Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1994 Jun 15;13(12):2897–2903. doi: 10.1002/j.1460-2075.1994.tb06584.x

Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein.

C Hagemeier 1, R Caswell 1, G Hayhurst 1, J Sinclair 1, T Kouzarides 1
PMCID: PMC395171  PMID: 8026474

Abstract

The 86 kDa immediate early IE2 protein of human cytomegalovirus (HCMV) can activate transcription of both viral and cellular genes and can repress transcription from its own promoter. Using two in vivo assays, we provide evidence of a functional interaction between IE2 and the retinoblastoma (RB) protein: IE2 alleviates RB-induced repression of a promoter bearing E2F binding sites and RB alleviates IE2-mediated repression of its own promoter. These functional effects are likely to be a result of a direct contact between IE2 and RB, which we can demonstrate both in vitro and in HCMV-infected cells. The interaction between IE2 and RB shows similar characteristics to the interaction between RB and E1A. First, binding to IE2 requires an intact RB pocket domain. Secondly, the binding is sensitive to the phosphorylation state of RB, because cyclin A-CDK-induced phosphorylation of RB diminishes IE2 binding. Thirdly, the IE2 domain required for RB binding is separate to the domains necessary for TBP and TFIIB binding. Our results demonstrate that large and small DNA viruses have a common interface with the host cell, namely the association with the RB tumour suppressor protein.

Full text

PDF
2897

Images in this article

2898Image
on p.2898
2899Image
on p.2899
2899Image
on p.2899
2899Image
on p.2899
2900Image
on p.2900
2900Image
on p.2900
2901Image
on p.2901

Selected References

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

  1. Bandara L. R., La Thangue N. B. Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor. Nature. 1991 Jun 6;351(6326):494–497. doi: 10.1038/351494a0. [DOI] [PubMed] [Google Scholar]
  2. Bannister A. J., Cook A., Kouzarides T. In vitro DNA binding activity of Fos/Jun and BZLF1 but not C/EBP is affected by redox changes. Oncogene. 1991 Jul;6(7):1243–1250. [PubMed] [Google Scholar]
  3. 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]
  4. Chellappan S. P., Hiebert S., Mudryj M., Horowitz J. M., Nevins J. R. The E2F transcription factor is a cellular target for the RB protein. Cell. 1991 Jun 14;65(6):1053–1061. doi: 10.1016/0092-8674(91)90557-f. [DOI] [PubMed] [Google Scholar]
  5. Cherrington J. M., Khoury E. L., Mocarski E. S. Human cytomegalovirus ie2 negatively regulates alpha gene expression via a short target sequence near the transcription start site. J Virol. 1991 Feb;65(2):887–896. doi: 10.1128/jvi.65.2.887-896.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chittenden T., Livingston D. M., Kaelin W. G., Jr The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. Cell. 1991 Jun 14;65(6):1073–1082. doi: 10.1016/0092-8674(91)90559-h. [DOI] [PubMed] [Google Scholar]
  7. Dalton S. Cell cycle regulation of the human cdc2 gene. EMBO J. 1992 May;11(5):1797–1804. doi: 10.1002/j.1460-2075.1992.tb05231.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DeCaprio J. A., Ludlow J. W., Figge J., Shew J. Y., Huang C. M., Lee W. H., Marsilio E., Paucha E., Livingston D. M. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell. 1988 Jul 15;54(2):275–283. doi: 10.1016/0092-8674(88)90559-4. [DOI] [PubMed] [Google Scholar]
  9. Dyson N., Guida P., McCall C., Harlow E. Adenovirus E1A makes two distinct contacts with the retinoblastoma protein. J Virol. 1992 Jul;66(7):4606–4611. doi: 10.1128/jvi.66.7.4606-4611.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dyson N., Howley P. M., Münger K., Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 1989 Feb 17;243(4893):934–937. doi: 10.1126/science.2537532. [DOI] [PubMed] [Google Scholar]
  11. Ewen M. E., Sluss H. K., Sherr C. J., Matsushime H., Kato J., Livingston D. M. Functional interactions of the retinoblastoma protein with mammalian D-type cyclins. Cell. 1993 May 7;73(3):487–497. doi: 10.1016/0092-8674(93)90136-e. [DOI] [PubMed] [Google Scholar]
  12. Gu W., Schneider J. W., Condorelli G., Kaushal S., Mahdavi V., Nadal-Ginard B. Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation. Cell. 1993 Feb 12;72(3):309–324. doi: 10.1016/0092-8674(93)90110-c. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Hagemeier C., Cook A., Kouzarides T. The retinoblastoma protein binds E2F residues required for activation in vivo and TBP binding in vitro. Nucleic Acids Res. 1993 Nov 11;21(22):4998–5004. doi: 10.1093/nar/21.22.4998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hagemeier C., Walker S. M., Sissons P. J., Sinclair J. H. The 72K IE1 and 80K IE2 proteins of human cytomegalovirus independently trans-activate the c-fos, c-myc and hsp70 promoters via basal promoter elements. J Gen Virol. 1992 Sep;73(Pt 9):2385–2393. doi: 10.1099/0022-1317-73-9-2385. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Helin K., Ed H. The retinoblastoma protein as a transcriptional repressor. Trends Cell Biol. 1993 Feb;3(2):43–46. doi: 10.1016/0962-8924(93)90150-y. [DOI] [PubMed] [Google Scholar]
  19. Helin K., Lees J. A., Vidal M., Dyson N., Harlow E., Fattaey A. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell. 1992 Jul 24;70(2):337–350. doi: 10.1016/0092-8674(92)90107-n. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Hiebert S. W., Chellappan S. P., Horowitz J. M., Nevins J. R. The interaction of RB with E2F coincides with an inhibition of the transcriptional activity of E2F. Genes Dev. 1992 Feb;6(2):177–185. doi: 10.1101/gad.6.2.177. [DOI] [PubMed] [Google Scholar]
  22. Hollingsworth R. E., Jr, Hensey C. E., Lee W. H. Retinoblastoma protein and the cell cycle. Curr Opin Genet Dev. 1993 Feb;3(1):55–62. doi: 10.1016/s0959-437x(05)80341-7. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Kaelin W. G., Jr, Krek W., Sellers W. R., DeCaprio J. A., Ajchenbaum F., Fuchs C. S., Chittenden T., Li Y., Farnham P. J., Blanar M. A. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 1992 Jul 24;70(2):351–364. doi: 10.1016/0092-8674(92)90108-o. [DOI] [PubMed] [Google Scholar]
  26. Klucher K. M., Rabert D. K., Spector D. H. Sequences in the human cytomegalovirus 2.7-kilobase RNA promoter which mediate its regulation as an early gene. J Virol. 1989 Dec;63(12):5334–5343. doi: 10.1128/jvi.63.12.5334-5343.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kothari S., Baillie J., Sissons J. G., Sinclair J. H. The 21bp repeat element of the human cytomegalovirus major immediate early enhancer is a negative regulator of gene expression in undifferentiated cells. Nucleic Acids Res. 1991 Apr 25;19(8):1767–1771. doi: 10.1093/nar/19.8.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kouzarides T. Transcriptional regulation by the retinoblastoma protein. Trends Cell Biol. 1993 Jul;3(7):211–213. doi: 10.1016/0962-8924(93)90113-f. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Liu B., Hermiston T. W., Stinski M. F. A cis-acting element in the major immediate-early (IE) promoter of human cytomegalovirus is required for negative regulation by IE2. J Virol. 1991 Feb;65(2):897–903. doi: 10.1128/jvi.65.2.897-903.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Rustgi A. K., Dyson N., Bernards R. Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product. Nature. 1991 Aug 8;352(6335):541–544. doi: 10.1038/352541a0. [DOI] [PubMed] [Google Scholar]
  34. Shan B., Zhu X., Chen P. L., Durfee T., Yang Y., Sharp D., Lee W. H. Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F. Mol Cell Biol. 1992 Dec;12(12):5620–5631. doi: 10.1128/mcb.12.12.5620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Spector D. J., Tevethia M. J. Identification of a human cytomegalovirus virus DNA segment that complements an adenovirus 5 immediate early mutant. Virology. 1986 Jun;151(2):329–338. doi: 10.1016/0042-6822(86)90053-x. [DOI] [PubMed] [Google Scholar]
  36. St Jeor S. C., Hutt R. Cell DNA replication as a function in the synthesis of human cytomegalovirus. J Gen Virol. 1977 Oct;37(1):65–73. doi: 10.1099/0022-1317-37-1-65. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. 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]
  40. 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]
  41. 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]
  42. Weinberg R. A. Tumor suppressor genes. Science. 1991 Nov 22;254(5035):1138–1146. doi: 10.1126/science.1659741. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Whyte P., Williamson N. M., Harlow E. Cellular targets for transformation by the adenovirus E1A proteins. Cell. 1989 Jan 13;56(1):67–75. doi: 10.1016/0092-8674(89)90984-7. [DOI] [PubMed] [Google Scholar]
  45. Zamanian M., La Thangue N. B. Adenovirus E1a prevents the retinoblastoma gene product from repressing the activity of a cellular transcription factor. EMBO J. 1992 Jul;11(7):2603–2610. doi: 10.1002/j.1460-2075.1992.tb05325.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES