1887

Abstract

Hepatitis B virus (HBV) genomes with deletions in the precore-core (preC-C) promoter have been detected in HBV infections without serological markers. To address whether the mutations are responsible for the reduced production of virus antigens, either an 8 bp (8d, position 1763 to 1770) or a 20 bp (20d, 1753 to 1772) deletion was created in a wild-type (wt) HBV clone. Both mutations cause premature termination of the overlapping X ORF. When introduced into HepG2 cells, both mutants produced reduced amounts of HBsAg, HBcAg and HBeAg, but released the same or more virion- associated DNA compared with the wt. A co-transfection of the 20d mutant with a small amount of intact X gene resulted in a 3-fold increase of HBcAg production compared to transfection with either the 20d or wt alone. When the promoter region was cloned into CAT plasmids, the 8d preC promoter showed weak activity and its initiation site was shifted 6 to 10 bp downstream. The preC promoter activity of 20d was not detectable by CAT ELISA and 5′ RACE. The levels of C transcripts of both mutants were higher than that of the wt, and their start sites were not altered. Therefore, the deletions cause the reduction of HBsAg, HBcAg and HBeAg although the mutant viruses can still replicate in cultured cells. The reduction of HBeAg is due to both the reduced preC promoter activity and the defect in HBx. The reduction of HBcAg is due to the disrupted X gene, despite augmented C promoter activity.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-78-6-1479
1997-06-01
2022-05-23
Loading full text...

Full text loading...

/deliver/fulltext/jgv/78/6/9191946.html?itemId=/content/journal/jgv/10.1099/0022-1317-78-6-1479&mimeType=html&fmt=ahah

References

  1. Aden D. P., Fogel A., Plotkin S., Damjanov I., Knowles B. B. 1979; Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature 282:615–616
    [Google Scholar]
  2. Alter H. J., Seeff L. B., Kaplan P. M., McAuliffe V. J., Wright E. C., Gerin J. L., Purcell R. H., Holland P. V., Zimmerman H. J. 1976; Type B hepatitis : the infectivity of blood positive for e antigen and DNA polymerase after accidental needle stick exposure. New England Journal of Medicine 295:909–913
    [Google Scholar]
  3. Bruss V., Gerlich W. H. 1988; Formation of transmembraneous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein. Virology 163:268–275
    [Google Scholar]
  4. 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]
  5. Chen I., Huang C., Ting L. 1995; Overlapping initiator and TATA box functions in the basal core promoter of hepatitis B virus. Journal of Virology 69:3647–3657
    [Google Scholar]
  6. Colgrove R., Simon G., Ganem D. 1989; Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication. Journal of Virology 63:4019–4026
    [Google Scholar]
  7. Edwards J. B. D. M., Delort J., Mallet J. 1991; Oligodeoxy-ribonucleotide ligation to single-stranded cDNAs : a new tool for cloning 5ʹ ends of mRNAs and for constructing cDNA libraries by in vitro amplification. Nucleic Acids Research 19:5227–5232
    [Google Scholar]
  8. Feitelson M. 1994; Biology of hepatitis B virus variants. Laboratory Investigation 71:324–349
    [Google Scholar]
  9. Feitelson M., Duan L. X., Horiike N., Clayton M. 1991; Hepatitis B X open reading frame deletion mutants isolated from atypical hepatitis B infections. Journal of Hepatology 13:S58–S60
    [Google Scholar]
  10. Feitelson M. A., Duan L. X., Guo J., Horiike N., McIntyre G., Blumberg B. S., Thomas H. C., Carman W. 1995a; Precore and X region mutants in hepatitis B virus infections among renal dialysis patients. Journal of Viral Hepatitis 2:19–31
    [Google Scholar]
  11. Feitelson M. A., Duan L. X., Guo J., Blumberg B. S. 1995b; X region deletion mutants associated with surface antigen-positive hepatitis B virus infections. Gastroenterology 108:1810–1819
    [Google Scholar]
  12. Jean-Jean O., Levrero M., Will H., Perricaudet M., Rossignol J. M. 1989; Expression mechanism of the hepatitis B virus (HBV) C gene and biosynthesis of HBe antigen. Virology 170:99–106
    [Google Scholar]
  13. Junker-Niepman M., Bartenschlager R., Schaller H. 1990; A short cis -acting sequence is required for hepatitis B virus pregenome encapsi- dation and sufficient for packaging of foreign RNA. EMBO Journal 9:3389–3396
    [Google Scholar]
  14. Kim S. H., Hong S. P., Kim S. K., Lee W. S., Rho H. M. 1992; Replication of a mutant hepatitis B virus with a fused X-C reading frame in hepatoma cells. Journal of General Virology 73:2421–2424
    [Google Scholar]
  15. Lamberts C., Nassal M., Velhagen I., Zentgraf H., Schroder C. H. 1993; Precore-mediated inhibition of hepatitis B virus progeny DNA synthesis. Journal of Virology 67:3756–3762
    [Google Scholar]
  16. Landt O., Grunert H. P., Hahn U. 1990; A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene 96:125–128
    [Google Scholar]
  17. Laskus T., Rakela J., Tong M. J., Nowicki M. J., Mosley J. W., Persing D. H. 1994; Naturally occurring hepatitis B virus mutants with deletions in the core promoter region. Journal of Hepatology 20:837–841
    [Google Scholar]
  18. Lo W. Y., Ting L. P. 1994; Repression of enhancer II activity by a negative regulatory element in the hepatitis B virus genome. Journal of Virology 68:1758–1764
    [Google Scholar]
  19. Mason W. S., Aldrich C., Summers J., Taylor J. M. 1982; Asymmetric replication of duck hepatitis B virus DNA in liver cells (free minus strand DNA). Proceedings of the National Academy of Sciences USA: 793997–4001
    [Google Scholar]
  20. Nakatake H., Chisaka O., Yamamoto S., Matsubara K., Koshy R. 1993; Effect of X protein on transactivation of hepatitis B virus promoters and on viral replication. Virology 195:305–314
    [Google Scholar]
  21. Nassal M., Junker-Niepman M., Schaller H. 1990; Translational inactivation of RNA function: discrimination against a subset of genomic transcripts during HBV nucleocapsid assembly. Cell 63:1357–1363
    [Google Scholar]
  22. Okada K., Kamiyama M., Inomata M., Imai Y., Miyakawa Y., Mayumi M. 1976; e antigen and anti-e in the serum of asymptomatic carrier mother as indicators of positive and negative transmission of hepatitis B virus to their infants. New England Journal of Medicine 294:746–749
    [Google Scholar]
  23. Okamoto H., Omi S., Wang Y., Imai M., Mayumi M. 1990; Trans complementation of the C gene of human and the P gene of woodchuck hepadnaviruses. Journal of General Virology 71:959–963
    [Google Scholar]
  24. Okamoto H., Tsuda F., Akahane Y., Sugai Y., Yoshiba M., Moriyama K., Tanaka T., Miyakawa Y., Mayumi M. 1994; Hepatitis B virus with mutations in the core promoter for an e antigen-negative phenotype in carriers with antibody to e antigen. Journal of Virology 68:8102–8110
    [Google Scholar]
  25. Ou J. H., Bao H., Shih C., Tahara S. M. 1990; Preferred translation of human hepatitis B virus polymerase from core protein - but not from precore protein-specific transcript. Journal of Virology 64:4578–4581
    [Google Scholar]
  26. Pollack J. R., Ganem D. 1993; An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation. Journal of Virology 67:3254–3263
    [Google Scholar]
  27. Preisler-Adams S., Schlayer H. J., Peters T., Hettler F., Gerok W., Rasenack J. 1993; Sequence analysis of hepatitis B virus DNA in immunologically negative infection. Archives of Virology 133:385–396
    [Google Scholar]
  28. Preisler-Adams S., Schlayer H., Peters T., Hiller B., Gerok W., Rasenack J. 1994; In vitro HBV gene expression of immunologically negative HBV variants. Gastroenterology 106:A965
    [Google Scholar]
  29. Repp R., Keller C., Borkhardt A., Csecke A., Schaefer S., Gerlich W. H., Lampert F. 1992; Detection of a hepatitis B virus variant with a truncated X gene and enhancer II. Archives of Virology 125:299–304
    [Google Scholar]
  30. Runkel L., Fischer M., Schaller H. 1993; Two-codon insertion mutations of the HBx define two separate regions necessary for its trans-activation function. Virology 197:529–536
    [Google Scholar]
  31. Schaller H., Fischer M. 1991; Transcription control of hepadnavirus gene expression. Current Topics in Microbiology and Immunology 168:21–39
    [Google Scholar]
  32. Selden R. F., Howie K. B., Rowe M. E., Goodman H. M., Moore D. D. 1986; Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Molecular and Cellular Biology 6:3173–3179
    [Google Scholar]
  33. Seeger C., Ganem D., Varmus H. E. 1986; Biochemical and genetic evidence for the hepatitis B virus replication strategy. Science 232:477–484
    [Google Scholar]
  34. Spandau D. F., Lee C. H. 1988; Trans -activation of viral enhancers by the hepatitis B virus X protein. Journal of Virology 62:427–434
    [Google Scholar]
  35. Summers J., Mason W. S. 1982; Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29:403–415
    [Google Scholar]
  36. Takahashi K., Machida A., Funatsu G., Nomura M., Usuda S., Aoyagi S., Tachibana K., Miyamoto H., Imai M., Nakamura T., Miyakawa Y., Mayumi M. 1983; Immunochemical structure of hepatitis B e antigen in the serum. Journal ofImmunology 130:2903–2907
    [Google Scholar]
  37. Takada S., Koike K. 1994; Three sites of hepatitis B virus X protein cooperatively interact with cellular proteins. Virology 205:503–510
    [Google Scholar]
  38. Trusnt R., Antunovic J., Greenblatt J., Prives C., Cromlish J. A. 1995; Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element-directed transactivation. Journal of Virology 69:1851–1859
    [Google Scholar]
  39. Twu J. S., Schloemer R. H. 1987; Transcriptional trans-activating function of hepatitis B virus. Journal of Virology 61:3448–3453
    [Google Scholar]
  40. Usuda S., Tsuda F., Gotanda T., Tachibana K., Nomura M., Okamoto H., Imai M., Nakamura T., Miyakawa Y., Mayumi M. 1986; A solidphase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies. Journal of Immunological Methods 87:203–210
    [Google Scholar]
  41. Wang Y., Chen P., Wu S., Sun A. L., Wang H., Zhu Y. A., Li Z. P. 1990; A new enhancer element, ENII, identified in the X gene of hepatitis B virus. Journal of Virology 64:3977–3981
    [Google Scholar]
  42. Weimer T., Salfeld J., Will H. 1987; Expression of the hepatitis B virus core gene in vitro and in vivo . Journal of Virology 61:3109–3113
    [Google Scholar]
  43. Yaginuma K., Koike K. 1989; Identification of a promoter region for 3·6-kilobase mRNA of hepatitis B virus and specific cellular binding protein. Journal of Virology 63:2914–2920
    [Google Scholar]
  44. Yee J. K. 1989; A liver-specific enhancer in the core promoter region of human hepatitis B virus. Science 246:658–661
    [Google Scholar]
  45. Yuh C. H., Chang Y. L., Ting L. P. 1992; Transcriptional regulation of precore and pregenomic RNAs of hepatitis B virus. Journal of Virology 66:4073–4084
    [Google Scholar]
  46. Zoulim F., Saputell J., Seeger C. 1994; Woodchuck hepatitis virus X protein is required for viral infection in vivo . Journal of Virology 68:2026–2030
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-78-6-1479
Loading
/content/journal/jgv/10.1099/0022-1317-78-6-1479
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error