Abstract
The Epstein-Barr virus BZLF1 immediate-early gene encodes a transcriptional activator protein, Zta, which acts as a key regulatory switch in the transition between the latent and lytic viral life cycle. In this work, full-length Zta was expressed at high levels in Escherichia coli and purified to homogeneity by DNA affinity chromatography. The bacterial protein bound to specific target sequences (Zta response elements) and activated transcription in vitro from an Epstein-Barr virus early target promoter (BHLF1). Zta bound to DNA as a dimer. The formation of a heterodimer with a Zta deletion mutant was detected by gel electrophoresis mobility shift assays. Footprinting analysis on the BHLF1, BZLF1, and simian virus 40 control regions revealed multiple binding sites with no simple consensus sequence. Zta bound upstream from its own promoter at low concentrations, while at high concentrations it bound at the transcription start site, suggesting that it may activate and then autoregulate its own expression. These results demonstrate that Zta is a sequence-specific DNA-binding transcription factor.
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- Arrand J. R., Rymo L. Characterization of the major Epstein-Barr virus-specific RNA in Burkitt lymphoma-derived cells. J Virol. 1982 Feb;41(2):376–389. doi: 10.1128/jvi.41.2.376-389.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baichwal V. R., Sugden B. Latency comes of age for herpesviruses. Cell. 1988 Mar 25;52(6):787–789. doi: 10.1016/0092-8674(88)90419-9. [DOI] [PubMed] [Google Scholar]
- Biggin M., Bodescot M., Perricaudet M., Farrell P. Epstein-Barr virus gene expression in P3HR1-superinfected Raji cells. J Virol. 1987 Oct;61(10):3120–3132. doi: 10.1128/jvi.61.10.3120-3132.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
- Chavrier P., Gruffat H., Chevallier-Greco A., Buisson M., Sergeant A. The Epstein-Barr virus (EBV) early promoter DR contains a cis-acting element responsive to the EBV transactivator EB1 and an enhancer with constitutive and inducible activities. J Virol. 1989 Feb;63(2):607–614. doi: 10.1128/jvi.63.2.607-614.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Dillner J., Kallin B., Alexander H., Ernberg I., Uno M., Ono Y., Klein G., Lerner R. A. An Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA5) partly encoded by the transformation-associated Bam WYH region of EBV DNA: preferential expression in lymphoblastoid cell lines. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6641–6645. doi: 10.1073/pnas.83.17.6641. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Faggioni A., Zompetta C., Grimaldi S., Barile G., Frati L., Lazdins J. Calcium modulation activates Epstein-Barr virus genome in latently infected cells. Science. 1986 Jun 20;232(4757):1554–1556. doi: 10.1126/science.3012779. [DOI] [PubMed] [Google Scholar]
- Falkner F. G., Zachau H. G. Correct transcription of an immunoglobulin kappa gene requires an upstream fragment containing conserved sequence elements. Nature. 1984 Jul 5;310(5972):71–74. doi: 10.1038/310071a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Fennewald S., van Santen V., Kieff E. Nucleotide sequence of an mRNA transcribed in latent growth-transforming virus infection indicates that it may encode a membrane protein. J Virol. 1984 Aug;51(2):411–419. doi: 10.1128/jvi.51.2.411-419.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gentz R., Rauscher F. J., 3rd, Abate C., Curran T. Parallel association of Fos and Jun leucine zippers juxtaposes DNA binding domains. Science. 1989 Mar 31;243(4899):1695–1699. doi: 10.1126/science.2494702. [DOI] [PubMed] [Google Scholar]
- Graves B. J., Johnson P. F., McKnight S. L. Homologous recognition of a promoter domain common to the MSV LTR and the HSV tk gene. Cell. 1986 Feb 28;44(4):565–576. doi: 10.1016/0092-8674(86)90266-7. [DOI] [PubMed] [Google Scholar]
- 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]
- Hennessy K., Fennewald S., Hummel M., Cole T., Kieff E. A membrane protein encoded by Epstein-Barr virus in latent growth-transforming infection. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7207–7211. doi: 10.1073/pnas.81.22.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hennessy K., Fennewald S., Kieff E. A third viral nuclear protein in lymphoblasts immortalized by Epstein-Barr virus. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5944–5948. doi: 10.1073/pnas.82.17.5944. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hennessy K., Kieff E. A second nuclear protein is encoded by Epstein-Barr virus in latent infection. Science. 1985 Mar 8;227(4691):1238–1240. doi: 10.1126/science.2983420. [DOI] [PubMed] [Google Scholar]
- Hennessy K., Kieff E. One of two Epstein-Barr virus nuclear antigens contains a glycine-alanine copolymer domain. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5665–5669. doi: 10.1073/pnas.80.18.5665. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hennessy K., Wang F., Bushman E. W., Kieff E. Definitive identification of a member of the Epstein-Barr virus nuclear protein 3 family. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5693–5697. doi: 10.1073/pnas.83.15.5693. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hope I. A., Struhl K. GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EMBO J. 1987 Sep;6(9):2781–2784. doi: 10.1002/j.1460-2075.1987.tb02573.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hudson G. S., Farrell P. J., Barrell B. G. Two related but differentially expressed potential membrane proteins encoded by the EcoRI Dhet region of Epstein-Barr virus B95-8. J Virol. 1985 Feb;53(2):528–535. doi: 10.1128/jvi.53.2.528-535.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jeang K. T., Hayward S. D. Organization of the Epstein-Barr virus DNA molecule. III. Location of the P3HR-1 deletion junction and characterization of the NotI repeat units that form part of the template for an abundant 12-O-tetradecanoylphorbol-13-acetate-induced mRNA transcript. J Virol. 1983 Oct;48(1):135–148. doi: 10.1128/jvi.48.1.135-148.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Joab I., Rowe D. T., Bodescot M., Nicolas J. C., Farrell P. J., Perricaudet M. Mapping of the gene coding for Epstein-Barr virus-determined nuclear antigen EBNA3 and its transient overexpression in a human cell line by using an adenovirus expression vector. J Virol. 1987 Oct;61(10):3340–3344. doi: 10.1128/jvi.61.10.3340-3344.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones K. A., Yamamoto K. R., Tjian R. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell. 1985 Sep;42(2):559–572. doi: 10.1016/0092-8674(85)90113-8. [DOI] [PubMed] [Google Scholar]
- Kadonaga J. T., Carner K. R., Masiarz F. R., Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987 Dec 24;51(6):1079–1090. doi: 10.1016/0092-8674(87)90594-0. [DOI] [PubMed] [Google Scholar]
- Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kallin B., Dillner J., Ernberg I., Ehlin-Henriksson B., Rosén A., Henle W., Henle G., Klein G. Four virally determined nuclear antigens are expressed in Epstein-Barr virus-transformed cells. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1499–1503. doi: 10.1073/pnas.83.5.1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kenney S., Kamine J., Holley-Guthrie E., Lin J. C., Mar E. C., Pagano J. The Epstein-Barr virus (EBV) BZLF1 immediate-early gene product differentially affects latent versus productive EBV promoters. J Virol. 1989 Apr;63(4):1729–1736. doi: 10.1128/jvi.63.4.1729-1736.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kouzarides T., Ziff E. The role of the leucine zipper in the fos-jun interaction. Nature. 1988 Dec 15;336(6200):646–651. doi: 10.1038/336646a0. [DOI] [PubMed] [Google Scholar]
- Landschulz W. H., Johnson P. F., McKnight S. L. The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite. Science. 1989 Mar 31;243(4899):1681–1688. doi: 10.1126/science.2494700. [DOI] [PubMed] [Google Scholar]
- Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
- Laux G., Freese U. K., Bornkamm G. W. Structure and evolution of two related transcription units of Epstein-Barr virus carrying small tandem repeats. J Virol. 1985 Dec;56(3):987–995. doi: 10.1128/jvi.56.3.987-995.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laux G., Perricaudet M., Farrell P. J. A spliced Epstein-Barr virus gene expressed in immortalized lymphocytes is created by circularization of the linear viral genome. EMBO J. 1988 Mar;7(3):769–774. doi: 10.1002/j.1460-2075.1988.tb02874.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lerner M. R., Andrews N. C., Miller G., Steitz J. A. Two small RNAs encoded by Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus. Proc Natl Acad Sci U S A. 1981 Feb;78(2):805–809. doi: 10.1073/pnas.78.2.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lieberman P. M., Hardwick J. M., Hayward S. D. Responsiveness of the Epstein-Barr virus NotI repeat promoter to the Z transactivator is mediated in a cell-type-specific manner by two independent signal regions. J Virol. 1989 Jul;63(7):3040–3050. doi: 10.1128/jvi.63.7.3040-3050.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lieberman P. M., Hardwick J. M., Sample J., Hayward G. S., Hayward S. D. The zta transactivator involved in induction of lytic cycle gene expression in Epstein-Barr virus-infected lymphocytes binds to both AP-1 and ZRE sites in target promoter and enhancer regions. J Virol. 1990 Mar;64(3):1143–1155. doi: 10.1128/jvi.64.3.1143-1155.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Manet E., Gruffat H., Trescol-Biemont M. C., Moreno N., Chambard P., Giot J. F., Sergeant A. Epstein-Barr virus bicistronic mRNAs generated by facultative splicing code for two transcriptional trans-activators. EMBO J. 1989 Jun;8(6):1819–1826. doi: 10.1002/j.1460-2075.1989.tb03576.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McLachlan A. D., Karn J. Periodic features in the amino acid sequence of nematode myosin rod. J Mol Biol. 1983 Mar 15;164(4):605–626. doi: 10.1016/0022-2836(83)90053-0. [DOI] [PubMed] [Google Scholar]
- 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]
- O'Shea E. K., Rutkowski R., Kim P. S. Evidence that the leucine zipper is a coiled coil. Science. 1989 Jan 27;243(4890):538–542. doi: 10.1126/science.2911757. [DOI] [PubMed] [Google Scholar]
- Parslow T. G., Blair D. L., Murphy W. J., Granner D. K. Structure of the 5' ends of immunoglobulin genes: a novel conserved sequence. Proc Natl Acad Sci U S A. 1984 May;81(9):2650–2654. doi: 10.1073/pnas.81.9.2650. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Petti L., Kieff E. A sixth Epstein-Barr virus nuclear protein (EBNA3B) is expressed in latently infected growth-transformed lymphocytes. J Virol. 1988 Jun;62(6):2173–2178. doi: 10.1128/jvi.62.6.2173-2178.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Petti L., Sample J., Wang F., Kieff E. A fifth Epstein-Barr virus nuclear protein (EBNA3C) is expressed in latently infected growth-transformed lymphocytes. J Virol. 1988 Apr;62(4):1330–1338. doi: 10.1128/jvi.62.4.1330-1338.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rawlins D. R., Milman G., Hayward S. D., Hayward G. S. Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region. Cell. 1985 Oct;42(3):859–868. doi: 10.1016/0092-8674(85)90282-x. [DOI] [PubMed] [Google Scholar]
- Rio D. C., Tjian R. SV40 T antigen binding site mutations that affect autoregulation. Cell. 1983 Apr;32(4):1227–1240. doi: 10.1016/0092-8674(83)90305-7. [DOI] [PubMed] [Google Scholar]
- Roberts M. S., Boundy A., O'Hare P., Pizzorno M. C., Ciufo D. M., Hayward G. S. Direct correlation between a negative autoregulatory response element at the cap site of the herpes simplex virus type 1 IE175 (alpha 4) promoter and a specific binding site for the IE175 (ICP4) protein. J Virol. 1988 Nov;62(11):4307–4320. doi: 10.1128/jvi.62.11.4307-4320.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Rosenberg M., Ho Y. S., Shatzman A. The use of pKc30 and its derivatives for controlled expression of genes. Methods Enzymol. 1983;101:123–138. doi: 10.1016/0076-6879(83)01009-5. [DOI] [PubMed] [Google Scholar]
- Schmidt M. C., Zhou Q., Berk A. J. Sp1 activates transcription without enhancing DNA-binding activity of the TATA box factor. Mol Cell Biol. 1989 Aug;9(8):3299–3307. doi: 10.1128/mcb.9.8.3299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Takada K., Shimizu N., Sakuma S., Ono Y. trans activation of the latent Epstein-Barr virus (EBV) genome after transfection of the EBV DNA fragment. J Virol. 1986 Mar;57(3):1016–1022. doi: 10.1128/jvi.57.3.1016-1022.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Turner R., Tjian R. Leucine repeats and an adjacent DNA binding domain mediate the formation of functional cFos-cJun heterodimers. Science. 1989 Mar 31;243(4899):1689–1694. doi: 10.1126/science.2494701. [DOI] [PubMed] [Google Scholar]
- 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]
- Vinson C. R., Sigler P. B., McKnight S. L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science. 1989 Nov 17;246(4932):911–916. doi: 10.1126/science.2683088. [DOI] [PubMed] [Google Scholar]
- Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
- Wu L., Rosser D. S., Schmidt M. C., Berk A. A TATA box implicated in E1A transcriptional activation of a simple adenovirus 2 promoter. Nature. 1987 Apr 2;326(6112):512–515. doi: 10.1038/326512a0. [DOI] [PubMed] [Google Scholar]
- Yoshinaga S. K., L'Etoile N. D., Berk A. J. Purification and characterization of transcription factor IIIC2. J Biol Chem. 1989 Jun 25;264(18):10726–10731. [PubMed] [Google Scholar]
- 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]