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. 1993 Mar;67(3):1472–1481. doi: 10.1128/jvi.67.3.1472-1481.1993

Characterization of functional Sp1 transcription factor binding sites in the hepatitis B virus nucleocapsid promoter.

P Zhang 1, A K Raney 1, A McLachlan 1
PMCID: PMC237517  PMID: 8437225

Abstract

The hepatitis B virus nucleocapsid minimal promoter contains sequence elements which are similar to the Sp1 transcription factor binding site consensus sequence. The interaction of these regulatory elements with Sp1 was examined by DNase I footprinting with purified Sp1 protein and DNase I footprinting and gel retardation analysis with nuclear extracts from human cell lines and was examined functionally with transient transfection assays in human hepatoma and Drosophila melanogaster Schneider line-2 cells. DNase I footprinting identified two regions of the nucleocapsid promoter, representing three recognition elements, that bound purified Sp1. Gel retardation analysis with Huh7 nuclear extracts demonstrated that each of the three recognition elements bound the same or similar transcription factor(s) as that recognized by the Sp1 consensus sequence recognition element. The function of the nucleocapsid promoter elements was examined by transient transfection assays in D. melanogaster Schneider line-2 cells by using these binding sites cloned into a minimal promoter element. Each of these regulatory regions transactivated transcription from the minimal promoter element in response to exogenously expressed Sp1. In addition, the second Sp1 site was shown to be an essential element of the nucleocapsid promoter in human hepatoma cells. This demonstrates that the hepatitis B virus nucleocapsid promoter contains three functional Sp1 binding sites which may contribute to the level of transcription from this promoter during viral infection.

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Selected References

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

  1. Antonucci T. K., Rutter W. J. Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner. J Virol. 1989 Feb;63(2):579–583. doi: 10.1128/jvi.63.2.579-583.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Araki K., Miyazaki J., Hino O., Tomita N., Chisaka O., Matsubara K., Yamamura K. Expression and replication of hepatitis B virus genome in transgenic mice. Proc Natl Acad Sci U S A. 1989 Jan;86(1):207–211. doi: 10.1073/pnas.86.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartenschlager R., Junker-Niepmann M., Schaller H. The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation. J Virol. 1990 Nov;64(11):5324–5332. doi: 10.1128/jvi.64.11.5324-5332.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baumhueter S., Mendel D. B., Conley P. B., Kuo C. J., Turk C., Graves M. K., Edwards C. A., Courtois G., Crabtree G. R. HNF-1 shares three sequence motifs with the POU domain proteins and is identical to LF-B1 and APF. Genes Dev. 1990 Mar;4(3):372–379. doi: 10.1101/gad.4.3.372. [DOI] [PubMed] [Google Scholar]
  5. Birkenmeier E. H., Gwynn B., Howard S., Jerry J., Gordon J. I., Landschulz W. H., McKnight S. L. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein. Genes Dev. 1989 Aug;3(8):1146–1156. doi: 10.1101/gad.3.8.1146. [DOI] [PubMed] [Google Scholar]
  6. Blumenfeld M., Maury M., Chouard T., Yaniv M., Condamine H. Hepatic nuclear factor 1 (HNF1) shows a wider distribution than products of its known target genes in developing mouse. Development. 1991 Oct;113(2):589–599. doi: 10.1242/dev.113.2.589. [DOI] [PubMed] [Google Scholar]
  7. Bond-Matthews B., Davidson N. Transcription from each of the Drosophila act5C leader exons is driven by a separate functional promoter. Gene. 1988;62(2):289–300. doi: 10.1016/0378-1119(88)90566-5. [DOI] [PubMed] [Google Scholar]
  8. Briggs M. R., Kadonaga J. T., Bell S. P., Tjian R. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science. 1986 Oct 3;234(4772):47–52. doi: 10.1126/science.3529394. [DOI] [PubMed] [Google Scholar]
  9. Cattaneo R., Will H., Hernandez N., Schaller H. Signals regulating hepatitis B surface antigen transcription. Nature. 1983 Sep 22;305(5932):336–338. doi: 10.1038/305336a0. [DOI] [PubMed] [Google Scholar]
  10. Cattaneo R., Will H., Schaller H. Hepatitis B virus transcription in the infected liver. EMBO J. 1984 Sep;3(9):2191–2196. doi: 10.1002/j.1460-2075.1984.tb02113.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Courey A. J., Holtzman D. A., Jackson S. P., Tjian R. Synergistic activation by the glutamine-rich domains of human transcription factor Sp1. Cell. 1989 Dec 1;59(5):827–836. doi: 10.1016/0092-8674(89)90606-5. [DOI] [PubMed] [Google Scholar]
  12. Courey A. J., Tjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell. 1988 Dec 2;55(5):887–898. doi: 10.1016/0092-8674(88)90144-4. [DOI] [PubMed] [Google Scholar]
  13. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dikstein R., Faktor O., Shaul Y. Hierarchic and cooperative binding of the rat liver nuclear protein C/EBP at the hepatitis B virus enhancer. Mol Cell Biol. 1990 Aug;10(8):4427–4430. doi: 10.1128/mcb.10.8.4427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dubois M. F., Pourcel C., Rousset S., Chany C., Tiollais P. Excretion of hepatitis B surface antigen particles from mouse cells transformed with cloned viral DNA. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4549–4553. doi: 10.1073/pnas.77.8.4549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Farza H., Hadchouel M., Scotto J., Tiollais P., Babinet C., Pourcel C. Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome. J Virol. 1988 Nov;62(11):4144–4152. doi: 10.1128/jvi.62.11.4144-4152.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Frain M., Swart G., Monaci P., Nicosia A., Stämpfli S., Frank R., Cortese R. The liver-specific transcription factor LF-B1 contains a highly diverged homeobox DNA binding domain. Cell. 1989 Oct 6;59(1):145–157. doi: 10.1016/0092-8674(89)90877-5. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Gidoni D., Kadonaga J. T., Barrera-Saldaña H., Takahashi K., Chambon P., Tjian R. Bidirectional SV40 transcription mediated by tandem Sp1 binding interactions. Science. 1985 Nov 1;230(4725):511–517. doi: 10.1126/science.2996137. [DOI] [PubMed] [Google Scholar]
  20. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hirsch R. C., Lavine J. E., Chang L. J., Varmus H. E., Ganem D. Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription. Nature. 1990 Apr 5;344(6266):552–555. doi: 10.1038/344552a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Hu K. Q., Siddiqui A. Regulation of the hepatitis B virus gene expression by the enhancer element I. Virology. 1991 Apr;181(2):721–726. doi: 10.1016/0042-6822(91)90906-r. [DOI] [PubMed] [Google Scholar]
  24. Imazeki F., Yaginuma K., Omata M., Okuda K., Kobayashi M., Koike K. RNA transcripts of hepatitis B virus in hepatocellular carcinoma. Hepatology. 1987 Jul-Aug;7(4):753–757. doi: 10.1002/hep.1840070423. [DOI] [PubMed] [Google Scholar]
  25. Jameel S., Siddiqui A. The human hepatitis B virus enhancer requires trans-acting cellular factor(s) for activity. Mol Cell Biol. 1986 Feb;6(2):710–715. doi: 10.1128/mcb.6.2.710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kaneko S., Miller R. H. X-region-specific transcript in mammalian hepatitis B virus-infected liver. J Virol. 1988 Nov;62(11):3979–3984. doi: 10.1128/jvi.62.11.3979-3984.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Karpen S., Banerjee R., Zelent A., Price P., Acs G. Identification of protein-binding sites in the hepatitis B virus enhancer and core promoter domains. Mol Cell Biol. 1988 Dec;8(12):5159–5165. doi: 10.1128/mcb.8.12.5159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. López-Cabrera M., Letovsky J., Hu K. Q., Siddiqui A. Transcriptional factor C/EBP binds to and transactivates the enhancer element II of the hepatitis B virus. Virology. 1991 Aug;183(2):825–829. doi: 10.1016/0042-6822(91)91019-d. [DOI] [PubMed] [Google Scholar]
  30. McLachlan A., Milich D. R., Raney A. K., Riggs M. G., Hughes J. L., Sorge J., Chisari F. V. Expression of hepatitis B virus surface and core antigens: influences of pre-S and precore sequences. J Virol. 1987 Mar;61(3):683–692. doi: 10.1128/jvi.61.3.683-692.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Pei D. Q., Shih C. H. Transcriptional activation and repression by cellular DNA-binding protein C/EBP. J Virol. 1990 Apr;64(4):1517–1522. doi: 10.1128/jvi.64.4.1517-1522.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Raney A. K., Easton A. J., Milich D. R., McLachlan A. Promoter-specific transactivation of hepatitis B virus transcription by a glutamine- and proline-rich domain of hepatocyte nuclear factor 1. J Virol. 1991 Nov;65(11):5774–5781. doi: 10.1128/jvi.65.11.5774-5781.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Raney A. K., Le H. B., McLachlan A. Regulation of transcription from the hepatitis B virus major surface antigen promoter by the Sp1 transcription factor. J Virol. 1992 Dec;66(12):6912–6921. doi: 10.1128/jvi.66.12.6912-6921.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Raney A. K., Milich D. R., Easton A. J., McLachlan A. Differentiation-specific transcriptional regulation of the hepatitis B virus large surface antigen gene in human hepatoma cell lines. J Virol. 1990 May;64(5):2360–2368. doi: 10.1128/jvi.64.5.2360-2368.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Raney A. K., Milich D. R., McLachlan A. Characterization of hepatitis B virus major surface antigen gene transcriptional regulatory elements in differentiated hepatoma cell lines. J Virol. 1989 Sep;63(9):3919–3925. doi: 10.1128/jvi.63.9.3919-3925.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Raney A. K., Milich D. R., McLachlan A. Complex regulation of transcription from the hepatitis B virus major surface antigen promoter in human hepatoma cell lines. J Virol. 1991 Sep;65(9):4805–4811. doi: 10.1128/jvi.65.9.4805-4811.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Su H., Yee J. K. Regulation of hepatitis B virus gene expression by its two enhancers. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2708–2712. doi: 10.1073/pnas.89.7.2708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Su T. S., Lui W. Y., Lin L. H., Han S. H., P'eng F. K. Analysis of hepatitis B virus transcripts in infected human livers. Hepatology. 1989 Feb;9(2):180–185. doi: 10.1002/hep.1840090203. [DOI] [PubMed] [Google Scholar]
  43. Su W., Jackson S., Tjian R., Echols H. DNA looping between sites for transcriptional activation: self-association of DNA-bound Sp1. Genes Dev. 1991 May;5(5):820–826. doi: 10.1101/gad.5.5.820. [DOI] [PubMed] [Google Scholar]
  44. Trujillo M. A., Letovsky J., Maguire H. F., Lopez-Cabrera M., Siddiqui A. Functional analysis of a liver-specific enhancer of the hepatitis B virus. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3797–3801. doi: 10.1073/pnas.88.9.3797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Waisman A., Aloni Y., Laub O. In vitro regulation of human hepatitis B virus core gene transcription. Virology. 1990 Aug;177(2):737–744. doi: 10.1016/0042-6822(90)90540-8. [DOI] [PubMed] [Google Scholar]
  46. Wang Y., Chen P., Wu X., Sun A. L., Wang H., Zhu Y. A., Li Z. P. A new enhancer element, ENII, identified in the X gene of hepatitis B virus. J Virol. 1990 Aug;64(8):3977–3981. doi: 10.1128/jvi.64.8.3977-3981.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Will H., Reiser W., Weimer T., Pfaff E., Büscher M., Sprengel R., Cattaneo R., Schaller H. Replication strategy of human hepatitis B virus. J Virol. 1987 Mar;61(3):904–911. doi: 10.1128/jvi.61.3.904-911.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Yaginuma K., Shirakata Y., Kobayashi M., Koike K. Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. Proc Natl Acad Sci U S A. 1987 May;84(9):2678–2682. doi: 10.1073/pnas.84.9.2678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Yokosuka O., Omata M., Imazeki F., Ito Y., Okuda K. Hepatitis B virus RNA transcripts and DNA in chronic liver disease. N Engl J Med. 1986 Nov 6;315(19):1187–1192. doi: 10.1056/NEJM198611063151903. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Zhang P., Raney A. K., McLachlan A. Characterization of the hepatitis B virus X- and nucleocapsid gene transcriptional regulatory elements. Virology. 1992 Nov;191(1):31–41. doi: 10.1016/0042-6822(92)90163-j. [DOI] [PubMed] [Google Scholar]
  54. Zhou D. X., Yen T. S. Differential regulation of the hepatitis B virus surface gene promoters by a second viral enhancer. J Biol Chem. 1990 Dec 5;265(34):20731–20734. [PubMed] [Google Scholar]
  55. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]

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