1887

Abstract

Besides the three essential genes encoding the envelope, core and polymerase proteins, all mammalian hepadnaviruses examined to date contain a fourth gene which is referred to as the -gene. This gene is believed to encode a transcriptional transactivator which positively regulates viral gene expression. Attempts to detect X-protein or in tissue culture lead to varying results. Whereas some groups could detect a protein of the expected size, other groups did not. To establish optimal conditions for the isolation of the human hepatitis B virus X-protein, we introduced a recognition site for protein kinase A into the -gene. Upon phosphorylation with radioactive ATP, this modified X-protein can be detected with very high specificity and sensitivity. Tissue culture experiments showed that X-protein expressed from a cytomegalovirus-driven plasmid is not soluble in non-ionic detergent but rather has to be extracted from the cell pellet by boiling with SDS at a slightly alkaline pH. This method was then used to examine the organs of several transgenic mouse lines which expressed the modified -gene under control of the authentic promoter. The data show that expression of the -gene and subsequent biosynthesis of the X-protein is not tissue-specific but rather can occur in most organs.

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1999-09-01
2020-01-28
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References

  1. Balsano, C., Billet, O., Bennoun, M., Cavard, C., Zider, A., Grimber, G., Natoli, G., Briand, P. & Levrero, M. ( 1994; ). Hepatitis B virus X gene product acts as a transactivator in vivo. Journal of Hepatology 21, 103-109.[CrossRef]
    [Google Scholar]
  2. Bartenschlager, R., Kuhn, C. & Schaller, H. ( 1991; ). Expression of the P-protein of the human hepatitis B virus in a vaccinia virus system and detection of the nucleocapsid-associated P-gene product by radiolabelling at newly introduced phosphorylation sites. Nucleic Acids Research 20, 195-202.
    [Google Scholar]
  3. Billet, O., Grimber, G., Levrero, M., Seye, K. A., Briand, P. & Joulin, V. ( 1995; ). In vivo activity of the hepatitis B virus core promoter: tissue specificity and temporal regulation. Journal of Virology 69, 5912-5916.
    [Google Scholar]
  4. Blum, H. E., Zhang, Z. S., Galun, E., von Weizsäcker, F., Garner, B., Liang, T. J. & Wands, J. R. ( 1992; ). Hepatitis B virus X protein is not central to the viral life cycle in vitro. Journal of Virology 66, 1223-1227.
    [Google Scholar]
  5. Chen, H. S., Kaneko, S., Girones, R., Anderson, R. W., Hornbuckle, W. E., Tennant, B. C., Cote, P. J., Gerin, J. L., Purcell, R. H. & Miller, R. H. ( 1993; ). The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks. Journal of Virology 67, 1218-1226.
    [Google Scholar]
  6. Ganem, D. & Varmus, H. E. ( 1987; ). The molecular biology of the hepatitis B viruses. Annual Review of Biochemistry 56, 661-693.
    [Google Scholar]
  7. Guo, W., Bell, K. D. & Ou, J.-H. ( 1991; ). Characterization of the hepatitis B virus EnhI enhancer and X promoter complex. Journal of Virology 65, 6686-6692.
    [Google Scholar]
  8. Hogan, B., Beddington, R., Costantini, F. & Lacy, E. (1994). Manipulating the Mouse Embryo: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  9. Junker, M., Galle, P. & Schaller, H. ( 1987; ). Expression and replication of the hepatitis B virus genome under foreign promoter control. Nucleic Acids Research 15, 10117-10132.[CrossRef]
    [Google Scholar]
  10. Kim, C. M., Koike, K., Saito, I., Miyamura, T. & Jay, G. ( 1991; ). HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature 351, 317-320.[CrossRef]
    [Google Scholar]
  11. Koike, K., Moriya, K., Iino, S., Yotsuyanagi, H., Endo, Y., Miyamura, T. & Kurokawa, K. ( 1994; ). High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice. Hepatology 19, 810-819.[CrossRef]
    [Google Scholar]
  12. Laub, O. & Treinin, M. ( 1987; ). Identification of a promoter element located upstream from the hepatitis B virus X gene. Molecular and Cellular Biology 7, 545-548.
    [Google Scholar]
  13. Lee, T.-H., Finegold, M. J., Shen, R.-F., DeMayo, J. L., Woo, S. L. C. & Butel, J. S. ( 1990; ). Hepatitis B virus transactivator X protein is not tumorigenic in transgenic mice. Journal of Virology 64, 5939-5947.
    [Google Scholar]
  14. Li, B.-L., Langer, J. A., Schwartz, B. & Pestka, S. ( 1989; ). Creation of phosphorylation sites in proteins: construction of a phosphorylatable human interferon alpha. Proceedings of the National Academy of Sciences, USA 86, 558-562.[CrossRef]
    [Google Scholar]
  15. Moriya, K., Matsukura, M., Kurokawa, K. & Koike, K. ( 1996; ). In vivo inhibition of hepatitis B virus gene expression by antisense phosphorothioate oligonucleotides. Biochemical and Biophysical Research Communications 218, 217-223.[CrossRef]
    [Google Scholar]
  16. Perfumo, S., Amicone, L., Colloca, S., Giorgio, M., Pozzi, L. & Tripodi, M. ( 1992; ). Recognition efficiency of the hepatitis B virus polyadenylation signals is tissue specific in transgenic mice. Journal of Virology 66, 6819-6823.
    [Google Scholar]
  17. Reifenberg, K., Löhler, J., Pudollek, H.-P., Schmitteckert, E., Spindler, G., Köck, J. & Schlicht, H.-J. ( 1997; ). Long-term expression of the hepatitis B virus core-e and X-proteins does not cause pathologic changes in transgenic mice. Journal of Hepatology 26, 119-130.[CrossRef]
    [Google Scholar]
  18. Rossner, M. T. ( 1992; ). Hepatitis B virus X-gene product: a promiscuous transcriptional activator. Journal of Medical Virology 36, 101-117.[CrossRef]
    [Google Scholar]
  19. Shaul, Y., Rutter, W. J. & Laub, O. ( 1985; ). A human hepatitis B viral enhancer element. EMBO Journal 4, 427-430.
    [Google Scholar]
  20. Slagle, B. L., Lee, T.-H., Medina, D., Finegold, M. J. & Butel, J. S. ( 1996; ). Increased sensitivity to the hepatocarcinogen diethylnitrosamine intransgenic mice carrying the hepatitis B virus X gene. Molecular Carcinogenesis 15, 261-269.[CrossRef]
    [Google Scholar]
  21. Su, Q., Schröder, C. H., Hofmann, W. J., Otto, G., Pichlmayr, R. & Bannasch, P. ( 1998; ). Expression of hepatitis B virus X protein in HBV-infected human livers and hepatocellular carcinomas. Hepatology 27, 1109-1120.[CrossRef]
    [Google Scholar]
  22. Summers, J. ( 1981; ). Three recently described animal virus models for human hepatitis B virus. Hepatology 1, 179-183.[CrossRef]
    [Google Scholar]
  23. Terradillos, O., Billet, O., Renard, C.-A., Levy, R., Molina, T., Briand, P. & Buendia, M. A. ( 1997; ). The hepatitis B virus X gene potentiates c-myc-induced liver oncogenesis in transgenic mice. Oncogene 14, 395-404.[CrossRef]
    [Google Scholar]
  24. Tognoni, A., Cattaneo, R., Serfling, E. & Schaffner, W. ( 1985; ). A novel expression selection approach allows precise mapping of the hepatitis B virus enhancer. Nucleic Acids Research 13, 7457-7472.[CrossRef]
    [Google Scholar]
  25. Towbin, H., Stahelin, T. & Gordon, J. ( 1979; ). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350-4354.[CrossRef]
    [Google Scholar]
  26. Wang, Y., Chen, P., Wu, X., Sun, A., Wang, H., Zhu, Y. & Li, Z. ( 1990; ). A new enhancer element, ENII, identified in the X gene of hepatitis B virus. Journal of Virology 64, 3977-3981.
    [Google Scholar]
  27. Yaginuma, K., Shirakata, Y., Kobayashi, M. & Koike, K. ( 1987; ). Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. Proceedings of the National Academy of Sciences, USA 84, 2678-2682.[CrossRef]
    [Google Scholar]
  28. Yuh, C. & Ting, L. ( 1990; ). The genome of hepatitis B virus contains a second enhancer: cooperation of two elements within this enhancer is required for its function. Journal of Virology 64, 4281-4287.
    [Google Scholar]
  29. Zoulim, F., Saputelli, J. & Seeger, C. ( 1994; ). Woodchuck hepatitis virus X protein is required for viral infection in vivo. Journal of Virology 68, 2026-2030.
    [Google Scholar]
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