Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1982 Feb;41(2):674–685. doi: 10.1128/jvi.41.2.674-685.1982

Integration and expression of viral DNA in cells transformed by host range mutants of adenovirus type 5.

M Ruben, S Bacchetti, F L Graham
PMCID: PMC256796  PMID: 7077750

Abstract

Group I host range (hr) mutants of adenovirus type 5 are unable to transform rat embryo or rat embryo brain cells but induce an abnormal transformation of baby rat kidney cells. We established several transformed rat kidney cell lines and characterized them with respect to the transformed phenotype and the structure of the integrated viral DNA. The hr mutant-transformed cells, unlike wild-type virus transformants, were fibroblastic rather than epithelial, failed to grow in soft agar, and were also less tumorigenic in nude mice. Studies on the structure of the integrated viral DNA sequences showed that hr-transformed cells always contained the left end of the adenovirus DNA, but the size of the integrated DNA fragment varied among different lines, and a high percentage of the lines contained the entire viral genome colinearly integrated. The patterns of integration were maintained after prolonged growth in culture and after subcloning. Attempts to rescue infectious virus from lines which contained the entire genome were unsuccessful. Using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we analyzed the viral proteins expressed in hr-transformed cells. Results of these studies indicated that, like wild type-transformed cells, hr transformants expressed E1B proteins of molecular weight 58,000 and 19,000.

Full text

PDF
674

Images in this article

677Image
on p.677
678Image
on p.678
679Image
on p.679
680Image
on p.680
682Image
on p.682

Selected References

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

  1. Berk A. J., Lee F., Harrison T., Williams J., Sharp P. A. Phenotypes of adenovirus-5 host-range mutants for early-mRNA synthesis. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):429–436. doi: 10.1101/sqb.1980.044.01.046. [DOI] [PubMed] [Google Scholar]
  2. Berk A. J., Lee F., Harrison T., Williams J., Sharp P. A. Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell. 1979 Aug;17(4):935–944. doi: 10.1016/0092-8674(79)90333-7. [DOI] [PubMed] [Google Scholar]
  3. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  4. Botchan M., Topp W., Sambrook J. The arrangement of simian virus 40 sequences in the DNA of transformed cells. Cell. 1976 Oct;9(2):269–287. doi: 10.1016/0092-8674(76)90118-5. [DOI] [PubMed] [Google Scholar]
  5. Boyd V. A., Butel J. S. Demonstration of infectious deoxyribonucleic acid in transformed cells. I. Recovery of simian virus 40 from yielder and nonyielder transformed cells. J Virol. 1972 Sep;10(3):399–409. doi: 10.1128/jvi.10.3.399-409.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chow L. T., Roberts J. M., Lewis J. B., Broker T. R. A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA:DNA hybrids. Cell. 1977 Aug;11(4):819–836. doi: 10.1016/0092-8674(77)90294-x. [DOI] [PubMed] [Google Scholar]
  7. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  8. Dorsch-Häsler K., Fisher P. B., Weinstein I. B., Ginsberg H. S. Patterns of viral DNA integration in cells transformed by wild type or DNA-binding protein mutants of adenovirus type 5 and effect of chemical carcinogens on integration. J Virol. 1980 May;34(2):305–314. doi: 10.1128/jvi.34.2.305-314.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frost E., Williams J. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology. 1978 Nov;91(1):39–50. doi: 10.1016/0042-6822(78)90353-7. [DOI] [PubMed] [Google Scholar]
  10. Gallimore P. H. Viral DNA in transformed cells. II. A study of the sequences of adenovirus 2 DNA IN NINE LINES OF TRANSFORMED RAT CELLS USING SPECIFIC FRAGMENTS OF THE VIRAL GENOME;. J Mol Biol. 1974 Oct 15;89(1):49–72. doi: 10.1016/0022-2836(74)90162-4. [DOI] [PubMed] [Google Scholar]
  11. Galos R. S., Williams J., Binger M. H., Flint S. J. Location of additional early gene sequences in the adenoviral chromosome. Cell. 1979 Aug;17(4):945–956. doi: 10.1016/0092-8674(79)90334-9. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Graham F. L., Abrahams P. J., Mulder C., Heijneker H. L., Warnaar S. O., De Vries F. A., Fiers W., Van Der Eb A. J. Studies on in vitro transformation by DNA and DNA fragments of human adenoviruses and simian virus 40. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):637–650. doi: 10.1101/sqb.1974.039.01.077. [DOI] [PubMed] [Google Scholar]
  14. Graham F. L., Harrison T., Williams J. Defective transforming capacity of adenovirus type 5 host-range mutants. Virology. 1978 May 1;86(1):10–21. doi: 10.1016/0042-6822(78)90003-x. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Grodzicker T., Anderson C., Sambrook J., Mathews M. B. The physical locations of structural genes in adenovirus DNA. Virology. 1977 Jul 1;80(1):111–126. doi: 10.1016/0042-6822(77)90384-1. [DOI] [PubMed] [Google Scholar]
  18. Harrison T., Graham F., Williams J. Host-range mutants of adenovirus type 5 defective for growth in HeLa cells. Virology. 1977 Mar;77(1):319–329. doi: 10.1016/0042-6822(77)90428-7. [DOI] [PubMed] [Google Scholar]
  19. Ibelgaufts H., Doerfler W., Scheidtmann K. H., Wechsler W. Adenovirus type 12-induced rat tumor cells of neuroepithelial origin: persistence and expression of the viral genome. J Virol. 1980 Jan;33(1):423–437. doi: 10.1128/jvi.33.1.423-437.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jones N., Shenk T. An adenovirus type 5 early gene function regulates expression of other early viral genes. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3665–3669. doi: 10.1073/pnas.76.8.3665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones N., Shenk T. Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells. Cell. 1979 Jul;17(3):683–689. doi: 10.1016/0092-8674(79)90275-7. [DOI] [PubMed] [Google Scholar]
  22. Ketner G., Kelly T. J., Jr Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1102–1106. doi: 10.1073/pnas.73.4.1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lassam N. J., Bayley S. T., Graham F. L. Transforming proteins of human adenovirus 5: studies with infected and transformed cells. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):477–491. doi: 10.1101/sqb.1980.044.01.051. [DOI] [PubMed] [Google Scholar]
  24. Lassam N. J., Bayley S. T., Graham F. L. Tumor antigens of human Ad5 in transformed cells and in cells infected with transformation-defective host-range mutants. Cell. 1979 Nov;18(3):781–791. doi: 10.1016/0092-8674(79)90131-4. [DOI] [PubMed] [Google Scholar]
  25. Levinson A. D., Levine A. J. The group C adenovirus tumor antigens: identification in infected and transformed cells and a peptide map analysis. Cell. 1977 Aug;11(4):871–879. doi: 10.1016/0092-8674(77)90298-7. [DOI] [PubMed] [Google Scholar]
  26. Lewis J. B., Mathews M. B. Control of adenovirus early gene expression: a class of immediate early products. Cell. 1980 Aug;21(1):303–313. doi: 10.1016/0092-8674(80)90138-5. [DOI] [PubMed] [Google Scholar]
  27. MACPHERSON I., MONTAGNIER L. AGAR SUSPENSION CULTURE FOR THE SELECTIVE ASSAY OF CELLS TRANSFORMED BY POLYOMA VIRUS. Virology. 1964 Jun;23:291–294. doi: 10.1016/0042-6822(64)90301-0. [DOI] [PubMed] [Google Scholar]
  28. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mayer A. J., Ginsberg H. S. Persistence of type 5 adenovirus DNA in cells transformed by temperature-sensitive mutant, H5ts125. Proc Natl Acad Sci U S A. 1977 Feb;74(2):785–788. doi: 10.1073/pnas.74.2.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pantelouris E. M. Absence of thymus in a mouse mutant. Nature. 1968 Jan 27;217(5126):370–371. doi: 10.1038/217370a0. [DOI] [PubMed] [Google Scholar]
  31. Pontecorvo G., Riddle P. N., Hales A. Time and mode of fusion of human fibroblasts treated with polyethylene glycol (PEG). Nature. 1977 Jan 20;265(5591):257–258. doi: 10.1038/265257a0. [DOI] [PubMed] [Google Scholar]
  32. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  33. Ross S. R., Flint S. J., Levine A. J. Identification of the adenovirus early proteins and their genomic map positions. Virology. 1980 Jan 30;100(2):419–432. doi: 10.1016/0042-6822(80)90533-4. [DOI] [PubMed] [Google Scholar]
  34. Russell W. C., Hayashi K., Sanderson P. J., Pereira H. G. Adenovirus antigens--a study of their properties and sequential development in infection. J Gen Virol. 1967 Oct;1(4):495–507. doi: 10.1099/0022-1317-1-4-495. [DOI] [PubMed] [Google Scholar]
  35. Saborio J. L., Oberg B. In vivo and in vitro synthesis of adenovirus type 2 early proteins. J Virol. 1976 Mar;17(3):865–875. doi: 10.1128/jvi.17.3.865-875.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sambrook J., Botchan M., Gallimore P., Ozanne B., Pettersson U., Williams J., Sharp P. A. Viral DNA sequences in cells transformed by simian virus 40, adenovirus type 2 and adenovirus type 5. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):615–632. doi: 10.1101/sqb.1974.039.01.075. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Stabel S., Doerfler W., Friis R. R. Integration sites of adenovirus type 12 DNA in transformed hamster cells and hamster tumor cells. J Virol. 1980 Oct;36(1):22–40. doi: 10.1128/jvi.36.1.22-40.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stillman B. W., Lewis J. B., Chow L. T., Mathews M. B., Smart J. E. Identification of the gene and mRNA for the adenovirus terminal protein precursor. Cell. 1981 Feb;23(2):497–508. doi: 10.1016/0092-8674(81)90145-8. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Van Ormondt H., Maat J., De Waard A., Van der Eb A. J. The nucleotide sequence of the transforming HpaI-E fragment of adenovirus type 5 DNA. Gene. 1978 Dec;4(4):309–328. doi: 10.1016/0378-1119(78)90048-3. [DOI] [PubMed] [Google Scholar]
  42. Visser L., van Maarschalkerweerd M. W., Rozijn T. H., Wassenaar A. D., Reemst A. M., Sussenbach J. S. Viral DNA sequences in adenovirus-transformed cells. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):541–550. doi: 10.1101/sqb.1980.044.01.056. [DOI] [PubMed] [Google Scholar]
  43. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Williams J., Galos R. S., Binger M. H., Flint S. J. Location of additional early regions within the left quarter of the adenoviral genome. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):353–365. doi: 10.1101/sqb.1980.044.01.040. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Wold W. S., Green M. Adenovirus type 2 early polypeptides immunoprecipitated by antisera to five lines of adenovirus-transformed rat cells. J Virol. 1979 Apr;30(1):297–310. doi: 10.1128/jvi.30.1.297-310.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. van der Eb A. J., van Ormondt H., Schrier P. I., Lupker J. H., Jochemsen H., van den Elsen P. J., DeLeys R. J., Maat J., van Beveren C. P., Dijkema R. Structure and function of the transforming genes of human adenoviruses and SV40. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):383–399. doi: 10.1101/sqb.1980.044.01.043. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES