1887

Abstract

Hepatitis B virus (HBV) has a 3.2 kb circular DNA genome. It employs four promoters in conjunction with a single polyadenylation signal to generate 3.5, 2.4, 2.1 and 0.7 kb co-terminal RNAs. The 3.5 kb RNA is subdivided into the precore RNA for e-antigen expression and pregenomic RNA for genome replication. When introduced to a genotype A clone, several core promoter mutations markedly enhanced HBV genome replication, but suppressed e-antigen expression through up-regulation of pregenomic RNA at the expense of precore RNA. In this study, we found such mutations also diminished envelope proteins and hepatitis B surface antigen, products of the 2.1 and 2.4 kb subgenomic RNAs. Indeed, Northern blot analysis revealed overall increase in 3.5 kb RNA, but reduction in all subgenomic RNAs. To validate transcriptional interference, we subcloned 1.1×, 0.7× and 0.6× HBV genome, respectively, to a vector with or without a cytomegalovirus (CMV) promoter at the 5′ end, so as to produce the pregenomic RNA, 2.4 kb RNA, and 2.1 kb RNA in large excess or not at all. Parallel transfection of the three pairs of constructs into a human hepatoma cell line confirmed the ability of pregenomic RNA to suppress all subgenomic transcripts and established the ability of the 2.4 and 2.1 kb RNAs to suppress the 0.7 kb RNA. Consistent with our findings, pregenomic RNA of the related duck HBV has been reported to interfere with transcription of the subgenomic RNAs. Transcriptional interference might explain why HBV produces so little 0.7 kb RNA and HBx protein despite a strong X promoter.

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2016-10-13
2019-12-05
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References

  1. Antonucci T. K., Rutter W. J..( 1989;). Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner. . J Virol 63: 579–583.[PubMed]
    [Google Scholar]
  2. Baptista M., Kramvis A., Kew M. C..( 1999;). High prevalence of 1762(T) 1764(A) mutations in the basic core promoter of hepatitis B virus isolated from black Africans with hepatocellular carcinoma compared with asymptomatic carriers. . Hepatology 29: 946–953. [CrossRef] [PubMed]
    [Google Scholar]
  3. Baumert T. F., Rogers S. A., Hasegawa K., Liang T. J..( 1996;). Two core promotor mutations identified in a hepatitis B virus strain associated with fulminant hepatitis result in enhanced viral replication. . J Clin Invest 98: 2268–2276. [CrossRef] [PubMed]
    [Google Scholar]
  4. Beckel-Mitchener A., Summers J..( 1997;). A novel transcriptional element in circular DNA monomers of the duck hepatitis B virus. . J Virol 71: 7917–7922.[PubMed]
    [Google Scholar]
  5. Boussadia O., Amiot F., Cases S., Triqueneaux G., Jacquemin-Sablon H., Dautry F..( 1997;). Transcription of unr (upstream of N-ras) down-modulates N-ras expression in vivo. . FEBS Lett 420: 20–24.[PubMed] [CrossRef]
    [Google Scholar]
  6. Buckwold V. E., Xu Z., Chen M., Yen T. S., Ou J. H..( 1996;). Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. . J Virol 70: 5845–5851.[PubMed]
    [Google Scholar]
  7. Chen C. H., Changchien C. S., Lee C. M., Hung C. H., Hu T. H., Wang J. H., Wang J. C., Lu S. N..( 2008;). Combined mutations in pre-S/surface and core promoter/precore regions of hepatitis B virus increase the risk of hepatocellular carcinoma: a case-control study. . J Infect Dis 198: 1634–1642. [CrossRef] [PubMed]
    [Google Scholar]
  8. Diamantis I. D., McGandy C. E., Chen T. J., Liaw Y. F., Gudat F., Bianchi L..( 1992;). Hepatitis B X-gene expression in hepatocellular carcinoma. . J Hepatol 15: 400–403. [CrossRef] [PubMed]
    [Google Scholar]
  9. Fang Z. L., Sabin C. A., Dong B. Q., Wei S. C., Chen Q. Y., Fang K. X., Yang J. Y., Huang J., Wang X. Y., Harrison T. J..( 2008;). Hepatitis B virus pre-S deletion mutations are a risk factor for hepatocellular carcinoma: a matched nested case-control study. . J Gen Virol 89: 2882–2890. [CrossRef] [PubMed]
    [Google Scholar]
  10. Garcia T., Li J., Sureau C., Ito K., Qin Y., Wands J., Tong S..( 2009;). Drastic reduction in the production of subviral particles does not impair hepatitis B virus virion secretion. . J Virol 83: 11152–11165. [CrossRef] [PubMed]
    [Google Scholar]
  11. Greger I. H., Demarchi F., Giacca M., Proudfoot N. J..( 1998;). Transcriptional interference perturbs the binding of Sp1 to the HIV-1 promoter. . Nucleic Acids Res 26: 1294–1301. [CrossRef] [PubMed]
    [Google Scholar]
  12. Guarnieri M., Kim K. H., Bang G., Li J., Zhou Y., Tang X., Wands J., Tong S..( 2006;). Point mutations upstream of hepatitis B virus core gene affect DNA replication at the step of core protein expression. . J Virol 80: 587–595. [CrossRef] [PubMed]
    [Google Scholar]
  13. Hsia C. C., Yuwen H., Tabor E..( 1996;). Hot-spot mutations in hepatitis B virus X gene in hepatocellular carcinoma. . Lancet 348: 625–626. [CrossRef] [PubMed]
    [Google Scholar]
  14. Huang H. P., Hsu H. Y., Chen C. L., Ni Y. H., Wang H. Y., Tsuei D. J., Chiang C. L., Tsai Y. C., Chen H. L., Chang M. H..( 2010;). Pre-S2 deletions of hepatitis B virus and hepatocellular carcinoma in children. . Pediatr Res 67: 90–94. [CrossRef] [PubMed]
    [Google Scholar]
  15. Huang M., Summers J..( 1994;). pet, a small sequence distal to the pregenome cap site, is required for expression of the duck hepatitis B virus pregenome. . J Virol 68: 1564–1572.[PubMed]
    [Google Scholar]
  16. Huang Y., Tong S., Tai A. W., Hussain M., Lok A. S..( 2011;). Hepatitis B virus core promoter mutations contribute to hepatocarcinogenesis by deregulating SKP2 and its target, p21. . Gastroenterology 141: 1412–1421. [CrossRef] [PubMed]
    [Google Scholar]
  17. Huang Y., Tai A. W., Tong S., Lok A. S..( 2013;). HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription. . J Hepatol 58: 1068–1073. [CrossRef] [PubMed]
    [Google Scholar]
  18. Jammeh S., Tavner F., Watson R., Thomas H. C., Karayiannis P..( 2008;). Effect of basal core promoter and pre-core mutations on hepatitis B virus replication. . J Gen Virol 89: 901–909. [CrossRef] [PubMed]
    [Google Scholar]
  19. Kao J. H., Chen P. J., Lai M. Y., Chen D. S..( 2003;). Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers. . Gastroenterology 124: 327–334. [CrossRef] [PubMed]
    [Google Scholar]
  20. Kramvis A., Kew M. C..( 1999;). The core promoter of hepatitis B virus. . J Viral Hepat 6: 415–427. [CrossRef] [PubMed]
    [Google Scholar]
  21. Kramvis A..( 2014;). Genotypes and genetic variability of hepatitis B virus. . Intervirology 57: 141–150. [CrossRef] [PubMed]
    [Google Scholar]
  22. Kuang S. Y., Jackson P. E., Wang J. B., Lu P. X., Muñoz A., Qian G. S., Kensler T. W., Groopman J. D..( 2004;). Specific mutations of hepatitis B virus in plasma predict liver cancer development. . Proc Natl Acad Sci U S A 101: 3575–3580. [CrossRef] [PubMed]
    [Google Scholar]
  23. Moriyama K., Okamoto H., Tsuda F., Mayumi M..( 1996;). Reduced precore transcription and enhanced core-pregenome transcription of hepatitis B virus DNA after replacement of the precore-core promoter with sequences associated with e antigen-seronegative persistent infections. . Virology 226: 269–280. [CrossRef] [PubMed]
    [Google Scholar]
  24. Nassal M..( 2015;). HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. . Gut 64: 1972–1984. [CrossRef] [PubMed]
    [Google Scholar]
  25. 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. . J Virol 68: 8102–8110.[PubMed]
    [Google Scholar]
  26. Ou J. H., Rutter W. J..( 1987;). Regulation of secretion of the hepatitis B virus major surface antigen by the preS-1 protein. . J Virol 61: 782–786.[PubMed]
    [Google Scholar]
  27. Parekh S., Zoulim F., Ahn S. H., Tsai A., Li J., Kawai S., Khan N., Trépo C., Wands J., Tong S..( 2003;). Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promoter mutants. . J Virol 77: 6601–6612. [CrossRef] [PubMed]
    [Google Scholar]
  28. Paterlini P., Poussin K., Kew M., Franco D., Brechot C..( 1995;). Selective accumulation of the X transcript of hepatitis B virus in patients negative for hepatitis B surface antigen with hepatocellular carcinoma. . Hepatology 21: 313–321.[PubMed]
    [Google Scholar]
  29. Persing D. H., Varmus H. E., Ganem D..( 1986;). Inhibition of secretion of hepatitis B surface antigen by a related presurface polypeptide. . Science 234: 1388–1391. [CrossRef] [PubMed]
    [Google Scholar]
  30. Proudfoot N. J..( 1986;). Transcriptional interference and termination between duplicated alpha-globin gene constructs suggests a novel mechanism for gene regulation. . Nature 322: 562–565. [CrossRef] [PubMed]
    [Google Scholar]
  31. Seeger C., Mason W. S..( 2015;). Molecular biology of hepatitis B virus infection. . Virology 479-480: 672–686. [CrossRef] [PubMed]
    [Google Scholar]
  32. Shearwin K. E., Callen B. P., Egan J. B..( 2005;). Transcriptional interference – a crash course. . Trends Genet 21: 339–345. [CrossRef] [PubMed]
    [Google Scholar]
  33. Slagle B. L., Bouchard M. J..( 2016;). Hepatitis B virus X and regulation of viral gene expression. . Cold Spring Harb Perspect Med 6: a021402. [CrossRef] [PubMed]
    [Google Scholar]
  34. Standring D. N., Ou J. H., Rutter W. J..( 1986;). Assembly of viral particles in Xenopus oocytes: pre-surface-antigens regulate secretion of the hepatitis B viral surface envelope particle. . Proc Natl Acad Sci U S A 83: 9338–9342. [CrossRef] [PubMed]
    [Google Scholar]
  35. Tong S., Li J., Wands J. R., Wen Y. M..( 2013;). Hepatitis B virus genetic variants: biological properties and clinical implications. . Emerg Microbes Infect 2:,e10. [CrossRef] [PubMed]
    [Google Scholar]
  36. Trépo C., Chan H. L., Lok A..( 2014;). Hepatitis B virus infection. . Lancet 384: 2053–2063. [CrossRef] [PubMed]
    [Google Scholar]
  37. Tsai A., Kawai S., Kwei K., Gewaily D., Hutter A., Tong D. R., Li J., Wands J. R., Tong S..( 2009;). Chimeric constructs between two hepatitis B virus genomes confirm transcriptional impact of core promoter mutations and reveal multiple effects of core gene mutations. . Virology 387: 364–372. [CrossRef] [PubMed]
    [Google Scholar]
  38. Wang H. C., Huang W., Lai M. D., Su I. J..( 2006;). Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis. . Cancer Sci 97: 683–688. [CrossRef] [PubMed]
    [Google Scholar]
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