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. 1995 Apr;69(4):2565–2573. doi: 10.1128/jvi.69.4.2565-2573.1995

Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus.

H J Kim 1, C Gatz 1, W Hillen 1, T R Jones 1
PMCID: PMC188934  PMID: 7884907

Abstract

The tetracycline repressor (TetR)-regulated gene expression system from Escherichia coli was used to control gene expression in recombinant human cytomegalovirus (HCMV). To adapt the TetR system in HCMV, derivatives of the viral US11 (early) gene promoter, which controls the beta-glucuronidase reporter gene, were constructed by systematic insertion of the tetracycline operator (tetO) sequences. Gene expression from constructs containing two or three appropriately placed tetO sequences adjacent to the TATA box were efficiently repressed by a TetR-VP16 fusion protein (tTA) in a transient expression system. Efficient repression (50- to 120-fold) also occurred in tTA-expressing stably transfected human cells which were infected with recombinant HCMV containing a US11 promoter surrounded by three tetO sequences. The tTA-mediated gene repression was relieved in the presence of 1 microgram of tetracycline per ml. The results of this study are significant in three respects. (i) This is the first demonstration that a TetR-derived protein can be used to efficiently repress gene expression in a mammalian system. (ii) Efficient repression was dependent on the presence of the transcriptional activation domain from the herpes simplex virus type 1 VP16 protein. (iii) The ability to regulate gene expression in a controlled fashion in order to elucidate viral gene function is an important development in the HCMV field. The tTA-mediated gene repression system may be extremely useful for creating host-range mutants in essential genes in order to study their role in the HCMV replicative cycle, a system that is otherwise exceedingly difficult to genetically dissect.

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Selected References

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

  1. Baim S. B., Labow M. A., Levine A. J., Shenk T. A chimeric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropyl beta-D-thiogalactopyranoside. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5072–5076. doi: 10.1073/pnas.88.12.5072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bertrand K. P., Postle K., Wray L. V., Jr, Reznikoff W. S. Overlapping divergent promoters control expression of Tn10 tetracycline resistance. Gene. 1983 Aug;23(2):149–156. doi: 10.1016/0378-1119(83)90046-x. [DOI] [PubMed] [Google Scholar]
  3. Brown M., Figge J., Hansen U., Wright C., Jeang K. T., Khoury G., Livingston D. M., Roberts T. M. lac repressor can regulate expression from a hybrid SV40 early promoter containing a lac operator in animal cells. Cell. 1987 Jun 5;49(5):603–612. doi: 10.1016/0092-8674(87)90536-8. [DOI] [PubMed] [Google Scholar]
  4. Browne H., Churcher M., Minson T. Construction and characterization of a human cytomegalovirus mutant with the UL18 (class I homolog) gene deleted. J Virol. 1992 Nov;66(11):6784–6787. doi: 10.1128/jvi.66.11.6784-6787.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Deuschle U., Pepperkok R., Wang F. B., Giordano T. J., McAllister W. T., Ansorge W., Bujard H. Regulated expression of foreign genes in mammalian cells under the control of coliphage T3 RNA polymerase and lac repressor. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5400–5404. doi: 10.1073/pnas.86.14.5400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dingermann T., Frank-Stoll U., Werner H., Wissmann A., Hillen W., Jacquet M., Marschalek R. RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system. EMBO J. 1992 Apr;11(4):1487–1492. doi: 10.1002/j.1460-2075.1992.tb05193.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dingermann T., Werner H., Schütz A., Zündorf I., Nerke K., Knecht D., Marschalek R. Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene. Mol Cell Biol. 1992 Sep;12(9):4038–4045. doi: 10.1128/mcb.12.9.4038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Figge J., Wright C., Collins C. J., Roberts T. M., Livingston D. M. Stringent regulation of stably integrated chloramphenicol acetyl transferase genes by E. coli lac repressor in monkey cells. Cell. 1988 Mar 11;52(5):713–722. doi: 10.1016/0092-8674(88)90409-6. [DOI] [PubMed] [Google Scholar]
  10. Frohberg C., Heins L., Gatz C. Characterization of the interaction of plant transcription factors using a bacterial repressor protein. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10470–10474. doi: 10.1073/pnas.88.23.10470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gatz C., Frohberg C., Wendenburg R. Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants. Plant J. 1992 May;2(3):397–404. doi: 10.1111/j.1365-313x.1992.00397.x. [DOI] [PubMed] [Google Scholar]
  12. Gatz C., Kaiser A., Wendenburg R. Regulation of a modified CaMV 35S promoter by the Tn10-encoded Tet repressor in transgenic tobacco. Mol Gen Genet. 1991 Jun;227(2):229–237. doi: 10.1007/BF00259675. [DOI] [PubMed] [Google Scholar]
  13. Gatz C., Quail P. H. Tn10-encoded tet repressor can regulate an operator-containing plant promoter. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1394–1397. doi: 10.1073/pnas.85.5.1394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ge H., Roeder R. G. Purification, cloning, and characterization of a human coactivator, PC4, that mediates transcriptional activation of class II genes. Cell. 1994 Aug 12;78(3):513–523. doi: 10.1016/0092-8674(94)90428-6. [DOI] [PubMed] [Google Scholar]
  15. Goodrich J. A., Hoey T., Thut C. J., Admon A., Tjian R. Drosophila TAFII40 interacts with both a VP16 activation domain and the basal transcription factor TFIIB. Cell. 1993 Nov 5;75(3):519–530. doi: 10.1016/0092-8674(93)90386-5. [DOI] [PubMed] [Google Scholar]
  16. Gossen M., Bonin A. L., Bujard H. Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem Sci. 1993 Dec;18(12):471–475. doi: 10.1016/0968-0004(93)90009-c. [DOI] [PubMed] [Google Scholar]
  17. Gossen M., Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547–5551. doi: 10.1073/pnas.89.12.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hillen W., Klock G., Kaffenberger I., Wray L. V., Reznikoff W. S. Purification of the TET repressor and TET operator from the transposon Tn10 and characterization of their interaction. J Biol Chem. 1982 Jun 10;257(11):6605–6613. [PubMed] [Google Scholar]
  19. Hillen W., Schollmeier K., Gatz C. Control of expression of the Tn10-encoded tetracycline resistance operon. II. Interaction of RNA polymerase and TET repressor with the tet operon regulatory region. J Mol Biol. 1984 Jan 15;172(2):185–201. doi: 10.1016/s0022-2836(84)80037-6. [DOI] [PubMed] [Google Scholar]
  20. Hu M. C., Davidson N. The inducible lac operator-repressor system is functional in mammalian cells. Cell. 1987 Feb 27;48(4):555–566. doi: 10.1016/0092-8674(87)90234-0. [DOI] [PubMed] [Google Scholar]
  21. Ingles C. J., Shales M., Cress W. D., Triezenberg S. J., Greenblatt J. Reduced binding of TFIID to transcriptionally compromised mutants of VP16. Nature. 1991 Jun 13;351(6327):588–590. doi: 10.1038/351588a0. [DOI] [PubMed] [Google Scholar]
  22. Jones T. R., Muzithras V. P. Fine mapping of transcripts expressed from the US6 gene family of human cytomegalovirus strain AD169. J Virol. 1991 Apr;65(4):2024–2036. doi: 10.1128/jvi.65.4.2024-2036.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jones T. R., Muzithras V. P., Gluzman Y. Replacement mutagenesis of the human cytomegalovirus genome: US10 and US11 gene products are nonessential. J Virol. 1991 Nov;65(11):5860–5872. doi: 10.1128/jvi.65.11.5860-5872.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jones T. R., Sun L., Bebernitz G. A., Muzithras V. P., Kim H. J., Johnston S. H., Baum E. Z. Proteolytic activity of human cytomegalovirus UL80 protease cleavage site mutants. J Virol. 1994 Jun;68(6):3742–3752. doi: 10.1128/jvi.68.6.3742-3752.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jorgensen R. A., Reznikoff W. S. Organization of structural and regulatory genes that mediate tetracycline resistance in transposon Tn10. J Bacteriol. 1979 Jun;138(3):705–714. doi: 10.1128/jb.138.3.705-714.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
  27. Kaye J., Browne H., Stoffel M., Minson T. The UL16 gene of human cytomegalovirus encodes a glycoprotein that is dispensable for growth in vitro. J Virol. 1992 Nov;66(11):6609–6615. doi: 10.1128/jvi.66.11.6609-6615.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kim T. K., Hashimoto S., Kelleher R. J., 3rd, Flanagan P. M., Kornberg R. D., Horikoshi M., Roeder R. G. Effects of activation-defective TBP mutations on transcription initiation in yeast. Nature. 1994 May 19;369(6477):252–255. doi: 10.1038/369252a0. [DOI] [PubMed] [Google Scholar]
  29. Kleinschmidt C., Tovar K., Hillen W., Porschke D. Dynamics of repressor-operator recognition: the Tn10-encoded tetracycline resistance control. Biochemistry. 1988 Feb 23;27(4):1094–1104. doi: 10.1021/bi00404a003. [DOI] [PubMed] [Google Scholar]
  30. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Labow M. A., Baim S. B., Shenk T., Levine A. J. Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells. Mol Cell Biol. 1990 Jul;10(7):3343–3356. doi: 10.1128/mcb.10.7.3343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Oehmichen R., Klock G., Altschmied L., Hillen W. Construction of an E. coli strain overproducing the Tn10-encoded TET repressor and its use for large scale purification. EMBO J. 1984 Mar;3(3):539–543. doi: 10.1002/j.1460-2075.1984.tb01843.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rodriguez J. F., Smith G. L. IPTG-dependent vaccinia virus: identification of a virus protein enabling virion envelopment by Golgi membrane and egress. Nucleic Acids Res. 1990 Sep 25;18(18):5347–5351. doi: 10.1093/nar/18.18.5347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rodriguez J. F., Smith G. L. Inducible gene expression from vaccinia virus vectors. Virology. 1990 Jul;177(1):239–250. doi: 10.1016/0042-6822(90)90477-9. [DOI] [PubMed] [Google Scholar]
  36. Roeder R. G. The complexities of eukaryotic transcription initiation: regulation of preinitiation complex assembly. Trends Biochem Sci. 1991 Nov;16(11):402–408. doi: 10.1016/0968-0004(91)90164-q. [DOI] [PubMed] [Google Scholar]
  37. Röder F. T., Schmülling T., Gatz C. Efficiency of the tetracycline-dependent gene expression system: complete suppression and efficient induction of the rolB phenotype in transgenic plants. Mol Gen Genet. 1994 Apr;243(1):32–38. doi: 10.1007/BF00283873. [DOI] [PubMed] [Google Scholar]
  38. Sadowski I., Ma J., Triezenberg S., Ptashne M. GAL4-VP16 is an unusually potent transcriptional activator. Nature. 1988 Oct 6;335(6190):563–564. doi: 10.1038/335563a0. [DOI] [PubMed] [Google Scholar]
  39. Spaete R. R., Mocarski E. S. Insertion and deletion mutagenesis of the human cytomegalovirus genome. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7213–7217. doi: 10.1073/pnas.84.20.7213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Takahashi M., Altschmied L., Hillen W. Kinetic and equilibrium characterization of the Tet repressor-tetracycline complex by fluorescence measurements. Evidence for divalent metal ion requirement and energy transfer. J Mol Biol. 1986 Feb 5;187(3):341–348. doi: 10.1016/0022-2836(86)90437-7. [DOI] [PubMed] [Google Scholar]
  42. Takekoshi M., Maeda-Takekoshi F., Ihara S., Sakuma S., Watanabe Y. Inducible expression of a foreign gene inserted into the human cytomegalovirus genome. J Gen Virol. 1993 Aug;74(Pt 8):1649–1652. doi: 10.1099/0022-1317-74-8-1649. [DOI] [PubMed] [Google Scholar]
  43. Takekoshi M., Maeda-Takekoshi F., Ihara S., Sakuma S., Watanabe Y. Site-specific stable insertion into the human cytomegalovirus genome of a foreign gene under control of the SV40 promoter. Gene. 1991 May 30;101(2):209–213. doi: 10.1016/0378-1119(91)90413-6. [DOI] [PubMed] [Google Scholar]
  44. Triezenberg S. J., Kingsbury R. C., McKnight S. L. Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev. 1988 Jun;2(6):718–729. doi: 10.1101/gad.2.6.718. [DOI] [PubMed] [Google Scholar]
  45. Weinmann P., Gossen M., Hillen W., Bujard H., Gatz C. A chimeric transactivator allows tetracycline-responsive gene expression in whole plants. Plant J. 1994 Apr;5(4):559–569. doi: 10.1046/j.1365-313x.1994.5040559.x. [DOI] [PubMed] [Google Scholar]
  46. Zawel L., Reinberg D. Advances in RNA polymerase II transcription. Curr Opin Cell Biol. 1992 Jun;4(3):488–495. doi: 10.1016/0955-0674(92)90016-6. [DOI] [PubMed] [Google Scholar]
  47. Zhang Y. F., Moss B. Inducer-dependent conditional-lethal mutant animal viruses. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1511–1515. doi: 10.1073/pnas.88.4.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]

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