Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1993 Jul;67(7):3756–3762. doi: 10.1128/jvi.67.7.3756-3762.1993

Precore-mediated inhibition of hepatitis B virus progeny DNA synthesis.

C Lamberts 1, M Nassal 1, I Velhagen 1, H Zentgraf 1, C H Schröder 1
PMCID: PMC237739  PMID: 8510204

Abstract

The capacities to induce the synthesis of hepatitis B virus (HBV) unit-length DNA were compared for two HBV DNAs with an overall sequence diversity of about 10%. They had been cloned from serum (DNA2) and from a hepatocellular carcinoma (DNA4), respectively. As a major difference, DNA4 carries a translational stop signal preventing the synthesis of precore protein. Progeny DNA yields obtained after transfection with respective pregenome transcription units allocated DNA2 to a low-replicator and DNA4 to a high-replicator phenotype. Cotransfection of DNA2 interfered with progeny DNA synthesis induced by DNA4. By mutual exchange of restriction fragments, the region on the viral genome responsible for the differing replicator phenotypes was confined to a sequence comprising the 3'-terminal part of the X gene, core promoter, encapsidation signal epsilon, precore/core gene, and 5'-terminal part of the pol gene. Point mutations in DNA2 abolishing proper expression of the precore gene strongly enhanced the yield of progeny DNA, whereas cotransfection of a precore expression plasmid with DNA4 or with the mutated DNA2 substantially lowered the amount of progeny DNA. Hence, precore expression acts as an inhibitory principle for HBV replication. The same stop mutation as in DNA4 has been found to arise frequently in virus carriers. Loss of precore expression and concomitant conversion to a more severe hepatitis, as observed in the course of a chronic infection, thus can be explained by a relaxation of replication-level control.

Full text

PDF
3756

Images in this article

3757Image
on p.3757
3758Image
on p.3758
3758Image
on p.3758
3758Image
on p.3758
3759Image
on p.3759
3759Image
on p.3759
3760Image
on p.3760

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. Bartenschlager R., Schaller H. Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. EMBO J. 1992 Sep;11(9):3413–3420. doi: 10.1002/j.1460-2075.1992.tb05420.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brunetto M. R., Giarin M. M., Oliveri F., Chiaberge E., Baldi M., Alfarano A., Serra A., Saracco G., Verme G., Will H. Wild-type and e antigen-minus hepatitis B viruses and course of chronic hepatitis. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4186–4190. doi: 10.1073/pnas.88.10.4186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bruss V., Gerlich W. H. Formation of transmembraneous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein. Virology. 1988 Apr;163(2):268–275. doi: 10.1016/0042-6822(88)90266-8. [DOI] [PubMed] [Google Scholar]
  5. Carman W. F., Jacyna M. R., Hadziyannis S., Karayiannis P., McGarvey M. J., Makris A., Thomas H. C. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet. 1989 Sep 9;2(8663):588–591. doi: 10.1016/s0140-6736(89)90713-7. [DOI] [PubMed] [Google Scholar]
  6. Chang C., Enders G., Sprengel R., Peters N., Varmus H. E., Ganem D. Expression of the precore region of an avian hepatitis B virus is not required for viral replication. J Virol. 1987 Oct;61(10):3322–3325. doi: 10.1128/jvi.61.10.3322-3325.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen H. S., Kew M. C., Hornbuckle W. E., Tennant B. C., Cote P. J., Gerin J. L., Purcell R. H., Miller R. H. The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host. J Virol. 1992 Sep;66(9):5682–5684. doi: 10.1128/jvi.66.9.5682-5684.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Garcia P. D., Ou J. H., Rutter W. J., Walter P. Targeting of the hepatitis B virus precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage translocation can be aborted and the product released into the cytoplasm. J Cell Biol. 1988 Apr;106(4):1093–1104. doi: 10.1083/jcb.106.4.1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Junker-Niepmann M., Bartenschlager R., Schaller H. A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA. EMBO J. 1990 Oct;9(10):3389–3396. doi: 10.1002/j.1460-2075.1990.tb07540.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Loncarevic I. F., Zentgraf H., Schröder C. H. Sequence of a replication competent hepatitis B virus genome with a preX open reading frame. Nucleic Acids Res. 1990 Aug 25;18(16):4940–4940. doi: 10.1093/nar/18.16.4940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Loncarević I. F., Schranz P., Zentgraf H., Liang X. H., Herrmann G., Tang Z. Y., Schröder C. H. Replication of hepatitis B virus in a hepatocellular carcinoma. Virology. 1990 Jan;174(1):158–168. doi: 10.1016/0042-6822(90)90064-x. [DOI] [PubMed] [Google Scholar]
  17. Milich D. R., Jones J. E., Hughes J. L., Price J., Raney A. K., McLachlan A. Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero? Proc Natl Acad Sci U S A. 1990 Sep;87(17):6599–6603. doi: 10.1073/pnas.87.17.6599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miyanohara A., Imamura T., Araki M., Sugawara K., Ohtomo N., Matsubara K. Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: synthesis of two polypeptides translated from different initiation codons. J Virol. 1986 Jul;59(1):176–180. doi: 10.1128/jvi.59.1.176-180.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nassal M., Junker-Niepmann M., Schaller H. Translational inactivation of RNA function: discrimination against a subset of genomic transcripts during HBV nucleocapsid assembly. Cell. 1990 Dec 21;63(6):1357–1363. doi: 10.1016/0092-8674(90)90431-d. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Nassal M. Total chemical synthesis of a gene for hepatitis B virus core protein and its functional characterization. Gene. 1988 Jun 30;66(2):279–294. doi: 10.1016/0378-1119(88)90364-2. [DOI] [PubMed] [Google Scholar]
  22. Okamoto H., Yotsumoto S., Akahane Y., Yamanaka T., Miyazaki Y., Sugai Y., Tsuda F., Tanaka T., Miyakawa Y., Mayumi M. Hepatitis B viruses with precore region defects prevail in persistently infected hosts along with seroconversion to the antibody against e antigen. J Virol. 1990 Mar;64(3):1298–1303. doi: 10.1128/jvi.64.3.1298-1303.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Omata M., Ehata T., Yokosuka O., Hosoda K., Ohto M. Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis. N Engl J Med. 1991 Jun 13;324(24):1699–1704. doi: 10.1056/NEJM199106133242404. [DOI] [PubMed] [Google Scholar]
  24. Ou J. H., Laub O., Rutter W. J. Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1578–1582. doi: 10.1073/pnas.83.6.1578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Schlicht H. J., Salfeld J., Schaller H. The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation. J Virol. 1987 Dec;61(12):3701–3709. doi: 10.1128/jvi.61.12.3701-3709.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schlicht H. J., Schaller H. The secretory core protein of human hepatitis B virus is expressed on the cell surface. J Virol. 1989 Dec;63(12):5399–5404. doi: 10.1128/jvi.63.12.5399-5404.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Standring D. N., Ou J. H., Masiarz F. R., Rutter W. J. A signal peptide encoded within the precore region of hepatitis B virus directs the secretion of a heterogeneous population of e antigens in Xenopus oocytes. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8405–8409. doi: 10.1073/pnas.85.22.8405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Takahashi K., Machida A., Funatsu G., Nomura M., Usuda S., Aoyagi S., Tachibana K., Miyamoto H., Imai M., Nakamura T. Immunochemical structure of hepatitis B e antigen in the serum. J Immunol. 1983 Jun;130(6):2903–2907. [PubMed] [Google Scholar]
  35. Tong S. P., Diot C., Gripon P., Li J., Vitvitski L., Trépo C., Guguen-Guillouzo C. In vitro replication competence of a cloned hepatitis B virus variant with a nonsense mutation in the distal pre-C region. Virology. 1991 Apr;181(2):733–737. doi: 10.1016/0042-6822(91)90908-t. [DOI] [PubMed] [Google Scholar]
  36. Tong S. P., Li J. S., Vitvitski L., Trépo C. Active hepatitis B virus replication in the presence of anti-HBe is associated with viral variants containing an inactive pre-C region. Virology. 1990 Jun;176(2):596–603. doi: 10.1016/0042-6822(90)90030-u. [DOI] [PubMed] [Google Scholar]
  37. Tong S. P., Li J. S., Vitvitski L., Trépo C. Replication capacities of natural and artificial precore stop codon mutants of hepatitis B virus: relevance of pregenome encapsidation signal. Virology. 1992 Nov;191(1):237–245. doi: 10.1016/0042-6822(92)90185-r. [DOI] [PubMed] [Google Scholar]
  38. Tur-Kaspa R., Teicher L., Laub O., Itin A., Dagan D., Bloom B. R., Shafritz D. A. Alpha interferon suppresses hepatitis B virus enhancer activity and reduces viral gene transcription. J Virol. 1990 Apr;64(4):1821–1824. doi: 10.1128/jvi.64.4.1821-1824.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Ulrich P. P., Bhat R. A., Kelly I., Brunetto M. R., Bonino F., Vyas G. N. A precore-defective mutant of hepatitis B virus associated with e antigen-negative chronic liver disease. J Med Virol. 1990 Oct;32(2):109–118. doi: 10.1002/jmv.1890320208. [DOI] [PubMed] [Google Scholar]
  41. Wasenauer G., Köck J., Schlicht H. J. A cysteine and a hydrophobic sequence in the noncleaved portion of the pre-C leader peptide determine the biophysical properties of the secretory core protein (HBe protein) of human hepatitis B virus. J Virol. 1992 Sep;66(9):5338–5346. doi: 10.1128/jvi.66.9.5338-5346.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Yee J. K. A liver-specific enhancer in the core promoter region of human hepatitis B virus. Science. 1989 Nov 3;246(4930):658–661. doi: 10.1126/science.2554495. [DOI] [PubMed] [Google Scholar]

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

RESOURCES