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. 1996 Sep;70(9):5845–5851. doi: 10.1128/jvi.70.9.5845-5851.1996

Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication.

V E Buckwold 1, Z Xu 1, M Chen 1, T S Yen 1, J H Ou 1
PMCID: PMC190601  PMID: 8709203

Abstract

The basal core promoter (BCP) of hepatitis B virus (HBV) controls the transcription of both the precore RNA and the core RNA. The precore RNA codes for the secreted e antigen, while the core RNA codes for the major core protein and the DNA polymerase and also is the pregenomic RNA. The double mutation of nucleotides 1762 and 1764 in the BCP from A and G to T and A, respectively, is frequently observed in HBV sequences isolated from chronic patients. Several papers have reported conflicting results regarding whether this double mutation is important for e antigen expression. In order to address this issue, we have introduced this double mutation into the HBV genome and studied its effects on HBV gene expression and replication. Our results indicate that the mutated BCP can no longer bind a liver-enriched transcription factor(s) and that the transcription of only precore RNA and, consequently, the expression of e antigen were reduced. The reduction of precore gene expression was accompanied by an increase in progeny virus production. This increase was found to occur at or immediately prior to the encapsidation of the pregenomic RNA. Thus, the results of our in vitro study resolve the discrepancy of previous clinical observations and indicate that this double mutation suppresses but does not abolish the e antigen phenotype. The implications of these findings in the pathogenesis of HBV are discussed.

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Selected References

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  1. Andrews N. C., Faller D. V. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991 May 11;19(9):2499–2499. doi: 10.1093/nar/19.9.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bruss V., Ganem D. The role of envelope proteins in hepatitis B virus assembly. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):1059–1063. doi: 10.1073/pnas.88.3.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Carman W., Thomas H., Domingo E. Viral genetic variation: hepatitis B virus as a clinical example. Lancet. 1993 Feb 6;341(8841):349–353. doi: 10.1016/0140-6736(93)90146-8. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Chen I. H., Huang C. J., Ting L. P. Overlapping initiator and TATA box functions in the basal core promoter of hepatitis B virus. J Virol. 1995 Jun;69(6):3647–3657. doi: 10.1128/jvi.69.6.3647-3657.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chisari F. V., Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol. 1995;13:29–60. doi: 10.1146/annurev.iy.13.040195.000333. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Guo W. T., Bell K. D., Ou J. H. Characterization of the hepatitis B virus EnhI enhancer and X promoter complex. J Virol. 1991 Dec;65(12):6686–6692. doi: 10.1128/jvi.65.12.6686-6692.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guo W., Chen M., Yen T. S., Ou J. H. Hepatocyte-specific expression of the hepatitis B virus core promoter depends on both positive and negative regulation. Mol Cell Biol. 1993 Jan;13(1):443–448. doi: 10.1128/mcb.13.1.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hawkins A. E., Gilson R. J., Bickerton E. A., Tedder R. S., Weller I. V. Conservation of precore and core sequences of hepatitis B virus in chronic viral carriers. J Med Virol. 1994 May;43(1):5–12. doi: 10.1002/jmv.1890430103. [DOI] [PubMed] [Google Scholar]
  13. Honigwachs J., Faktor O., Dikstein R., Shaul Y., Laub O. Liver-specific expression of hepatitis B virus is determined by the combined action of the core gene promoter and the enhancer. J Virol. 1989 Feb;63(2):919–924. doi: 10.1128/jvi.63.2.919-924.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lamberts C., Nassal M., Velhagen I., Zentgraf H., Schröder C. H. Precore-mediated inhibition of hepatitis B virus progeny DNA synthesis. J Virol. 1993 Jul;67(7):3756–3762. doi: 10.1128/jvi.67.7.3756-3762.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laskus T., Rakela J., Nowicki M. J., Persing D. H. Hepatitis B virus core promoter sequence analysis in fulminant and chronic hepatitis B. Gastroenterology. 1995 Nov;109(5):1618–1623. doi: 10.1016/0016-5085(95)90651-7. [DOI] [PubMed] [Google Scholar]
  16. Lenhoff R. J., Summers J. Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus. J Virol. 1994 Jul;68(7):4565–4571. doi: 10.1128/jvi.68.7.4565-4571.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. López-Cabrera M., Letovsky J., Hu K. Q., Siddiqui A. Multiple liver-specific factors bind to the hepatitis B virus core/pregenomic promoter: trans-activation and repression by CCAAT/enhancer binding protein. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5069–5073. doi: 10.1073/pnas.87.13.5069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  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. Okada K., Kamiyama I., Inomata M., Imai M., Miyakawa Y. e antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants. N Engl J Med. 1976 Apr 1;294(14):746–749. doi: 10.1056/NEJM197604012941402. [DOI] [PubMed] [Google Scholar]
  21. Okamoto H., Tsuda F., Akahane Y., Sugai Y., Yoshiba M., Moriyama K., Tanaka T., Miyakawa Y., Mayumi M. Hepatitis B virus with mutations in the core promoter for an e antigen-negative phenotype in carriers with antibody to e antigen. J Virol. 1994 Dec;68(12):8102–8110. doi: 10.1128/jvi.68.12.8102-8110.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Ou J. H., Yen T. S., Wang Y. F., Kam W. K., Rutter W. J. Cloning and characterization of a human ribosomal protein gene with enhanced expression in fetal and neoplastic cells. Nucleic Acids Res. 1987 Nov 11;15(21):8919–8934. doi: 10.1093/nar/15.21.8919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Raimondo G., Meucci G., Sardo M. A., Rodinò G., Campo S., Vecchi M., Pernice M., Rumi M. G., Tatarella M., de Franchis R. Persistence of "wild-type" and "e-minus" hepatitis B virus infection in chronic healthy HBsAg/anti-HBe positive carriers. J Hepatol. 1994 Jan;20(1):148–151. doi: 10.1016/s0168-8278(05)80482-9. [DOI] [PubMed] [Google Scholar]
  25. Sato S., Suzuki K., Akahane Y., Akamatsu K., Akiyama K., Yunomura K., Tsuda F., Tanaka T., Okamoto H., Miyakawa Y. Hepatitis B virus strains with mutations in the core promoter in patients with fulminant hepatitis. Ann Intern Med. 1995 Feb 15;122(4):241–248. doi: 10.7326/0003-4819-122-4-199502150-00001. [DOI] [PubMed] [Google Scholar]
  26. Sen R., Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell. 1986 Aug 29;46(5):705–716. doi: 10.1016/0092-8674(86)90346-6. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Takahashi K., Aoyama K., Ohno N., Iwata K., Akahane Y., Baba K., Yoshizawa H., Mishiro S. The precore/core promoter mutant (T1762A1764) of hepatitis B virus: clinical significance and an easy method for detection. J Gen Virol. 1995 Dec;76(Pt 12):3159–3164. doi: 10.1099/0022-1317-76-12-3159. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Wang J., Lee A. S., Ou J. H. Proteolytic conversion of hepatitis B virus e antigen precursor to end product occurs in a postendoplasmic reticulum compartment. J Virol. 1991 Sep;65(9):5080–5083. doi: 10.1128/jvi.65.9.5080-5083.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yaginuma K., Koike K. Identification of a promoter region for 3.6-kilobase mRNA of hepatitis B virus and specific cellular binding protein. J Virol. 1989 Jul;63(7):2914–2920. doi: 10.1128/jvi.63.7.2914-2920.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yeh C. T., Wong S. W., Fung Y. K., Ou J. H. Cell cycle regulation of nuclear localization of hepatitis B virus core protein. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6459–6463. doi: 10.1073/pnas.90.14.6459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yuan T. T., Faruqi A., Shih J. W., Shih C. The mechanism of natural occurrence of two closely linked HBV precore predominant mutations. Virology. 1995 Aug 1;211(1):144–156. doi: 10.1006/viro.1995.1387. [DOI] [PubMed] [Google Scholar]
  35. Yuh C. H., Chang Y. L., Ting L. P. Transcriptional regulation of precore and pregenomic RNAs of hepatitis B virus. J Virol. 1992 Jul;66(7):4073–4084. doi: 10.1128/jvi.66.7.4073-4084.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhang P., McLachlan A. Differentiation-specific transcriptional regulation of the hepatitis B virus nucleocapsid gene in human hepatoma cell lines. Virology. 1994 Jul;202(1):430–440. doi: 10.1006/viro.1994.1359. [DOI] [PubMed] [Google Scholar]
  37. Zheng Y. W., Yen T. S. Negative regulation of hepatitis B virus gene expression and replication by oxidative stress. J Biol Chem. 1994 Mar 25;269(12):8857–8862. [PubMed] [Google Scholar]

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