Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1996 Jun;70(6):3894–3901. doi: 10.1128/jvi.70.6.3894-3901.1996

Characterization of the ZI domains in the Epstein-Barr virus BZLF1 gene promoter: role in phorbol ester induction.

A M Borras 1, J L Strominger 1, S H Speck 1
PMCID: PMC190267  PMID: 8648726

Abstract

Induction of the Epstein-Barr virus lytic cycle is mediated through the immediate-early BZLF1 gene and the coordinately regulated BRLF1 gene. The BZLF1 gene product, Zta, transactivates its own promoter, as well as the promoters of a number of lytic genes, thereby initiating a cascade of viral gene expression. Previous work identified four related elements (ZIA, ZIB, ZIC, and ZID) and a cyclic AMP response element binding-AP-1 element (ZII) that are involved in the induction of the BZLF1 promoter (Zp) by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (E. Flemington and S. H. Speck, J. Virol. 64:1217-1226, 1990). Here we report a detailed characterization of TPA induction mediated by the ZI domains. Mutation of individual ZI domains within the context of the intact promoter significantly diminished TPA induction. Cloning of individual ZI domains upstream of a minimal promoter demonstrated that the ZIA, ZIC, and ZID domains, but not the ZIB domain, are TPA responsive. Furthermore, cloning of the ZII domain downstream of the ZI domains significantly augmented TPA induction. The critical regions within the ZIA and ZIC elements involved in binding of cellular factors were identified by using methylation interference and electrophoretic mobility shift analyses of ZI domain mutants. Four specific complexes were observed with the ZIA and ZID domains, all of which could be specifically competed for by either the ZIA or ZID domain. Methylation interference analyses of bound complexes revealed the presence of two overlapping binding sites for cellular factors in the ZIA domain, and functional studies provided evidence that both of these sites are involved in TPA induction. Functional analyses of the ZIC domain revealed that the 5' region of this domain is largely responsible for mediating TPA induction. Binding data correlated well with functional activity and revealed that the ZIC domain binds only a subset of the cellular factors that bind to the ZIA and ZID domains. Analysis of factor binding to the ZIB domain revealed only a single shifted complex, which correlated with the most slowly migrating complex observed with the ZIA and ZID domains. These data provide a direct demonstration of TPA induction mediated by the ZIA, ZIC, and ZID domains and also provide the first evidence that the ZI domains exhibit distinct functional characteristics.

Full Text

The Full Text of this article is available as a PDF (514.9 KB).

Selected References

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

  1. Bauer G., Höfler P., Zur Hausen H. Epstein-Barr virus induction by a serum factor. I. Induction and cooperation with additional inducers. Virology. 1982 Aug;121(1):184–194. doi: 10.1016/0042-6822(82)90128-3. [DOI] [PubMed] [Google Scholar]
  2. Cayrol C., Flemington E. K. Identification of cellular target genes of the Epstein-Barr virus transactivator Zta: activation of transforming growth factor beta igh3 (TGF-beta igh3) and TGF-beta 1. J Virol. 1995 Jul;69(7):4206–4212. doi: 10.1128/jvi.69.7.4206-4212.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang Y. N., Dong D. L., Hayward G. S., Hayward S. D. The Epstein-Barr virus Zta transactivator: a member of the bZIP family with unique DNA-binding specificity and a dimerization domain that lacks the characteristic heptad leucine zipper motif. J Virol. 1990 Jul;64(7):3358–3369. doi: 10.1128/jvi.64.7.3358-3369.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chevallier-Greco A., Manet E., Chavrier P., Mosnier C., Daillie J., Sergeant A. Both Epstein-Barr virus (EBV)-encoded trans-acting factors, EB1 and EB2, are required to activate transcription from an EBV early promoter. EMBO J. 1986 Dec 1;5(12):3243–3249. doi: 10.1002/j.1460-2075.1986.tb04635.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Countryman J., Jenson H., Seibl R., Wolf H., Miller G. Polymorphic proteins encoded within BZLF1 of defective and standard Epstein-Barr viruses disrupt latency. J Virol. 1987 Dec;61(12):3672–3679. doi: 10.1128/jvi.61.12.3672-3679.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Countryman J., Miller G. Activation of expression of latent Epstein-Barr herpesvirus after gene transfer with a small cloned subfragment of heterogeneous viral DNA. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4085–4089. doi: 10.1073/pnas.82.12.4085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Daibata M., Speck S. H., Mulder C., Sairenji T. Regulation of the BZLF1 promoter of Epstein-Barr virus by second messengers in anti-immunoglobulin-treated B cells. Virology. 1994 Feb;198(2):446–454. doi: 10.1006/viro.1994.1056. [DOI] [PubMed] [Google Scholar]
  8. Farrell P. J., Rowe D. T., Rooney C. M., Kouzarides T. Epstein-Barr virus BZLF1 trans-activator specifically binds to a consensus AP-1 site and is related to c-fos. EMBO J. 1989 Jan;8(1):127–132. doi: 10.1002/j.1460-2075.1989.tb03356.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Flemington E. K., Borras A. M., Lytle J. P., Speck S. H. Characterization of the Epstein-Barr virus BZLF1 protein transactivation domain. J Virol. 1992 Feb;66(2):922–929. doi: 10.1128/jvi.66.2.922-929.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Flemington E. K., Goldfeld A. E., Speck S. H. Efficient transcription of the Epstein-Barr virus immediate-early BZLF1 and BRLF1 genes requires protein synthesis. J Virol. 1991 Dec;65(12):7073–7077. doi: 10.1128/jvi.65.12.7073-7077.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Flemington E. K., Lytle J. P., Cayrol C., Borras A. M., Speck S. H. DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities. Mol Cell Biol. 1994 May;14(5):3041–3052. doi: 10.1128/mcb.14.5.3041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Flemington E., Speck S. H. Autoregulation of Epstein-Barr virus putative lytic switch gene BZLF1. J Virol. 1990 Mar;64(3):1227–1232. doi: 10.1128/jvi.64.3.1227-1232.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Flemington E., Speck S. H. Epstein-Barr virus BZLF1 trans activator induces the promoter of a cellular cognate gene, c-fos. J Virol. 1990 Sep;64(9):4549–4552. doi: 10.1128/jvi.64.9.4549-4552.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Flemington E., Speck S. H. Evidence for coiled-coil dimer formation by an Epstein-Barr virus transactivator that lacks a heptad repeat of leucine residues. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9459–9463. doi: 10.1073/pnas.87.23.9459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Flemington E., Speck S. H. Identification of phorbol ester response elements in the promoter of Epstein-Barr virus putative lytic switch gene BZLF1. J Virol. 1990 Mar;64(3):1217–1226. doi: 10.1128/jvi.64.3.1217-1226.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Foss K., McClain W. H. Rapid site-specific mutagenesis in plasmids. Gene. 1987;59(2-3):285–290. doi: 10.1016/0378-1119(87)90336-2. [DOI] [PubMed] [Google Scholar]
  17. Goldfeld A. E., Liu P., Liu S., Flemington E. K., Strominger J. L., Speck S. H. Cyclosporin A and FK506 block induction of the Epstein-Barr virus lytic cycle by anti-immunoglobulin. Virology. 1995 May 10;209(1):225–229. doi: 10.1006/viro.1995.1247. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Grogan E., Jenson H., Countryman J., Heston L., Gradoville L., Miller G. Transfection of a rearranged viral DNA fragment, WZhet, stably converts latent Epstein-Barr viral infection to productive infection in lymphoid cells. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1332–1336. doi: 10.1073/pnas.84.5.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hardwick J. M., Lieberman P. M., Hayward S. D. A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen. J Virol. 1988 Jul;62(7):2274–2284. doi: 10.1128/jvi.62.7.2274-2284.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kallin B., Luka J., Klein G. Immunochemical characterization of Epstein-Barr virus-associated early and late antigens in n-butyrate-treated P3HR-1 cells. J Virol. 1979 Dec;32(3):710–716. doi: 10.1128/jvi.32.3.710-716.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kouzarides T., Packham G., Cook A., Farrell P. J. The BZLF1 protein of EBV has a coiled coil dimerisation domain without a heptad leucine repeat but with homology to the C/EBP leucine zipper. Oncogene. 1991 Feb;6(2):195–204. [PubMed] [Google Scholar]
  23. Lieberman P. M., Berk A. J. In vitro transcriptional activation, dimerization, and DNA-binding specificity of the Epstein-Barr virus Zta protein. J Virol. 1990 Jun;64(6):2560–2568. doi: 10.1128/jvi.64.6.2560-2568.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Luka J., Kallin B., Klein G. Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by n-butyrate. Virology. 1979 Apr 15;94(1):228–231. doi: 10.1016/0042-6822(79)90455-0. [DOI] [PubMed] [Google Scholar]
  25. Miller G., Rabson M., Heston L. Epstein-Barr virus with heterogeneous DNA disrupts latency. J Virol. 1984 Apr;50(1):174–182. doi: 10.1128/jvi.50.1.174-182.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Packham G., Economou A., Rooney C. M., Rowe D. T., Farrell P. J. Structure and function of the Epstein-Barr virus BZLF1 protein. J Virol. 1990 May;64(5):2110–2116. doi: 10.1128/jvi.64.5.2110-2116.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shimizu N., Takada K. Analysis of the BZLF1 promoter of Epstein-Barr virus: identification of an anti-immunoglobulin response sequence. J Virol. 1993 Jun;67(6):3240–3245. doi: 10.1128/jvi.67.6.3240-3245.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Singh H., Sen R., Baltimore D., Sharp P. A. A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature. 1986 Jan 9;319(6049):154–158. doi: 10.1038/319154a0. [DOI] [PubMed] [Google Scholar]
  29. Takada K., Ono Y. Synchronous and sequential activation of latently infected Epstein-Barr virus genomes. J Virol. 1989 Jan;63(1):445–449. doi: 10.1128/jvi.63.1.445-449.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Taylor N., Flemington E., Kolman J. L., Baumann R. P., Speck S. H., Miller G. ZEBRA and a Fos-GCN4 chimeric protein differ in their DNA-binding specificities for sites in the Epstein-Barr virus BZLF1 promoter. J Virol. 1991 Aug;65(8):4033–4041. doi: 10.1128/jvi.65.8.4033-4041.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tovey M. G., Lenoir G., Begon-Lours J. Activation of latent Epstein-Barr virus by antibody to human IgM. Nature. 1978 Nov 16;276(5685):270–272. doi: 10.1038/276270a0. [DOI] [PubMed] [Google Scholar]
  32. Urier G., Buisson M., Chambard P., Sergeant A. The Epstein-Barr virus early protein EB1 activates transcription from different responsive elements including AP-1 binding sites. EMBO J. 1989 May;8(5):1447–1453. doi: 10.1002/j.1460-2075.1989.tb03527.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zimber-Strobl U., Kremmer E., Grässer F., Marschall G., Laux G., Bornkamm G. W. The Epstein-Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter. EMBO J. 1993 Jan;12(1):167–175. doi: 10.1002/j.1460-2075.1993.tb05642.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. zur Hausen H., O'Neill F. J., Freese U. K., Hecker E. Persisting oncogenic herpesvirus induced by the tumour promotor TPA. Nature. 1978 Mar 23;272(5651):373–375. doi: 10.1038/272373a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES