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. 1984 Jul;4(7):1354–1362. doi: 10.1128/mcb.4.7.1354

Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

D F Klessig, D E Brough, V Cleghon
PMCID: PMC368918  PMID: 6542172

Abstract

The DNA-binding protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central role in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by DBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DBP is toxic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50- to 200-fold, and within 8 h its synthesis from the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells.

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Selected References

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  1. Anderson C. W., Hardy M. M., Dunn J. J., Klessig D. F. Independent, spontaneous mutants of adenovirus type 2-simian virus 40 hybrid Ad2+ND3 that grow efficiently in monkey cells possess indentical mutations in the adenovirus type 2 DNA-binding protein gene. J Virol. 1983 Oct;48(1):31–39. doi: 10.1128/jvi.48.1.31-39.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson C. W. Spontaneous mutants of the adenovirus-simian virus 40 hybrid, Ad2+ND3, that grow efficiently in monkey cells. Virology. 1981 May;111(1):263–269. doi: 10.1016/0042-6822(81)90670-x. [DOI] [PubMed] [Google Scholar]
  3. Anderson K. P., Klessig D. F. Posttranscriptional block to synthesis of a human adenovirus capsid protein in abortively infected monkey cells. J Mol Appl Genet. 1983;2(1):31–43. [PubMed] [Google Scholar]
  4. Anderson K. P., Klessig D. F. Synthesis of human adenovirus early RNA species is similar in productive and abortive infections of monkey and human cells. J Virol. 1982 May;42(2):748–754. doi: 10.1128/jvi.42.2.748-754.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ariga H., Klein H., Levine A. J., Horwitz M. S. A cleavage product of the adenovirus DNA binding protein is active in DNA replication in vitro. Virology. 1980 Feb;101(1):307–310. doi: 10.1016/0042-6822(80)90510-3. [DOI] [PubMed] [Google Scholar]
  6. Babich A., Nevins J. R. The stability of early adenovirus mRNA is controlled by the viral 72 kd DNA-binding protein. Cell. 1981 Nov;26(3 Pt 1):371–379. doi: 10.1016/0092-8674(81)90206-3. [DOI] [PubMed] [Google Scholar]
  7. Berk A. J., Sharp P. A. Structure of the adenovirus 2 early mRNAs. Cell. 1978 Jul;14(3):695–711. doi: 10.1016/0092-8674(78)90252-0. [DOI] [PubMed] [Google Scholar]
  8. Buetti E., Diggelmann H. Cloned mouse mammary tumor virus DNA is biologically active in transfected mouse cells and its expression is stimulated by glucocorticoid hormones. Cell. 1981 Feb;23(2):335–345. doi: 10.1016/0092-8674(81)90129-x. [DOI] [PubMed] [Google Scholar]
  9. Carter T. H., Blanton R. A. Autoregulation of adenovirus type 5 early gene expression II. Effect of temperature-sensitive early mutations on virus RNA accumulation. J Virol. 1978 Nov;28(2):450–456. doi: 10.1128/jvi.28.2.450-456.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carter T. H., Blanton R. A. Possible role of the 72,000 dalton DNA-binding protein in regulation of adenovirus type 5 early gene expression. J Virol. 1978 Feb;25(2):664–674. doi: 10.1128/jvi.25.2.664-674.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chapman A. B., Costello M. A., Lee F., Ringold G. M. Amplification and hormone-regulated expression of a mouse mammary tumor virus-Eco gpt fusion plasmid in mouse 3T6 cells. Mol Cell Biol. 1983 Aug;3(8):1421–1429. doi: 10.1128/mcb.3.8.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chow L. T., Broker T. R., Lewis J. B. Complex splicing patterns of RNAs from the early regions of adenovirus-2. J Mol Biol. 1979 Oct 25;134(2):265–303. doi: 10.1016/0022-2836(79)90036-6. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Farber M. S., Baum S. G. Transcription of adenovirus RNA in permissive and nonpermissive infections. J Virol. 1978 Jul;27(1):136–148. doi: 10.1128/jvi.27.1.136-148.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fraenkel-Conrat H., Singer B. Effect of introduction of small alkyl groups on mRNA function. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1983–1985. doi: 10.1073/pnas.77.4.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Friefeld B. R., Krevolin M. D., Horwitz M. S. Effects of the adenovirus H5ts125 and H5ts107 DNA binding proteins on DNA replication in vitro. Virology. 1983 Jan 30;124(2):380–389. doi: 10.1016/0042-6822(83)90354-9. [DOI] [PubMed] [Google Scholar]
  17. Ginsberg H. S., Ensinger M. J., Kauffman R. S., Mayer A. J., Lundholm U. Cell transformation: a study of regulation with types 5 and 12 adenovirus temperature-sensitive mutants. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):419–426. doi: 10.1101/sqb.1974.039.01.054. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Graham F. L., van der Eb A. J. Transformation of rat cells by DNA of human adenovirus 5. Virology. 1973 Aug;54(2):536–539. doi: 10.1016/0042-6822(73)90163-3. [DOI] [PubMed] [Google Scholar]
  20. Grodzicker T., Anderson C., Sharp P. A., Sambrook J. Conditional lethal mutants of adenovirus 2-simian virus 40 hybrids. I. Host range mutants of Ad2+ND1. J Virol. 1974 Jun;13(6):1237–1244. doi: 10.1128/jvi.13.6.1237-1244.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Horwitz M. S. Temperature-sensitive replication of H5ts125 adenovirus DNA in vitro. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4291–4295. doi: 10.1073/pnas.75.9.4291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huang A. L., Ostrowski M. C., Berard D., Hager G. L. Glucocorticoid regulation of the Ha-MuSV p21 gene conferred by sequences from mouse mammary tumor virus. Cell. 1981 Dec;27(2 Pt 1):245–255. doi: 10.1016/0092-8674(81)90408-6. [DOI] [PubMed] [Google Scholar]
  23. Hynes N. E., Kennedy N., Rahmsdorf U., Groner B. Hormone-responsive expression of an endogenous proviral gene of mouse mammary tumor virus after molecular cloning and gene transfer into cultured cells. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2038–2042. doi: 10.1073/pnas.78.4.2038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Khoury G., May E. Regulation of early and late simian virus 40 transcription: overproduction of early viral RNA in the absence of a functional T-antigen. J Virol. 1977 Jul;23(1):167–176. doi: 10.1128/jvi.23.1.167-176.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kitchingman G. R., Lai S. P., Westphal H. Loop structures in hybrids of early RNA and the separated strands of adenovirus DNA. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4392–4395. doi: 10.1073/pnas.74.10.4392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klein H., Maltzman W., Levine A. J. Structure-function relationships of the adenovirus DNA-binding protein. J Biol Chem. 1979 Nov 10;254(21):11051–11060. [PubMed] [Google Scholar]
  27. Klessig D. F., Anderson C. W. Block to multiplication of adenovirus serotype 2 in monkey cells. J Virol. 1975 Dec;16(6):1650–1668. doi: 10.1128/jvi.16.6.1650-1668.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Klessig D. F., Chow L. T. Incomplete splicing and deficient accumulation of the fiber messenger RNA in monkey cells infected by human adenovirus type 2. J Mol Biol. 1980 May 15;139(2):221–242. doi: 10.1016/0022-2836(80)90306-x. [DOI] [PubMed] [Google Scholar]
  29. Klessig D. F., Grodzicker T. Mutations that allow human Ad2 and Ad5 to express late genes in monkey cells map in the viral gene encoding the 72K DNA binding protein. Cell. 1979 Aug;17(4):957–966. doi: 10.1016/0092-8674(79)90335-0. [DOI] [PubMed] [Google Scholar]
  30. Klessig D. F. Isolation of a variant of human adenovirus serotype 2 that multiplies efficiently on monkey cells. J Virol. 1977 Mar;21(3):1243–1246. doi: 10.1128/jvi.21.3.1243-1246.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Klessig D. F., Quinlan M. P. Genetic evidence for separate functional domains on the human adenovirus specified, 72 kd, DNA binding protein. J Mol Appl Genet. 1982;1(4):263–272. [PubMed] [Google Scholar]
  32. Kruijer W., Van Schaik F. M., Sussenbach J. S. Nucleotide sequence of the gene encoding adenovirus type 2 DNA binding protein. Nucleic Acids Res. 1982 Aug 11;10(15):4493–4500. doi: 10.1093/nar/10.15.4493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kruijer W., van Schaik F. M., Sussenbach J. S. Structure and organization of the gene coding for the DNA binding protein of adenovirus type 5. Nucleic Acids Res. 1981 Sep 25;9(18):4439–4457. doi: 10.1093/nar/9.18.4439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lee F., Mulligan R., Berg P., Ringold G. Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids. Nature. 1981 Nov 19;294(5838):228–232. doi: 10.1038/294228a0. [DOI] [PubMed] [Google Scholar]
  35. Majors J., Varmus H. E. A small region of the mouse mammary tumor virus long terminal repeat confers glucocorticoid hormone regulation on a linked heterologous gene. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5866–5870. doi: 10.1073/pnas.80.19.5866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mayo K. E., Palmiter R. D. Glucocorticoid regulation of metallothionein-I mRNA synthesis in cultured mouse cells. J Biol Chem. 1981 Mar 25;256(6):2621–2624. [PubMed] [Google Scholar]
  37. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nicolas J. C., Ingrand D., Sarnow P., Levine A. J. A mutation in the adenovirus type 5 DNA binding protein that fails to autoregulate the production of the DNA binding protein. Virology. 1982 Oct 30;122(2):481–485. doi: 10.1016/0042-6822(82)90249-5. [DOI] [PubMed] [Google Scholar]
  39. Nicolas J. C., Sarnow P., Girard M., Levine A. J. Host range temperature-conditional mutants in the adenovirus DNA binding protein are defective in the assembly of infectious virus. Virology. 1983 Apr 15;126(1):228–239. doi: 10.1016/0042-6822(83)90474-9. [DOI] [PubMed] [Google Scholar]
  40. PISCATOR M. OM KADMIUM I NORMALA MAENNISKONJURAR SAMT REDOGOERELSE FOER ISOLERING AV METALLOTHIONEIN UR LEVER FRAN KADMIUMEXPONERADE KANINER. Nord Hyg Tidskr. 1964;45:76–82. [PubMed] [Google Scholar]
  41. Parks W. P., Scolnick E. M., Kozikowski E. H. Dexamethasone stimulation of murine mammary tumor virus expression: a tissue culture source of virus. Science. 1974 Apr 12;184(4133):158–160. doi: 10.1126/science.184.4133.158. [DOI] [PubMed] [Google Scholar]
  42. Reed S. I., Stark G. R., Alwine J. C. Autoregulation of simian virus 40 gene A by T antigen. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3083–3087. doi: 10.1073/pnas.73.9.3083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Reich P. R., Baum S. G., Rose J. A., Rowe W. P., Weissman S. M. Nucleic acid homology studies of adenovirus type 7-SV40 interactions. Proc Natl Acad Sci U S A. 1966 Feb;55(2):336–341. doi: 10.1073/pnas.55.2.336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rice S. A., Klessig D. F. The function(s) provided by the adenovirus-specified, DNA-binding protein required for viral late gene expression is independent of the role of the protein in viral DNA replication. J Virol. 1984 Jan;49(1):35–49. doi: 10.1128/jvi.49.1.35-49.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ringold G. M., Shank P. R., Varmus H. E., Ring J., Yamamoto K. R. Integration and transcription of mouse mammary tumor virus DNA in rat hepatoma cells. Proc Natl Acad Sci U S A. 1979 Feb;76(2):665–669. doi: 10.1073/pnas.76.2.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ringold G. M., Yamamoto K. R., Tomkins G. M., Bishop M., Varmus H. E. Dexamethasone-mediated induction of mouse mammary tumor virus RNA: a system for studying glucocorticoid action. Cell. 1975 Nov;6(3):299–305. doi: 10.1016/0092-8674(75)90181-6. [DOI] [PubMed] [Google Scholar]
  47. Rubenstein F. E., Ginsberg H. S. Transformation characteristics of temperature-sensitive mutants of type 12 adenovirus. Intervirology. 1974;3(3):170–174. doi: 10.1159/000149753. [DOI] [PubMed] [Google Scholar]
  48. Shapiro S. G., Squibb K. S., Markowitz L. A., Cousins R. J. Cell-free synthesis of metallothionein directed by rat liver polyadenylated messenger ribonucleic acid. Biochem J. 1978 Dec 1;175(3):833–840. doi: 10.1042/bj1750833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  50. Tegtmeyer P., Schwartz M., Collins J. K., Rundell K. Regulation of tumor antigen synthesis by simain virus 40 gene A. J Virol. 1975 Jul;16(1):168–178. doi: 10.1128/jvi.16.1.168-178.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Thomas G. P., Mathews M. B. DNA replication and the early to late transition in adenovirus infection. Cell. 1980 Nov;22(2 Pt 2):523–533. doi: 10.1016/0092-8674(80)90362-1. [DOI] [PubMed] [Google Scholar]
  52. Van Der Vliet P. C., Levine A. J., Ensinger M. J., Ginsberg H. S. Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovrius defective in viral DNA synthesis. J Virol. 1975 Feb;15(2):348–354. doi: 10.1128/jvi.15.2.348-354.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Vliet P. C., Sussenbach J. S. An adenovirus type 5 gene function required for initiation of viral DNA replication. Virology. 1975 Oct;67(2):415–426. doi: 10.1016/0042-6822(75)90443-2. [DOI] [PubMed] [Google Scholar]
  54. Willians J. F., Young C. S., Austin P. E. Genetic analysis of human adenovirus type 5 in permissive and nonpermissive cells. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):427–437. doi: 10.1101/sqb.1974.039.01.055. [DOI] [PubMed] [Google Scholar]

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