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. 1997 Apr;71(4):3307–3311. doi: 10.1128/jvi.71.4.3307-3311.1997

Conditional repression of the E2 transcription unit in E1-E3-deleted adenovirus vectors is correlated with a strong reduction in viral DNA replication and late gene expression in vitro.

K Rittner 1, H Schultz 1, A Pavirani 1, M Mehtali 1
PMCID: PMC191469  PMID: 9060700

Abstract

An E1-E3-deleted recombinant adenovirus vector expressing the hybrid protein TetR-KRAB has been produced. In this virus, AdTG9562, the E2 transcription is regulated by TetR-KRAB and tetO sequences inserted in cis. In the absence of tetracycline, a strong reduction in E2A gene expression, viral DNA replication, and late gene expression was observed in noncomplementing A549 cells, and a reduction in viral growth was seen in the E1-expressing 293 cells. In contrast, there was no repression in the presence of the regulator tetracycline. We propose that regulation by TetR-KRAB is a valuable tool with which to study the effects of viral gene expression in vitro.

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Selected References

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  1. Brough D. E., Droguett G., Horwitz M. S., Klessig D. F. Multiple functions of the adenovirus DNA-binding protein are required for efficient viral DNA synthesis. Virology. 1993 Sep;196(1):269–281. doi: 10.1006/viro.1993.1475. [DOI] [PubMed] [Google Scholar]
  2. Challberg M. D., Kelly T. J. Animal virus DNA replication. Annu Rev Biochem. 1989;58:671–717. doi: 10.1146/annurev.bi.58.070189.003323. [DOI] [PubMed] [Google Scholar]
  3. Chang L. S., Shenk T. The adenovirus DNA-binding protein stimulates the rate of transcription directed by adenovirus and adeno-associated virus promoters. J Virol. 1990 May;64(5):2103–2109. doi: 10.1128/jvi.64.5.2103-2109.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chartier C., Degryse E., Gantzer M., Dieterle A., Pavirani A., Mehtali M. Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J Virol. 1996 Jul;70(7):4805–4810. doi: 10.1128/jvi.70.7.4805-4810.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chroboczek J., Bieber F., Jacrot B. The sequence of the genome of adenovirus type 5 and its comparison with the genome of adenovirus type 2. Virology. 1992 Jan;186(1):280–285. doi: 10.1016/0042-6822(92)90082-z. [DOI] [PubMed] [Google Scholar]
  6. Deuschle U., Meyer W. K., Thiesen H. J. Tetracycline-reversible silencing of eukaryotic promoters. Mol Cell Biol. 1995 Apr;15(4):1907–1914. doi: 10.1128/mcb.15.4.1907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dix I., Leppard K. N. Regulated splicing of adenovirus type 5 E4 transcripts and regulated cytoplasmic accumulation of E4 mRNA. J Virol. 1993 Jun;67(6):3226–3231. doi: 10.1128/jvi.67.6.3226-3231.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Engelhardt J. F., Simon R. H., Yang Y., Zepeda M., Weber-Pendleton S., Doranz B., Grossman M., Wilson J. M. Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: biological efficacy study. Hum Gene Ther. 1993 Dec;4(6):759–769. doi: 10.1089/hum.1993.4.6-759. [DOI] [PubMed] [Google Scholar]
  9. Engelhardt J. F., Ye X., Doranz B., Wilson J. M. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6196–6200. doi: 10.1073/pnas.91.13.6196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ensinger M. J., Ginsberg H. S. Selection and preliminary characterization of temperature-sensitive mutants of type 5 adenovirus. J Virol. 1972 Sep;10(3):328–339. doi: 10.1128/jvi.10.3.328-339.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fang B., Wang H., Gordon G., Bellinger D. A., Read M. S., Brinkhous K. M., Woo S. L., Eisensmith R. C. Lack of persistence of E1- recombinant adenoviral vectors containing a temperature-sensitive E2A mutation in immunocompetent mice and hemophilia B dogs. Gene Ther. 1996 Mar;3(3):217–222. [PubMed] [Google Scholar]
  12. Gorziglia M. I., Kadan M. J., Yei S., Lim J., Lee G. M., Luthra R., Trapnell B. C. Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy. J Virol. 1996 Jun;70(6):4173–4178. doi: 10.1128/jvi.70.6.4173-4178.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  15. Hay R. T., Freeman A., Leith I., Monaghan A., Webster A. Molecular interactions during adenovirus DNA replication. Curr Top Microbiol Immunol. 1995;199(Pt 2):31–48. doi: 10.1007/978-3-642-79499-5_2. [DOI] [PubMed] [Google Scholar]
  16. Helin K., Harlow E. Heterodimerization of the transcription factors E2F-1 and DP-1 is required for binding to the adenovirus E4 (ORF6/7) protein. J Virol. 1994 Aug;68(8):5027–5035. doi: 10.1128/jvi.68.8.5027-5035.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Henry L. J., Xia D., Wilke M. E., Deisenhofer J., Gerard R. D. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli. J Virol. 1994 Aug;68(8):5239–5246. doi: 10.1128/jvi.68.8.5239-5246.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krougliak V., Graham F. L. Development of cell lines capable of complementing E1, E4, and protein IX defective adenovirus type 5 mutants. Hum Gene Ther. 1995 Dec;6(12):1575–1586. doi: 10.1089/hum.1995.6.12-1575. [DOI] [PubMed] [Google Scholar]
  19. Lathe R., Vilotte J. L., Clark A. J. Plasmid and bacteriophage vectors for excision of intact inserts. Gene. 1987;57(2-3):193–201. doi: 10.1016/0378-1119(87)90122-3. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Margolin J. F., Friedman J. R., Meyer W. K., Vissing H., Thiesen H. J., Rauscher F. J., 3rd Krüppel-associated boxes are potent transcriptional repression domains. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4509–4513. doi: 10.1073/pnas.91.10.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neill S. D., Hemstrom C., Virtanen A., Nevins J. R. An adenovirus E4 gene product trans-activates E2 transcription and stimulates stable E2F binding through a direct association with E2F. Proc Natl Acad Sci U S A. 1990 Mar;87(5):2008–2012. doi: 10.1073/pnas.87.5.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nevins J. R., Winkler J. J. Regulation of early adenovirus transcription: a protein product of early region 2 specifically represses region 4 transcription. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1893–1897. doi: 10.1073/pnas.77.4.1893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pengue G., Lania L. Krüppel-associated box-mediated repression of RNA polymerase II promoters is influenced by the arrangement of basal promoter elements. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1015–1020. doi: 10.1073/pnas.93.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reich N. C., Sarnow P., Duprey E., Levine A. J. Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein. Virology. 1983 Jul 30;128(2):480–484. doi: 10.1016/0042-6822(83)90274-x. [DOI] [PubMed] [Google Scholar]
  26. Shockett P., Difilippantonio M., Hellman N., Schatz D. G. A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6522–6526. doi: 10.1073/pnas.92.14.6522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Simon R. H., Engelhardt J. F., Yang Y., Zepeda M., Weber-Pendleton S., Grossman M., Wilson J. M. Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: toxicity study. Hum Gene Ther. 1993 Dec;4(6):771–780. doi: 10.1089/hum.1993.4.6-771. [DOI] [PubMed] [Google Scholar]
  28. Smith C. D., Craft D. W., Shiromoto R. S., Yan P. O. Alternative cell line for virus isolation. J Clin Microbiol. 1986 Aug;24(2):265–268. doi: 10.1128/jcm.24.2.265-268.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Swaminathan S., Thimmapaya B. Regulation of adenovirus E2 transcription unit. Curr Top Microbiol Immunol. 1995;199(Pt 3):177–194. doi: 10.1007/978-3-642-79586-2_9. [DOI] [PubMed] [Google Scholar]
  30. Wang Q., Jia X. C., Finer M. H. A packaging cell line for propagation of recombinant adenovirus vectors containing two lethal gene-region deletions. Gene Ther. 1995 Dec;2(10):775–783. [PubMed] [Google Scholar]
  31. Yang Y., Nunes F. A., Berencsi K., Furth E. E., Gönczöl E., Wilson J. M. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4407–4411. doi: 10.1073/pnas.91.10.4407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yang Y., Nunes F. A., Berencsi K., Gönczöl E., Engelhardt J. F., Wilson J. M. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat Genet. 1994 Jul;7(3):362–369. doi: 10.1038/ng0794-362. [DOI] [PubMed] [Google Scholar]
  33. Yeh P., Dedieu J. F., Orsini C., Vigne E., Denefle P., Perricaudet M. Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit. J Virol. 1996 Jan;70(1):559–565. doi: 10.1128/jvi.70.1.559-565.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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