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. 1993 Jan;67(1):323–331. doi: 10.1128/jvi.67.1.323-331.1993

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.

D Lang 1, T Stamminger 1
PMCID: PMC237366  PMID: 8380080

Abstract

The 86-kDa IE-2 protein of human cytomegalovirus is able to autoregulate its own expression via a short nucleotide sequence, termed the cis repression signal (CRS), that is located between the TATA box and the cap site of the IE-1/2 enhancer-promoter. Here we report that the 86-kDa IE-2 protein can interact directly with the CRS, thus demonstrating that IE-2 is a DNA-binding protein. This could be shown by both DNase I protection and gel retardation experiments using a procaryotically expressed IE-2 protein that was purified to near homogeneity. The interaction was sequence specific since a mutated form of the CRS that had previously been reported to be defective in mediating negative regulation could no longer compete for binding in DNase I protection experiments. In addition, an IE-2-reactive monoclonal antibody was able to elicit a supershift in gel retardation experiments, thus proving the presence of IE-2 within the protein-DNA complex. These results suggest that formation of a specific complex between an IE protein and a target sequence located near the cap site of its own gene promoter may be a common mechanism used by both alphaherpesviruses and betaherpesviruses to autoregulate IE gene transcription, although the sequence requirements differ between the two herpesviral subgroups.

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Selected References

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

  1. Akrigg A., Wilkinson G. W., Oram J. D. The structure of the major immediate early gene of human cytomegalovirus strain AD169. Virus Res. 1985 Mar;2(2):107–121. doi: 10.1016/0168-1702(85)90242-4. [DOI] [PubMed] [Google Scholar]
  2. Andreoni M., Faircloth M., Vugler L., Britt W. J. A rapid microneutralization assay for the measurement of neutralizing antibody reactive with human cytomegalovirus. J Virol Methods. 1989 Feb;23(2):157–167. doi: 10.1016/0166-0934(89)90129-8. [DOI] [PubMed] [Google Scholar]
  3. Biegalke B. J., Geballe A. P. Sequence requirements for activation of the HIV-1 LTR by human cytomegalovirus. Virology. 1991 Jul;183(1):381–385. doi: 10.1016/0042-6822(91)90151-z. [DOI] [PubMed] [Google Scholar]
  4. Boshart M., Weber F., Jahn G., Dorsch-Häsler K., Fleckenstein B., Schaffner W. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell. 1985 Jun;41(2):521–530. doi: 10.1016/s0092-8674(85)80025-8. [DOI] [PubMed] [Google Scholar]
  5. Buratowski S., Hahn S., Guarente L., Sharp P. A. Five intermediate complexes in transcription initiation by RNA polymerase II. Cell. 1989 Feb 24;56(4):549–561. doi: 10.1016/0092-8674(89)90578-3. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Demarchi J. M. Human cytomegalovirus DNA: restriction enzyme cleavage maps and map locations for immediate-early, early, and late RNAs. Virology. 1981 Oct 15;114(1):23–38. doi: 10.1016/0042-6822(81)90249-x. [DOI] [PubMed] [Google Scholar]
  8. DiDonato J. A., Muller M. T. DNA binding and gene regulation by the herpes simplex virus type 1 protein ICP4 and involvement of the TATA element. J Virol. 1989 Sep;63(9):3737–3747. doi: 10.1128/jvi.63.9.3737-3747.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dostatni N., Lambert P. F., Sousa R., Ham J., Howley P. M., Yaniv M. The functional BPV-1 E2 trans-activating protein can act as a repressor by preventing formation of the initiation complex. Genes Dev. 1991 Sep;5(9):1657–1671. doi: 10.1101/gad.5.9.1657. [DOI] [PubMed] [Google Scholar]
  10. Everett R. D. The regulation of transcription of viral and cellular genes by herpesvirus immediate-early gene products (review). Anticancer Res. 1987 Jul-Aug;7(4A):589–604. [PubMed] [Google Scholar]
  11. Felser J. M., Kinchington P. R., Inchauspe G., Straus S. E., Ostrove J. M. Cell lines containing varicella-zoster virus open reading frame 62 and expressing the "IE" 175 protein complement ICP4 mutants of herpes simplex virus type 1. J Virol. 1988 Jun;62(6):2076–2082. doi: 10.1128/jvi.62.6.2076-2082.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ghazal P., Young J., Giulietti E., DeMattei C., Garcia J., Gaynor R., Stenberg R. M., Nelson J. A. A discrete cis element in the human immunodeficiency virus long terminal repeat mediates synergistic trans activation by cytomegalovirus immediate-early proteins. J Virol. 1991 Dec;65(12):6735–6742. doi: 10.1128/jvi.65.12.6735-6742.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Hochuli E., Döbeli H., Schacher A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr. 1987 Dec 18;411:177–184. doi: 10.1016/s0021-9673(00)93969-4. [DOI] [PubMed] [Google Scholar]
  17. Hoffmann A., Roeder R. G. Purification of his-tagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Res. 1991 Nov 25;19(22):6337–6338. doi: 10.1093/nar/19.22.6337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ihara S., Feldman L., Watanabe S., Ben-Porat T. Characterization of the immediate-early functions of pseudorabies virus. Virology. 1983 Dec;131(2):437–454. doi: 10.1016/0042-6822(83)90510-x. [DOI] [PubMed] [Google Scholar]
  19. Jahn G., Knust E., Schmolla H., Sarre T., Nelson J. A., McDougall J. K., Fleckenstein B. Predominant immediate-early transcripts of human cytomegalovirus AD 169. J Virol. 1984 Feb;49(2):363–370. doi: 10.1128/jvi.49.2.363-370.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Janknecht R., de Martynoff G., Lou J., Hipskind R. A., Nordheim A., Stunnenberg H. G. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8972–8976. doi: 10.1073/pnas.88.20.8972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lang D., Fickenscher H., Stamminger T. Analysis of proteins binding to the proximal promoter region of the human cytomegalovirus IE-1/2 enhancer/promoter reveals both consensus and aberrant recognition sequences for transcription factors Sp1 and CREB. Nucleic Acids Res. 1992 Jul 11;20(13):3287–3295. doi: 10.1093/nar/20.13.3287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Mazeron M. C., Jahn G., Plachter B. Monoclonal antibody E-13 (M-810) to human cytomegalovirus recognizes an epitope encoded by exon 2 of the major immediate early gene. J Gen Virol. 1992 Oct;73(Pt 10):2699–2703. doi: 10.1099/0022-1317-73-10-2699. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Puchtler E., Stamminger T. An inducible promoter mediates abundant expression from the immediate-early 2 gene region of human cytomegalovirus at late times after infection. J Virol. 1991 Nov;65(11):6301–6306. doi: 10.1128/jvi.65.11.6301-6306.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stamminger T., Fickenscher H., Fleckenstein B. Cell type-specific induction of the major immediate early enhancer of human cytomegalovirus by cyclic AMP. J Gen Virol. 1990 Jan;71(Pt 1):105–113. doi: 10.1099/0022-1317-71-1-105. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. Tedder D. G., Pizer L. I. Role for DNA-protein interaction in activation of the herpes simplex virus glycoprotein D gene. J Virol. 1988 Dec;62(12):4661–4672. doi: 10.1128/jvi.62.12.4661-4672.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tenney D. J., Colberg-Poley A. M. Human cytomegalovirus UL36-38 and US3 immediate-early genes: temporally regulated expression of nuclear, cytoplasmic, and polysome-associated transcripts during infection. J Virol. 1991 Dec;65(12):6724–6734. doi: 10.1128/jvi.65.12.6724-6734.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Thomsen D. R., Stenberg R. M., Goins W. F., Stinski M. F. Promoter-regulatory region of the major immediate early gene of human cytomegalovirus. Proc Natl Acad Sci U S A. 1984 Feb;81(3):659–663. doi: 10.1073/pnas.81.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Villarejo M. R., Zabin I. Beta-galactosidase from termination and deletion mutant strains. J Bacteriol. 1974 Oct;120(1):466–474. doi: 10.1128/jb.120.1.466-474.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Walker S., Hagemeier C., Sissons J. G., Sinclair J. H. A 10-base-pair element of the human immunodeficiency virus type 1 long terminal repeat (LTR) is an absolute requirement for transactivation by the human cytomegalovirus 72-kilodalton IE1 protein but can be compensated for by other LTR regions in transactivation by the 80-kilodalton IE2 protein. J Virol. 1992 Mar;66(3):1543–1550. doi: 10.1128/jvi.66.3.1543-1550.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Wilcox K. W., Kohn A., Sklyanskaya E., Roizman B. Herpes simplex virus phosphoproteins. I. Phosphate cycles on and off some viral polypeptides and can alter their affinity for DNA. J Virol. 1980 Jan;33(1):167–182. doi: 10.1128/jvi.33.1.167-182.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wilkinson G. W., Akrigg A., Greenaway P. J. Transcription of the immediate early genes of human cytomegalovirus strain AD169. Virus Res. 1984;1(2):101–106. doi: 10.1016/0168-1702(84)90067-4. [DOI] [PubMed] [Google Scholar]
  48. Wu C. L., Wilcox K. W. The conserved DNA-binding domains encoded by the herpes simplex virus type 1 ICP4, pseudorabies virus IE180, and varicella-zoster virus ORF62 genes recognize similar sites in the corresponding promoters. J Virol. 1991 Mar;65(3):1149–1159. doi: 10.1128/jvi.65.3.1149-1159.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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