Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Feb;71(2):1310–1316. doi: 10.1128/jvi.71.2.1310-1316.1997

In vitro model for the nuclear transport of the hepadnavirus genome.

M Kann 1, A Bischof 1, W H Gerlich 1
PMCID: PMC191186  PMID: 8995655

Abstract

Hepadnaviruses contain a DNA genome, but they replicate via an RNA intermediate, synthesized by the cellular RNA polymerase II in the nucleus of the infected cell. Thus, nuclear transport of the viral DNA is required in the viral life cycle. Protein-free DNA is only poorly imported into the nucleus, so one or more of the viral proteins must be involved in the transport of the viral genome. In order to identify these viral proteins, we purified woodchuck hepadnavirus (WHV) core particles from infected woodchuck liver, isolated WHV DNA, and extracted the covalent complex of viral polymerase from the particles using urea. Intact core particles, the polymerase-DNA complex, or protein-free WHV DNA from core particles was added to digitonin-permeabilized HuH-7 cells, in which the cytosol was substituted by rabbit reticulocyte lysate (RRL) and an ATP-generating system. The distribution of the viral genome was analyzed by semiquantitative PCR or by hybridization in total nuclei, RRL, nuclear membranes, and nucleoplasm. The polymerase-DNA complex was efficiently transported into the nucleus, as indicated by the resistance of the nucleus-associated DNA to a short-term treatment with DNase I of the intact nuclei. The DNA within core particles stayed mainly in the cytosol and remained protected against DNase I. A minor part of the encapsidated DNA was bound to nuclei. It was protected against DNase I but became accessible after disruption of the nuclei. Deproteinized viral DNA completely remained in the cytosol. These data show that the viral polymerase is probably sufficient for mediating the transport of a hepadnavirus genome into the nucleus and that the viral core particles may release the genome at the nuclear membrane.

Full Text

The Full Text of this article is available as a PDF (360.1 KB).

Selected References

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

  1. Adam S. A., Marr R. S., Gerace L. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol. 1990 Sep;111(3):807–816. doi: 10.1083/jcb.111.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Albin C., Robinson W. S. Protein kinase activity in hepatitis B virus. J Virol. 1980 Apr;34(1):297–302. doi: 10.1128/jvi.34.1.297-302.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartenschlager R., Kuhn C., Schaller H. 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 Res. 1992 Jan 25;20(2):195–202. doi: 10.1093/nar/20.2.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartenschlager R., Schaller H. The amino-terminal domain of the hepadnaviral P-gene encodes the terminal protein (genome-linked protein) believed to prime reverse transcription. EMBO J. 1988 Dec 20;7(13):4185–4192. doi: 10.1002/j.1460-2075.1988.tb03315.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birnbaum F., Nassal M. Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein. J Virol. 1990 Jul;64(7):3319–3330. doi: 10.1128/jvi.64.7.3319-3330.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bishop N., Civitico G., Wang Y. Y., Guo K. J., Birch C., Gust I., Locarnini S. Antiviral strategies in chronic hepatitis B virus infection: I. Establishment of an in vitro system using the duck hepatitis B virus model. J Med Virol. 1990 Jun;31(2):82–89. doi: 10.1002/jmv.1890310204. [DOI] [PubMed] [Google Scholar]
  7. Civitico G. M., Locarnini S. A. The half-life of duck hepatitis B virus supercoiled DNA in congenitally infected primary hepatocyte cultures. Virology. 1994 Aug 15;203(1):81–89. doi: 10.1006/viro.1994.1457. [DOI] [PubMed] [Google Scholar]
  8. Crowther R. A., Kiselev N. A., Böttcher B., Berriman J. A., Borisova G. P., Ose V., Pumpens P. Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy. Cell. 1994 Jun 17;77(6):943–950. doi: 10.1016/0092-8674(94)90142-2. [DOI] [PubMed] [Google Scholar]
  9. Eckhardt S. G., Milich D. R., McLachlan A. Hepatitis B virus core antigen has two nuclear localization sequences in the arginine-rich carboxyl terminus. J Virol. 1991 Feb;65(2):575–582. doi: 10.1128/jvi.65.2.575-582.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Foster G. R., Ackrill A. M., Goldin R. D., Kerr I. M., Thomas H. C., Stark G. R. Expression of the terminal protein region of hepatitis B virus inhibits cellular responses to interferons alpha and gamma and double-stranded RNA. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2888–2892. doi: 10.1073/pnas.88.7.2888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gerlich W. H., Goldmann U., Müller R., Stibbe W., Wolff W. Specificity and localization of the hepatitis B virus-associated protein kinase. J Virol. 1982 Jun;42(3):761–766. doi: 10.1128/jvi.42.3.761-766.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gerlich W. H., Robinson W. S. Hepatitis B virus contains protein attached to the 5' terminus of its complete DNA strand. Cell. 1980 Oct;21(3):801–809. doi: 10.1016/0092-8674(80)90443-2. [DOI] [PubMed] [Google Scholar]
  13. Greber U. F., Willetts M., Webster P., Helenius A. Stepwise dismantling of adenovirus 2 during entry into cells. Cell. 1993 Nov 5;75(3):477–486. doi: 10.1016/0092-8674(93)90382-z. [DOI] [PubMed] [Google Scholar]
  14. Guidotti L. G., Martinez V., Loh Y. T., Rogler C. E., Chisari F. V. Hepatitis B virus nucleocapsid particles do not cross the hepatocyte nuclear membrane in transgenic mice. J Virol. 1994 Sep;68(9):5469–5475. doi: 10.1128/jvi.68.9.5469-5475.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Görlich D., Prehn S., Laskey R. A., Hartmann E. Isolation of a protein that is essential for the first step of nuclear protein import. Cell. 1994 Dec 2;79(5):767–778. doi: 10.1016/0092-8674(94)90067-1. [DOI] [PubMed] [Google Scholar]
  16. Hatton T., Zhou S., Standring D. N. RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication. J Virol. 1992 Sep;66(9):5232–5241. doi: 10.1128/jvi.66.9.5232-5241.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Heinzinger N. K., Bukrinsky M. I., Haggerty S. A., Ragland A. M., Kewalramani V., Lee M. A., Gendelman H. E., Ratner L., Stevenson M., Emerman M. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7311–7315. doi: 10.1073/pnas.91.15.7311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kann M., Gerlich W. H. Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus. J Virol. 1994 Dec;68(12):7993–8000. doi: 10.1128/jvi.68.12.7993-8000.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kann M., Thomssen R., Köchel H. G., Gerlich W. H. Characterization of the endogenous protein kinase activity of the hepatitis B virus. Arch Virol Suppl. 1993;8:53–62. doi: 10.1007/978-3-7091-9312-9_6. [DOI] [PubMed] [Google Scholar]
  20. Kemler I., Whittaker G., Helenius A. Nuclear import of microinjected influenza virus ribonucleoproteins. Virology. 1994 Aug 1;202(2):1028–1033. doi: 10.1006/viro.1994.1432. [DOI] [PubMed] [Google Scholar]
  21. Kenney J. M., von Bonsdorff C. H., Nassal M., Fuller S. D. Evolutionary conservation in the hepatitis B virus core structure: comparison of human and duck cores. Structure. 1995 Oct 15;3(10):1009–1019. doi: 10.1016/s0969-2126(01)00237-4. [DOI] [PubMed] [Google Scholar]
  22. Köck J., Schlicht H. J. Analysis of the earliest steps of hepadnavirus replication: genome repair after infectious entry into hepatocytes does not depend on viral polymerase activity. J Virol. 1993 Aug;67(8):4867–4874. doi: 10.1128/jvi.67.8.4867-4874.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Martin K., Helenius A. Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell. 1991 Oct 4;67(1):117–130. doi: 10.1016/0092-8674(91)90576-k. [DOI] [PubMed] [Google Scholar]
  24. Molnar-Kimber K. L., Summers J., Taylor J. M., Mason W. S. Protein covalently bound to minus-strand DNA intermediates of duck hepatitis B virus. J Virol. 1983 Jan;45(1):165–172. doi: 10.1128/jvi.45.1.165-172.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moore M. S., Blobel G. The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. Cell. 1992 Jun 12;69(6):939–950. doi: 10.1016/0092-8674(92)90613-h. [DOI] [PubMed] [Google Scholar]
  26. Nassal M. The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol. 1992 Jul;66(7):4107–4116. doi: 10.1128/jvi.66.7.4107-4116.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ou J. H., Yeh C. T., Yen T. S. Transport of hepatitis B virus precore protein into the nucleus after cleavage of its signal peptide. J Virol. 1989 Dec;63(12):5238–5243. doi: 10.1128/jvi.63.12.5238-5243.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Qiao M., Macnaughton T. B., Gowans E. J. Adsorption and penetration of hepatitis B virus in a nonpermissive cell line. Virology. 1994 Jun;201(2):356–363. doi: 10.1006/viro.1994.1301. [DOI] [PubMed] [Google Scholar]
  29. Seifer M., Zhou S., Standring D. N. A micromolar pool of antigenically distinct precursors is required to initiate cooperative assembly of hepatitis B virus capsids in Xenopus oocytes. J Virol. 1993 Jan;67(1):249–257. doi: 10.1128/jvi.67.1.249-257.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]
  33. Wu T. T., Coates L., Aldrich C. E., Summers J., Mason W. S. In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway. Virology. 1990 Mar;175(1):255–261. doi: 10.1016/0042-6822(90)90206-7. [DOI] [PubMed] [Google Scholar]
  34. Yeh C. T., Liaw Y. F., Ou J. H. The arginine-rich domain of hepatitis B virus precore and core proteins contains a signal for nuclear transport. J Virol. 1990 Dec;64(12):6141–6147. doi: 10.1128/jvi.64.12.6141-6147.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zhou S., Standring D. N. Hepatitis B virus capsid particles are assembled from core-protein dimer precursors. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10046–10050. doi: 10.1073/pnas.89.21.10046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zyzik E., Gerlich W. H., Uy A., Köchel H., Thomssen R. Assay of hepatitis B virus genome titers in sera of infected subjects. Eur J Clin Microbiol. 1986 Jun;5(3):330–335. doi: 10.1007/BF02017791. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES