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. 1994 Oct;68(10):6223–6231. doi: 10.1128/jvi.68.10.6223-6231.1994

Transactivation by the human cytomegalovirus IE2 86-kilodalton protein requires a domain that binds to both the TATA box-binding protein and the retinoblastoma protein.

M H Sommer 1, A L Scully 1, D H Spector 1
PMCID: PMC237042  PMID: 8083962

Abstract

The human cytomegalovirus major immediate-early (IE) proteins play an indispensable role in regulating viral gene expression. One of these gene products, the IE2 86-kDa protein (IE2 86), is a potent activator of both homologous and heterologous promoters and can form a complex with a component of the basal transcription apparatus, the TATA box-binding protein (TBP). In this report, we show that when IE2 86 is expressed as a glutathione S-transferase (GST)-IE2 86 fusion protein, there are three independent regions that can interact with TBP and with another important cellular regulatory protein, the retinoblastoma gene product (RB). One of these three regions, as well as a domain at the carboxy terminus, contain consensus sites for casein kinase phosphorylation and negatively regulate binding of in vitro-translated IE2 86 to GST-TBP or GST-RB. The dimerization domain of IE2 86 must be present for the interaction of the in vitro-translated protein with GST-TBP and GST-RB. Analysis of IE2 86 mutants in vivo demonstrates that one of the strong binding regions is required for the protein to function as a transactivator. Our results also indicate that domains other than those that interact with TBP and RB are required for the activation function of this protein.

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Selected References

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  1. Bandara L. R., Adamczewski J. P., Hunt T., La Thangue N. B. Cyclin A and the retinoblastoma gene product complex with a common transcription factor. Nature. 1991 Jul 18;352(6332):249–251. doi: 10.1038/352249a0. [DOI] [PubMed] [Google Scholar]
  2. Caswell R., Hagemeier C., Chiou C. J., Hayward G., Kouzarides T., Sinclair J. The human cytomegalovirus 86K immediate early (IE) 2 protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via regions of IE2 required for transcriptional regulation. J Gen Virol. 1993 Dec;74(Pt 12):2691–2698. doi: 10.1099/0022-1317-74-12-2691. [DOI] [PubMed] [Google Scholar]
  3. Cherrington J. M., Mocarski E. S. Human cytomegalovirus ie1 transactivates the alpha promoter-enhancer via an 18-base-pair repeat element. J Virol. 1989 Mar;63(3):1435–1440. doi: 10.1128/jvi.63.3.1435-1440.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chiou C. J., Zong J., Waheed I., Hayward G. S. Identification and mapping of dimerization and DNA-binding domains in the C terminus of the IE2 regulatory protein of human cytomegalovirus. J Virol. 1993 Oct;67(10):6201–6214. doi: 10.1128/jvi.67.10.6201-6214.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Furnari B. A., Poma E., Kowalik T. F., Huong S. M., Huang E. S. Human cytomegalovirus immediate-early gene 2 protein interacts with itself and with several novel cellular proteins. J Virol. 1993 Aug;67(8):4981–4991. doi: 10.1128/jvi.67.8.4981-4991.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  7. Hagemeier C., Bannister A. J., Cook A., Kouzarides T. The activation domain of transcription factor PU.1 binds the retinoblastoma (RB) protein and the transcription factor TFIID in vitro: RB shows sequence similarity to TFIID and TFIIB. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1580–1584. doi: 10.1073/pnas.90.4.1580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hagemeier C., Walker S., Caswell R., Kouzarides T., Sinclair J. The human cytomegalovirus 80-kilodalton but not the 72-kilodalton immediate-early protein transactivates heterologous promoters in a TATA box-dependent mechanism and interacts directly with TFIID. J Virol. 1992 Jul;66(7):4452–4456. doi: 10.1128/jvi.66.7.4452-4456.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hateboer G., Timmers H. T., Rustgi A. K., Billaud M., van 't Veer L. J., Bernards R. TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8489–8493. doi: 10.1073/pnas.90.18.8489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hermiston T. W., Malone C. L., Stinski M. F. Human cytomegalovirus immediate-early two protein region involved in negative regulation of the major immediate-early promoter. J Virol. 1990 Jul;64(7):3532–3536. doi: 10.1128/jvi.64.7.3532-3536.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Horikoshi N., Maguire K., Kralli A., Maldonado E., Reinberg D., Weinmann R. Direct interaction between adenovirus E1A protein and the TATA box binding transcription factor IID. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5124–5128. doi: 10.1073/pnas.88.12.5124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jeor S. C., Albrecht T. B., Funk F. D., Rapp F. Stimulation of cellular DNA synthesis by human cytomegalovirus. J Virol. 1974 Feb;13(2):353–362. doi: 10.1128/jvi.13.2.353-362.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jupp R., Hoffmann S., Stenberg R. M., Nelson J. A., Ghazal P. Human cytomegalovirus IE86 protein interacts with promoter-bound TATA-binding protein via a specific region distinct from the autorepression domain. J Virol. 1993 Dec;67(12):7539–7546. doi: 10.1128/jvi.67.12.7539-7546.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaelin W. G., Jr, Ewen M. E., Livingston D. M. Definition of the minimal simian virus 40 large T antigen- and adenovirus E1A-binding domain in the retinoblastoma gene product. Mol Cell Biol. 1990 Jul;10(7):3761–3769. doi: 10.1128/mcb.10.7.3761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klucher K. M., Sommer M., Kadonaga J. T., Spector D. H. In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins. Mol Cell Biol. 1993 Feb;13(2):1238–1250. doi: 10.1128/mcb.13.2.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lai H. C., Qian B., Tu C. P. Characterization of a variant rat glutathione S-transferase by cDNA expression in Escherichia coli. Arch Biochem Biophys. 1989 Sep;273(2):423–432. doi: 10.1016/0003-9861(89)90501-8. [DOI] [PubMed] [Google Scholar]
  17. Lai J. S., Herr W. Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6958–6962. doi: 10.1073/pnas.89.15.6958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lang D., Stamminger T. The 86-kilodalton IE-2 protein of human cytomegalovirus is a sequence-specific DNA-binding protein that interacts directly with the negative autoregulatory response element located near the cap site of the IE-1/2 enhancer-promoter. J Virol. 1993 Jan;67(1):323–331. doi: 10.1128/jvi.67.1.323-331.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee W. S., Kao C. C., Bryant G. O., Liu X., Berk A. J. Adenovirus E1A activation domain binds the basic repeat in the TATA box transcription factor. Cell. 1991 Oct 18;67(2):365–376. doi: 10.1016/0092-8674(91)90188-5. [DOI] [PubMed] [Google Scholar]
  20. Lin Y. S., Ha I., Maldonado E., Reinberg D., Green M. R. Binding of general transcription factor TFIIB to an acidic activating region. Nature. 1991 Oct 10;353(6344):569–571. doi: 10.1038/353569a0. [DOI] [PubMed] [Google Scholar]
  21. Liu F., Green M. R. A specific member of the ATF transcription factor family can mediate transcription activation by the adenovirus E1a protein. Cell. 1990 Jun 29;61(7):1217–1224. doi: 10.1016/0092-8674(90)90686-9. [DOI] [PubMed] [Google Scholar]
  22. Macias M. P., Stinski M. F. An in vitro system for human cytomegalovirus immediate early 2 protein (IE2)-mediated site-dependent repression of transcription and direct binding of IE2 to the major immediate early promoter. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):707–711. doi: 10.1073/pnas.90.2.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Malone C. L., Vesole D. H., Stinski M. F. Transactivation of a human cytomegalovirus early promoter by gene products from the immediate-early gene IE2 and augmentation by IE1: mutational analysis of the viral proteins. J Virol. 1990 Apr;64(4):1498–1506. doi: 10.1128/jvi.64.4.1498-1506.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McDonough S. H., Spector D. H. Transcription in human fibroblasts permissively infected by human cytomegalovirus strain AD169. Virology. 1983 Feb;125(1):31–46. doi: 10.1016/0042-6822(83)90061-2. [DOI] [PubMed] [Google Scholar]
  25. McKnight J. L., Kristie T. M., Roizman B. Binding of the virion protein mediating alpha gene induction in herpes simplex virus 1-infected cells to its cis site requires cellular proteins. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7061–7065. doi: 10.1073/pnas.84.20.7061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. O'Hare P., Goding C. R. Herpes simplex virus regulatory elements and the immunoglobulin octamer domain bind a common factor and are both targets for virion transactivation. Cell. 1988 Feb 12;52(3):435–445. doi: 10.1016/s0092-8674(88)80036-9. [DOI] [PubMed] [Google Scholar]
  27. Pizzorno M. C., Mullen M. A., Chang Y. N., Hayward G. S. The functionally active IE2 immediate-early regulatory protein of human cytomegalovirus is an 80-kilodalton polypeptide that contains two distinct activator domains and a duplicated nuclear localization signal. J Virol. 1991 Jul;65(7):3839–3852. doi: 10.1128/jvi.65.7.3839-3852.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pizzorno M. C., O'Hare P., Sha L., LaFemina R. L., Hayward G. S. trans-activation and autoregulation of gene expression by the immediate-early region 2 gene products of human cytomegalovirus. J Virol. 1988 Apr;62(4):1167–1179. doi: 10.1128/jvi.62.4.1167-1179.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Staprans S. I., Rabert D. K., Spector D. H. Identification of sequence requirements and trans-acting functions necessary for regulated expression of a human cytomegalovirus early gene. J Virol. 1988 Sep;62(9):3463–3473. doi: 10.1128/jvi.62.9.3463-3473.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stenberg R. M., Fortney J., Barlow S. W., Magrane B. P., Nelson J. A., Ghazal P. Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains. J Virol. 1990 Apr;64(4):1556–1565. doi: 10.1128/jvi.64.4.1556-1565.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stenberg R. M., Thomsen D. R., Stinski M. F. Structural analysis of the major immediate early gene of human cytomegalovirus. J Virol. 1984 Jan;49(1):190–199. doi: 10.1128/jvi.49.1.190-199.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stenberg R. M., Witte P. R., Stinski M. F. Multiple spliced and unspliced transcripts from human cytomegalovirus immediate-early region 2 and evidence for a common initiation site within immediate-early region 1. J Virol. 1985 Dec;56(3):665–675. doi: 10.1128/jvi.56.3.665-675.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stringer K. F., Ingles C. J., Greenblatt J. Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID. Nature. 1990 Jun 28;345(6278):783–786. doi: 10.1038/345783a0. [DOI] [PubMed] [Google Scholar]
  34. Toung Y. P., Hsieh T. S., Tu C. P. The Drosophila glutathione S-transferase 1-1 is encoded by an intronless gene at 87B. Biochem Biophys Res Commun. 1991 Aug 15;178(3):1205–1211. doi: 10.1016/0006-291x(91)91021-4. [DOI] [PubMed] [Google Scholar]
  35. Wade M., Kowalik T. F., Mudryj M., Huang E. S., Azizkhan J. C. E2F mediates dihydrofolate reductase promoter activation and multiprotein complex formation in human cytomegalovirus infection. Mol Cell Biol. 1992 Oct;12(10):4364–4374. doi: 10.1128/mcb.12.10.4364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wang C. Y., Petryniak B., Thompson C. B., Kaelin W. G., Leiden J. M. Regulation of the Ets-related transcription factor Elf-1 by binding to the retinoblastoma protein. Science. 1993 May 28;260(5112):1330–1335. doi: 10.1126/science.8493578. [DOI] [PubMed] [Google Scholar]
  37. Wathen M. W., Stinski M. F. Temporal patterns of human cytomegalovirus transcription: mapping the viral RNAs synthesized at immediate early, early, and late times after infection. J Virol. 1982 Feb;41(2):462–477. doi: 10.1128/jvi.41.2.462-477.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Welch P. J., Wang J. Y. A C-terminal protein-binding domain in the retinoblastoma protein regulates nuclear c-Abl tyrosine kinase in the cell cycle. Cell. 1993 Nov 19;75(4):779–790. doi: 10.1016/0092-8674(93)90497-e. [DOI] [PubMed] [Google Scholar]
  39. Whyte P., Buchkovich K. J., Horowitz J. M., Friend S. H., Raybuck M., Weinberg R. A., Harlow E. Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature. 1988 Jul 14;334(6178):124–129. doi: 10.1038/334124a0. [DOI] [PubMed] [Google Scholar]

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