coding region are present. The entire preC/C gene is deleted. In transient cotransfection assays using Chang liver cells (CCL 13), pM1 DNA exerts a 6- to 10-fold trans-activating effect on the expression of the pSV2CAT reporter plasmid. The trans-activation occurs by stimulation of transcription and is dependent on the simian virus 40 enhancer in the reporter plasmid. Deletion analysis of pM1 subclones reveals that the trans-activator is encoded by preS/S and not by X sequences. A frameshift mutation within the preS2 open reading frame shows that this portion is indispensable for the trans-activating function. Initiation of transcription has been mapped to the S1 promoter. A comparable trans-activating effect is also observed with cloned wild-type hepatitis B virus sequences similarly truncated. These results show that a transcriptional trans-activator function not present in the intact gene is generated by 3' truncation of integrated hepatitis B virus DNA preS/S sequences.

Full text

PDF
2970

Images in this article

2971Image
on p.2971
2971Image
on p.2971
2971Image
on p.2971
2972Image
on p.2972
2973Image
on p.2973
2973Image
on p.2973
2973Image
on p.2973

Selected References

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

  1. Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987 Jun 19;49(6):729–739. doi: 10.1016/0092-8674(87)90611-8. [DOI] [PubMed] [Google Scholar]
  2. Beasley R. P. Hepatitis B virus. The major etiology of hepatocellular carcinoma. Cancer. 1988 May 15;61(10):1942–1956. doi: 10.1002/1097-0142(19880515)61:10<1942::aid-cncr2820611003>3.0.co;2-j. [DOI] [PubMed] [Google Scholar]
  3. Berger I., Shaul Y. Integration of hepatitis B virus: analysis of unoccupied sites. J Virol. 1987 Apr;61(4):1180–1186. doi: 10.1128/jvi.61.4.1180-1186.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bulla G. A., Siddiqui A. Negative regulation of the hepatitis B virus pre-S1 promoter by internal DNA sequences. Virology. 1989 May;170(1):251–260. doi: 10.1016/0042-6822(89)90373-5. [DOI] [PubMed] [Google Scholar]
  5. CHANG R. S. M. Continuous subcultivation of epithelial-like cells from normal human tissues. Proc Soc Exp Biol Med. 1954 Nov;87(2):440–443. doi: 10.3181/00379727-87-21406. [DOI] [PubMed] [Google Scholar]
  6. Cattaneo R., Will H., Hernandez N., Schaller H. Signals regulating hepatitis B surface antigen transcription. Nature. 1983 Sep 22;305(5932):336–338. doi: 10.1038/305336a0. [DOI] [PubMed] [Google Scholar]
  7. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  8. De-Medina T., Faktor O., Shaul Y. The S promoter of hepatitis B virus is regulated by positive and negative elements. Mol Cell Biol. 1988 Jun;8(6):2449–2455. doi: 10.1128/mcb.8.6.2449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dejean A., Bougueleret L., Grzeschik K. H., Tiollais P. Hepatitis B virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma. Nature. 1986 Jul 3;322(6074):70–72. doi: 10.1038/322070a0. [DOI] [PubMed] [Google Scholar]
  10. Eble B. E., MacRae D. R., Lingappa V. R., Ganem D. Multiple topogenic sequences determine the transmembrane orientation of the hepatitis B surface antigen. Mol Cell Biol. 1987 Oct;7(10):3591–3601. doi: 10.1128/mcb.7.10.3591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. Galibert F., Mandart E., Fitoussi F., Tiollais P., Charnay P. Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature. 1979 Oct 25;281(5733):646–650. doi: 10.1038/281646a0. [DOI] [PubMed] [Google Scholar]
  13. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  15. Huh N., Utakoji T. Production of HBs-antigen by two new human hepatoma cell lines and its enhancement by dexamethasone. Gan. 1981 Feb;72(1):178–179. [PubMed] [Google Scholar]
  16. Imai M., Hoshi Y., Okamoto H., Matsui T., Tsurimoto T., Matsubara K., Miyakawa Y., Mayumi M. Free and integrated forms of hepatitis B virus DNA in human hepatocellular carcinoma cells (PLC/342) propagated in nude mice. J Virol. 1987 Nov;61(11):3555–3560. doi: 10.1128/jvi.61.11.3555-3560.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones N. C., Rigby P. W., Ziff E. B. Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev. 1988 Mar;2(3):267–281. doi: 10.1101/gad.2.3.267. [DOI] [PubMed] [Google Scholar]
  18. Kaneko S., Miller R. H. X-region-specific transcript in mammalian hepatitis B virus-infected liver. J Virol. 1988 Nov;62(11):3979–3984. doi: 10.1128/jvi.62.11.3979-3984.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kekulé A. S., Lauer U., Meyer M., Caselmann W. H., Hofschneider P. H., Koshy R. The preS2/S region of integrated hepatitis B virus DNA encodes a transcriptional transactivator. Nature. 1990 Feb 1;343(6257):457–461. doi: 10.1038/343457a0. [DOI] [PubMed] [Google Scholar]
  20. Koshy R., Koch S., von Loringhoven A. F., Kahmann R., Murray K., Hofschneider P. H. Integration of hepatitis B virus DNA: evidence for integration in the single-stranded gap. Cell. 1983 Aug;34(1):215–223. doi: 10.1016/0092-8674(83)90152-6. [DOI] [PubMed] [Google Scholar]
  21. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nagaya T., Nakamura T., Tokino T., Tsurimoto T., Imai M., Mayumi T., Kamino K., Yamamura K., Matsubara K. The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma. Genes Dev. 1987 Oct;1(8):773–782. doi: 10.1101/gad.1.8.773. [DOI] [PubMed] [Google Scholar]
  23. Nelson-Rees W. A., Daniels D. W., Flandermeyer R. R. Cross-contamination of cells in culture. Science. 1981 Apr 24;212(4493):446–452. doi: 10.1126/science.6451928. [DOI] [PubMed] [Google Scholar]
  24. Neurath A. R., Kent S. B. The pre-S region of hepadnavirus envelope proteins. Adv Virus Res. 1988;34:65–142. doi: 10.1016/s0065-3527(08)60516-3. [DOI] [PubMed] [Google Scholar]
  25. Ono Y., Onda H., Sasada R., Igarashi K., Sugino Y., Nishioka K. The complete nucleotide sequences of the cloned hepatitis B virus DNA; subtype adr and adw. Nucleic Acids Res. 1983 Mar 25;11(6):1747–1757. doi: 10.1093/nar/11.6.1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ou J., Rutter W. J. Hybrid hepatitis B virus-host transcripts in a human hepatoma cell. Proc Natl Acad Sci U S A. 1985 Jan;82(1):83–87. doi: 10.1073/pnas.82.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Persing D. H., Varmus H. E., Ganem D. Inhibition of secretion of hepatitis B surface antigen by a related presurface polypeptide. Science. 1986 Dec 12;234(4782):1388–1391. doi: 10.1126/science.3787251. [DOI] [PubMed] [Google Scholar]
  28. Persing D. H., Varmus H. E., Ganem D. The preS1 protein of hepatitis B virus is acylated at its amino terminus with myristic acid. J Virol. 1987 May;61(5):1672–1677. doi: 10.1128/jvi.61.5.1672-1677.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pontisso P., Petit M. A., Bankowski M. J., Peeples M. E. Human liver plasma membranes contain receptors for the hepatitis B virus pre-S1 region and, via polymerized human serum albumin, for the pre-S2 region. J Virol. 1989 May;63(5):1981–1988. doi: 10.1128/jvi.63.5.1981-1988.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Saito I., Oya Y., Shimojo H. Novel RNA family structure of hepatitis B virus expressed in human cells, using a helper-free adenovirus vector. J Virol. 1986 May;58(2):554–560. doi: 10.1128/jvi.58.2.554-560.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Seto E., Yen T. S., Peterlin B. M., Ou J. H. Trans-activation of the human immunodeficiency virus long terminal repeat by the hepatitis B virus X protein. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8286–8290. doi: 10.1073/pnas.85.21.8286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shaul Y., Rutter W. J., Laub O. A human hepatitis B viral enhancer element. EMBO J. 1985 Feb;4(2):427–430. doi: 10.1002/j.1460-2075.1985.tb03646.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Siddiqui A., Jameel S., Mapoles J. Transcriptional control elements of hepatitis B surface antigen gene. Proc Natl Acad Sci U S A. 1986 Feb;83(3):566–570. doi: 10.1073/pnas.83.3.566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  35. Spandau D. F., Lee C. H. trans-activation of viral enhancers by the hepatitis B virus X protein. J Virol. 1988 Feb;62(2):427–434. doi: 10.1128/jvi.62.2.427-434.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Su T. S., Lai C. J., Huang J. L., Lin L. H., Yauk Y. K., Chang C. M., Lo S. J., Han S. H. Hepatitis B virus transcript produced by RNA splicing. J Virol. 1989 Sep;63(9):4011–4018. doi: 10.1128/jvi.63.9.4011-4018.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Treinin M., Laub O. Identification of a promoter element located upstream from the hepatitis B virus X gene. Mol Cell Biol. 1987 Jan;7(1):545–548. doi: 10.1128/mcb.7.1.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Twu J. S., Schloemer R. H. Transcriptional trans-activating function of hepatitis B virus. J Virol. 1987 Nov;61(11):3448–3453. doi: 10.1128/jvi.61.11.3448-3453.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wollersheim M., Debelka U., Hofschneider P. H. A transactivating function encoded in the hepatitis B virus X gene is conserved in the integrated state. Oncogene. 1988 Nov;3(5):545–552. [PubMed] [Google Scholar]
  41. Zahm P., Hofschneider P. H., Koshy R. The HBV X-ORF encodes a transactivator: a potential factor in viral hepatocarcinogenesis. Oncogene. 1988 Aug;3(2):169–177. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES