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. 1993 Oct;67(10):6201–6214. doi: 10.1128/jvi.67.10.6201-6214.1993

Identification and mapping of dimerization and DNA-binding domains in the C terminus of the IE2 regulatory protein of human cytomegalovirus.

C J Chiou 1, J Zong 1, I Waheed 1, G S Hayward 1
PMCID: PMC238042  PMID: 8396676

Abstract

The 80-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediate-early (MIE) gene behaves both as a nonspecific transactivator of heterologous reporter genes and as a specific repressor of its own promoter-enhancer region. To begin to examine the biochemical properties of the IE2 protein, we prepared panels of N-terminal and C-terminal truncation mutants by in vitro translation procedures. In cross-linking experiments, the C-terminal half of IE2 (which is sufficient for down-regulation) formed dimers but N-terminal segments did not do so. Cotranslated Oct2/IE2 fusion proteins containing the same IE2 C-terminal region from codons 266 to 579 also formed mixed-subunit DNA-bound oligomeric complexes in gel mobility shift assays. Furthermore, an IE2 domain bounded by codons 388 to 542 proved to immunoprecipitate as heterodimers with cotranslated subunits containing known epitopes for specific antibodies. Deletion up to codon 428 or truncation back to codon 504 prevented this interaction. In direct gel shift DNA-binding assays, a bacterial GST/IE2(346-579) fusion protein bound to a 30-mer oligonucleotide probe encompassing the major immediate-early gene negative cis-regulatory target DNA sequence but failed to bind to a single-base-pair insertion mutant probe (delta CRS). This specific DNA-binding activity was abolished by further deletion up to codon 388 on the N-terminal side or by truncation at codon 542 on the C-terminal side. Therefore, the minimal DNA-binding domain requires additional amino acid motifs on both sides of the dimerization domain. This segment of IE2 is functionally important for both transactivation and down-regulation and contains several highly conserved amino acid motifs that are shared amongst the equivalent HCMV, simian CMV, mouse CMV, rat CMV, and human herpesvirus 6 proteins from other betaherpesviruses.

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Selected References

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

  1. Ambinder R. F., Mullen M. A., Chang Y. N., Hayward G. S., Hayward S. D. Functional domains of Epstein-Barr virus nuclear antigen EBNA-1. J Virol. 1991 Mar;65(3):1466–1478. doi: 10.1128/jvi.65.3.1466-1478.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baracchini E., Glezer E., Fish K., Stenberg R. M., Nelson J. A., Ghazal P. An isoform variant of the cytomegalovirus immediate-early auto repressor functions as a transcriptional activator. Virology. 1992 Jun;188(2):518–529. doi: 10.1016/0042-6822(92)90506-k. [DOI] [PubMed] [Google Scholar]
  3. Batchelor A. H., O'Hare P. Regulation and cell-type-specific activity of a promoter located upstream of the latency-associated transcript of herpes simplex virus type 1. J Virol. 1990 Jul;64(7):3269–3279. doi: 10.1128/jvi.64.7.3269-3279.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benson J. D., Huang E. S. Human cytomegalovirus induces expression of cellular topoisomerase II. J Virol. 1990 Jan;64(1):9–15. doi: 10.1128/jvi.64.1.9-15.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Chee M. S., Bankier A. T., Beck S., Bohni R., Brown C. M., Cerny R., Horsnell T., Hutchison C. A., 3rd, Kouzarides T., Martignetti J. A. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 1990;154:125–169. doi: 10.1007/978-3-642-74980-3_6. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Clerc R. G., Corcoran L. M., LeBowitz J. H., Baltimore D., Sharp P. A. The B-cell-specific Oct-2 protein contains POU box- and homeo box-type domains. Genes Dev. 1988 Dec;2(12A):1570–1581. doi: 10.1101/gad.2.12a.1570. [DOI] [PubMed] [Google Scholar]
  9. Colberg-Poley A. M., Santomenna L. D., Harlow P. P., Benfield P. A., Tenney D. J. Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression. J Virol. 1992 Jan;66(1):95–105. doi: 10.1128/jvi.66.1.95-105.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dasmahapatra B. Cell-free expression vector: use of insect virus translational initiation signal for in vitro gene expression. Methods Enzymol. 1993;217:143–151. doi: 10.1016/0076-6879(93)17060-i. [DOI] [PubMed] [Google Scholar]
  11. Davis M. G., Kenney S. C., Kamine J., Pagano J. S., Huang E. S. Immediate-early gene region of human cytomegalovirus trans-activates the promoter of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8642–8646. doi: 10.1073/pnas.84.23.8642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Hermiston T. W., Malone C. L., Witte P. R., Stinski M. F. Identification and characterization of the human cytomegalovirus immediate-early region 2 gene that stimulates gene expression from an inducible promoter. J Virol. 1987 Oct;61(10):3214–3221. doi: 10.1128/jvi.61.10.3214-3221.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Keil G. M., Ebeling-Keil A., Koszinowski U. H. Sequence and structural organization of murine cytomegalovirus immediate-early gene 1. J Virol. 1987 Jun;61(6):1901–1908. doi: 10.1128/jvi.61.6.1901-1908.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Koszinowski U. H., Keil G. M., Volkmer H., Fibi M. R., Ebeling-Keil A., Münch K. The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription. J Virol. 1986 Apr;58(1):59–66. doi: 10.1128/jvi.58.1.59-66.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. LaFemina R., Hayward G. S. Constitutive and retinoic acid-inducible expression of cytomegalovirus immediate-early genes in human teratocarcinoma cells. J Virol. 1986 May;58(2):434–440. doi: 10.1128/jvi.58.2.434-440.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lafemina R. L., Hayward G. S. Differences in cell-type-specific blocks to immediate early gene expression and DNA replication of human, simian and murine cytomegalovirus. J Gen Virol. 1988 Feb;69(Pt 2):355–374. doi: 10.1099/0022-1317-69-2-355. [DOI] [PubMed] [Google Scholar]
  18. Lafemina R. L., Pizzorno M. C., Mosca J. D., Hayward G. S. Expression of the acidic nuclear immediate-early protein (IE1) of human cytomegalovirus in stable cell lines and its preferential association with metaphase chromosomes. Virology. 1989 Oct;172(2):584–600. doi: 10.1016/0042-6822(89)90201-8. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Lieberman P. M., Berk A. J. The Zta trans-activator protein stabilizes TFIID association with promoter DNA by direct protein-protein interaction. Genes Dev. 1991 Dec;5(12B):2441–2454. doi: 10.1101/gad.5.12b.2441. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lillie J. W., Green M. R. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. doi: 10.1038/338039a0. [DOI] [PubMed] [Google Scholar]
  24. Lin Y. S., Green M. R. Mechanism of action of an acidic transcriptional activator in vitro. Cell. 1991 Mar 8;64(5):971–981. doi: 10.1016/0092-8674(91)90321-o. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Liu B., Stinski M. F. Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements. J Virol. 1992 Jul;66(7):4434–4444. doi: 10.1128/jvi.66.7.4434-4444.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Messerle M., Bühler B., Keil G. M., Koszinowski U. H. Structural organization, expression, and functional characterization of the murine cytomegalovirus immediate-early gene 3. J Virol. 1992 Jan;66(1):27–36. doi: 10.1128/jvi.66.1.27-36.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mosca J. D., Bednarik D. P., Raj N. B., Rosen C. A., Sodroski J. G., Haseltine W. A., Hayward G. S., Pitha P. M. Activation of human immunodeficiency virus by herpesvirus infection: identification of a region within the long terminal repeat that responds to a trans-acting factor encoded by herpes simplex virus 1. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7408–7412. doi: 10.1073/pnas.84.21.7408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nakabeppu Y., Ryder K., Nathans D. DNA binding activities of three murine Jun proteins: stimulation by Fos. Cell. 1988 Dec 2;55(5):907–915. doi: 10.1016/0092-8674(88)90146-8. [DOI] [PubMed] [Google Scholar]
  33. O'Hare P., Hayward G. S. Expression of recombinant genes containing herpes simplex virus delayed-early and immediate-early regulatory regions and trans activation by herpesvirus infection. J Virol. 1984 Nov;52(2):522–531. doi: 10.1128/jvi.52.2.522-531.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pizzorno M. C., Hayward G. S. The IE2 gene products of human cytomegalovirus specifically down-regulate expression from the major immediate-early promoter through a target sequence located near the cap site. J Virol. 1990 Dec;64(12):6154–6165. doi: 10.1128/jvi.64.12.6154-6165.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Sandford G. R., Ho K., Burns W. H. Characterization of the major locus of immediate-early genes of rat cytomegalovirus. J Virol. 1993 Jul;67(7):4093–4103. doi: 10.1128/jvi.67.7.4093-4103.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Santomenna L. D., Colberg-Poley A. M. Induction of cellular hsp70 expression by human cytomegalovirus. J Virol. 1990 May;64(5):2033–2040. doi: 10.1128/jvi.64.5.2033-2040.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Spaete R. R., Mocarski E. S. Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts. J Virol. 1985 Oct;56(1):135–143. doi: 10.1128/jvi.56.1.135-143.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Stamminger T., Puchtler E., Fleckenstein B. Discordant expression of the immediate-early 1 and 2 gene regions of human cytomegalovirus at early times after infection involves posttranscriptional processing events. J Virol. 1991 May;65(5):2273–2282. doi: 10.1128/jvi.65.5.2273-2282.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stasiak P. C., Mocarski E. S. Transactivation of the cytomegalovirus ICP36 gene promoter requires the alpha gene product TRS1 in addition to IE1 and IE2. J Virol. 1992 Feb;66(2):1050–1058. doi: 10.1128/jvi.66.2.1050-1058.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stenberg R. M., Depto A. S., Fortney J., Nelson J. A. Regulated expression of early and late RNAs and proteins from the human cytomegalovirus immediate-early gene region. J Virol. 1989 Jun;63(6):2699–2708. doi: 10.1128/jvi.63.6.2699-2708.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. 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]
  47. Tenney D. J., Colberg-Poley A. M. RNA analysis and isolation of cDNAs derived from the human cytomegalovirus immediate-early region at 0.24 map units. Intervirology. 1990;31(2-4):203–214. doi: 10.1159/000150155. [DOI] [PubMed] [Google Scholar]
  48. Tevethia M. J., Spector D. J., Leisure K. M., Stinski M. F. Participation of two human cytomegalovirus immediate early gene regions in transcriptional activation of adenovirus promoters. Virology. 1987 Dec;161(2):276–285. doi: 10.1016/0042-6822(87)90119-x. [DOI] [PubMed] [Google Scholar]
  49. 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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