Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Jan 15;90(2):707–711. doi: 10.1073/pnas.90.2.707

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.

M P Macias 1, M F Stinski 1
PMCID: PMC45734  PMID: 8380646

Abstract

In vivo, negative autoregulation of the strong major immediate early promoter (MIEP) of human cytomegalovirus requires the viral immediate early 2 protein (IE2) and a cis element located from position -13 through position -1 relative to the transcription start site. We have established an in vitro transcription system that reproduces the specificity of IE2-mediated negative autoregulation. The carboxyl-terminal 290-amino acid fragment of IE2 was purified as a bacterial fusion protein. Addition of this chimeric protein to the cell-free system specifically repressed transcription from the MIEP containing the wild-type cis-acting repressor element but not from a mutated template in which the cis element had been replaced by heterologous DNA. Control protein and a mutant IE2 fusion protein containing two specific amino acid substitutions in a putative zinc finger motif did not repress the MIEP in vitro. Using conditions defined by this functional assay, we demonstrated by mobility-shift experiments that IE2 binds directly and specifically to DNA bearing the cis-acting repressor element. In addition, IE2 bound to the MIEP in the in vitro transcription reaction mixture.

Full text

PDF
707

Images in this article

708Image
on p.708
708Image
on p.708
709Image
on p.709
709Image
on p.709
710Image
on p.710

Selected References

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

  1. 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]
  2. Ghazal P., Lubon H., Fleckenstein B., Hennighausen L. Binding of transcription factors and creation of a large nucleoprotein complex on the human cytomegalovirus enhancer. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3658–3662. doi: 10.1073/pnas.84.11.3658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. Keleher C. A., Goutte C., Johnson A. D. The yeast cell-type-specific repressor alpha 2 acts cooperatively with a non-cell-type-specific protein. Cell. 1988 Jun 17;53(6):927–936. doi: 10.1016/s0092-8674(88)90449-7. [DOI] [PubMed] [Google Scholar]
  7. Lee K. A., Green M. R. Small-scale preparation of extracts from radiolabeled cells efficient in pre-mRNA splicing. Methods Enzymol. 1990;181:20–30. doi: 10.1016/0076-6879(90)81108-7. [DOI] [PubMed] [Google Scholar]
  8. Li Y. C., Ross J., Scheppler J. A., Franza B. R., Jr An in vitro transcription analysis of early responses of the human immunodeficiency virus type 1 long terminal repeat to different transcriptional activators. Mol Cell Biol. 1991 Apr;11(4):1883–1893. doi: 10.1128/mcb.11.4.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Ohkuma Y., Horikoshi M., Roeder R. G., Desplan C. Binding site-dependent direct activation and repression of in vitro transcription by Drosophila homeodomain proteins. Cell. 1990 May 4;61(3):475–484. doi: 10.1016/0092-8674(90)90529-n. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. Rio D., Robbins A., Myers R., Tjian R. Regulation of simian virus 40 early transcription in vitro by a purified tumor antigen. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5706–5710. doi: 10.1073/pnas.77.10.5706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. Stinski M. F., Roehr T. J. Activation of the major immediate early gene of human cytomegalovirus by cis-acting elements in the promoter-regulatory sequence and by virus-specific trans-acting components. J Virol. 1985 Aug;55(2):431–441. doi: 10.1128/jvi.55.2.431-441.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES