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. 1994 Apr 11;22(7):1172–1178. doi: 10.1093/nar/22.7.1172

Characterization of the DNA-binding site repertoire for the Epstein-Barr virus transcription factor R.

H Gruffat 1, A Sergeant 1
PMCID: PMC523639  PMID: 8165130

Abstract

The Epstein-Barr virus gene BRLF1 encodes the transcription factor R, which is a sequence-specific DNA-binding protein important for the switch from latency to a productive cycle. We have defined a repertoire of specific R-binding sites using a GST-R fusion protein and a pool of 23 bp random DNA sequences. The R-bound sequences were selected by several rounds of Electrophoretic Mobility Shift Assay (EMSA) and amplification by PCR. Among the 45 sites selected, some positions in the sequences were highly conserved, i.e., 5'-GTGCC N7GTGGTG-3'. The guanine methylation assay revealed that R simultaneously contacts guanines in the two conserved cores, defining the consensus binding site 5'-GNCC N9 GGNG-3', and 30 sites among the 45 selected have this sequence. This last result also suggests that R binds two adjacent major grooves of the DNA. As shown by EMSA assay, R binds to all the sites tested with a comparable affinity, and they all mediate R-induced transcriptional activation in a transient expression assay.

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Selected References

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

  1. Blackwell T. K., Kretzner L., Blackwood E. M., Eisenman R. N., Weintraub H. Sequence-specific DNA binding by the c-Myc protein. Science. 1990 Nov 23;250(4984):1149–1151. doi: 10.1126/science.2251503. [DOI] [PubMed] [Google Scholar]
  2. Buisson M., Manet E., Trescol-Biemont M. C., Gruffat H., Durand B., Sergeant A. The Epstein-Barr virus (EBV) early protein EB2 is a posttranscriptional activator expressed under the control of EBV transcription factors EB1 and R. J Virol. 1989 Dec;63(12):5276–5284. doi: 10.1128/jvi.63.12.5276-5284.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carey M., Kakidani H., Leatherwood J., Mostashari F., Ptashne M. An amino-terminal fragment of GAL4 binds DNA as a dimer. J Mol Biol. 1989 Oct 5;209(3):423–432. doi: 10.1016/0022-2836(89)90007-7. [DOI] [PubMed] [Google Scholar]
  4. Chevallier-Greco A., Gruffat H., Manet E., Calender A., Sergeant A. The Epstein-Barr virus (EBV) DR enhancer contains two functionally different domains: domain A is constitutive and cell specific, domain B is transactivated by the EBV early protein R. J Virol. 1989 Feb;63(2):615–623. doi: 10.1128/jvi.63.2.615-623.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  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. Cox M. A., Leahy J., Hardwick J. M. An enhancer within the divergent promoter of Epstein-Barr virus responds synergistically to the R and Z transactivators. J Virol. 1990 Jan;64(1):313–321. doi: 10.1128/jvi.64.1.313-321.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Czerny T., Schaffner G., Busslinger M. DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site. Genes Dev. 1993 Oct;7(10):2048–2061. doi: 10.1101/gad.7.10.2048. [DOI] [PubMed] [Google Scholar]
  9. Fixman E. D., Hayward G. S., Hayward S. D. trans-acting requirements for replication of Epstein-Barr virus ori-Lyt. J Virol. 1992 Aug;66(8):5030–5039. doi: 10.1128/jvi.66.8.5030-5039.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Furnari F. B., Adams M. D., Pagano J. S. Regulation of the Epstein-Barr virus DNA polymerase gene. J Virol. 1992 May;66(5):2837–2845. doi: 10.1128/jvi.66.5.2837-2845.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Giot J. F., Mikaelian I., Buisson M., Manet E., Joab I., Nicolas J. C., Sergeant A. Transcriptional interference between the EBV transcription factors EB1 and R: both DNA-binding and activation domains of EB1 are required. Nucleic Acids Res. 1991 Mar 25;19(6):1251–1258. doi: 10.1093/nar/19.6.1251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gruffat H., Duran N., Buisson M., Wild F., Buckland R., Sergeant A. Characterization of an R-binding site mediating the R-induced activation of the Epstein-Barr virus BMLF1 promoter. J Virol. 1992 Jan;66(1):46–52. doi: 10.1128/jvi.66.1.46-52.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gruffat H., Manet E., Rigolet A., Sergeant A. The enhancer factor R of Epstein-Barr virus (EBV) is a sequence-specific DNA binding protein. Nucleic Acids Res. 1990 Dec 11;18(23):6835–6843. doi: 10.1093/nar/18.23.6835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gruffat H., Moreno N., Sergeant A. The Epstein-Barr virus (EBV) ORI1yt enhancer is not B-cell specific and does not respond synergistically to the EBV transcription factors R and Z. J Virol. 1990 Jun;64(6):2810–2818. doi: 10.1128/jvi.64.6.2810-2818.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hammerschmidt W., Sugden B. Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein-Barr virus. Cell. 1988 Nov 4;55(3):427–433. doi: 10.1016/0092-8674(88)90028-1. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Hardwick J. M., Tse L., Applegren N., Nicholas J., Veliuona M. A. The Epstein-Barr virus R transactivator (Rta) contains a complex, potent activation domain with properties different from those of VP16. J Virol. 1992 Sep;66(9):5500–5508. doi: 10.1128/jvi.66.9.5500-5508.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Holley-Guthrie E. A., Quinlivan E. B., Mar E. C., Kenney S. The Epstein-Barr virus (EBV) BMRF1 promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLF1 and BZLF1, in a cell-specific manner. J Virol. 1990 Aug;64(8):3753–3759. doi: 10.1128/jvi.64.8.3753-3759.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kenney S., Holley-Guthrie E., Mar E. C., Smith M. The Epstein-Barr virus BMLF1 promoter contains an enhancer element that is responsive to the BZLF1 and BRLF1 transactivators. J Virol. 1989 Sep;63(9):3878–3883. doi: 10.1128/jvi.63.9.3878-3883.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Manet E., Allera C., Gruffat H., Mikaelian I., Rigolet A., Sergeant A. The acidic activation domain of the Epstein-Barr virus transcription factor R interacts in vitro with both TBP and TFIIB and is cell-specifically potentiated by a proline-rich region. Gene Expr. 1993;3(1):49–59. [PMC free article] [PubMed] [Google Scholar]
  21. Manet E., Rigolet A., Gruffat H., Giot J. F., Sergeant A. Domains of the Epstein-Barr virus (EBV) transcription factor R required for dimerization, DNA binding and activation. Nucleic Acids Res. 1991 May 25;19(10):2661–2667. doi: 10.1093/nar/19.10.2661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Nicholas J., Coles L. S., Newman C., Honess R. W. Regulation of the herpesvirus saimiri (HVS) delayed-early 110-kilodalton promoter by HVS immediate-early gene products and a homolog of the Epstein-Barr virus R trans activator. J Virol. 1991 May;65(5):2457–2466. doi: 10.1128/jvi.65.5.2457-2466.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Quinlivan E. B., Holley-Guthrie E. A., Norris M., Gutsch D., Bachenheimer S. L., Kenney S. C. Direct BRLF1 binding is required for cooperative BZLF1/BRLF1 activation of the Epstein-Barr virus early promoter, BMRF1. Nucleic Acids Res. 1993 Jul 11;21(14):1999–2007. doi: 10.1093/nar/21.8.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rooney C. M., Rowe D. T., Ragot T., Farrell P. J. The spliced BZLF1 gene of Epstein-Barr virus (EBV) transactivates an early EBV promoter and induces the virus productive cycle. J Virol. 1989 Jul;63(7):3109–3116. doi: 10.1128/jvi.63.7.3109-3116.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schepers A., Pich D., Hammerschmidt W. A transcription factor with homology to the AP-1 family links RNA transcription and DNA replication in the lytic cycle of Epstein-Barr virus. EMBO J. 1993 Oct;12(10):3921–3929. doi: 10.1002/j.1460-2075.1993.tb06070.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sinclair A. J., Brimmell M., Shanahan F., Farrell P. J. Pathways of activation of the Epstein-Barr virus productive cycle. J Virol. 1991 May;65(5):2237–2244. doi: 10.1128/jvi.65.5.2237-2244.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Struhl K. The DNA-binding domains of the jun oncoprotein and the yeast GCN4 transcriptional activator protein are functionally homologous. Cell. 1987 Sep 11;50(6):841–846. doi: 10.1016/0092-8674(87)90511-3. [DOI] [PubMed] [Google Scholar]
  29. Wright W. E., Funk W. D. CASTing for multicomponent DNA-binding complexes. Trends Biochem Sci. 1993 Mar;18(3):77–80. doi: 10.1016/0968-0004(93)90156-h. [DOI] [PubMed] [Google Scholar]
  30. Zalani S., Holley-Guthrie E. A., Gutsch D. E., Kenney S. C. The Epstein-Barr virus immediate-early promoter BRLF1 can be activated by the cellular Sp1 transcription factor. J Virol. 1992 Dec;66(12):7282–7292. doi: 10.1128/jvi.66.12.7282-7292.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. van Santen V. L. Characterization of a bovine herpesvirus 4 immediate-early RNA encoding a homolog of the Epstein-Barr virus R transactivator. J Virol. 1993 Feb;67(2):773–784. doi: 10.1128/jvi.67.2.773-784.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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