Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1987 Mar;61(3):673–682. doi: 10.1128/jvi.61.3.673-682.1987

Adenovirus E1A 12S protein induces DNA synthesis and proliferation in primary epithelial cells in both the presence and absence of serum.

M P Quinlan, T Grodzicker
PMCID: PMC254006  PMID: 3027395

Abstract

Infection of primary baby rat kidney (BRK) cells with an adenovirus that carries an E1A 12S cDNA in place of the normal E1A region (adenovirus 5 [Ad5] 12S) resulted in the induction of cellular DNA synthesis and proliferation of the epithelial cells in the population, even in the absence of serum. Increased cellular DNA synthesis was first detectable by 12 h after infection and was maintained at a 10- to 20-fold higher level than in mock-infected cells. By 5 days after infection there was a 10-fold-greater number of 12S virus-infected BRK cells. These infected BRK cells retained many of their normal epithelial cell characteristics and were not transformed. The expression of the E1A 12S protein(s) occurred early after infection. There was no induction of adenoviral gene expression or viral DNA replication in these cells. The early effects of a fully transforming gene product(s) were also examined. The Ad5-simian virus 40 hybrid virus, Ad5.SVR4, in which the early region of simian virus 40 has replaced the E1 region of Ad5, was used to infect BRK cells. The kinetics of expression of the T antigens were similar to those of the 12S polypeptides. Infection with Ad5.SV4 also resulted in the induction of cellular DNA synthesis and cell proliferation at levels similar to those observed with the 12S virus. However, infection with Ad5.SVR4 resulted in cells that had lost some of their epithelial cell characteristics and were fully transformed. Thus, although the early cellular events induced by the two genes were similar, they did not yield the same final cellular phenotype.

Full text

PDF
673

Images in this article

675Image
on p.675
676Image
on p.676
676Image
on p.676
677Image
on p.677
678Image
on p.678
679Image
on p.679
679Image
on p.679

Selected References

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

  1. Bellett A. J., Li P., David E. T., Mackey E. J., Braithwaite A. W., Cutt J. R. Control functions of adenovirus transformation region E1A gene products in rat and human cells. Mol Cell Biol. 1985 Aug;5(8):1933–1939. doi: 10.1128/mcb.5.8.1933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bellett A. J., Waldron-Stevens L. K., Braithwaite A. W., Cheetham B. F. Spermine and aminoguanidine protect cells from chromosome aberrations induced by adenovirus during the G2 phase of the cell cycle. Chromosoma. 1982;84(4):571–583. doi: 10.1007/BF00292856. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Borrelli E., Hen R., Chambon P. Adenovirus-2 E1A products repress enhancer-induced stimulation of transcription. Nature. 1984 Dec 13;312(5995):608–612. doi: 10.1038/312608a0. [DOI] [PubMed] [Google Scholar]
  6. Borrelli E., Hen R., Wasylyk C., Wasylyk B., Chambon P. The immunoglobulin heavy chain enhancer is stimulated by the adenovirus type 2 E1A products in mouse fibroblasts. Proc Natl Acad Sci U S A. 1986 May;83(9):2846–2849. doi: 10.1073/pnas.83.9.2846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Braithwaite A. W., Cheetham B. F., Li P., Parish C. R., Waldron-Stevens L. K., Bellett A. J. Adenovirus-induced alterations of the cell growth cycle: a requirement for expression of E1A but not of E1B. J Virol. 1983 Jan;45(1):192–199. doi: 10.1128/jvi.45.1.192-199.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Braithwaite A. W., Murray J. D., Bellett A. J. Alterations to controls of cellular DNA synthesis by adenovirus infection. J Virol. 1981 Aug;39(2):331–340. doi: 10.1128/jvi.39.2.331-340.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carlock L. R., Jones N. C. Transformation-defective mutant of adenovirus type 5 containing a single altered E1a mRNA species. J Virol. 1981 Dec;40(3):657–664. doi: 10.1128/jvi.40.3.657-664.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cheetham B. F., Bellett A. J. A biochemical investigation of the adenovirus-induced G1 to S phase progression: thymidine kinase, ornithine decarboxylase, and inhibitors of polyamine biosynthesis. J Cell Physiol. 1982 Feb;110(2):114–122. doi: 10.1002/jcp.1041100203. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Esche H., Mathews M. B., Lewis J. B. Proteins and messenger RNAs of the transforming region of wild-type and mutant adenoviruses. J Mol Biol. 1980 Sep 25;142(3):399–417. doi: 10.1016/0022-2836(80)90279-x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Gaynor R. B., Hillman D., Berk A. J. Adenovirus early region 1A protein activates transcription of a nonviral gene introduced into mammalian cells by infection or transfection. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1193–1197. doi: 10.1073/pnas.81.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Green M. R., Treisman R., Maniatis T. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products. Cell. 1983 Nov;35(1):137–148. doi: 10.1016/0092-8674(83)90216-7. [DOI] [PubMed] [Google Scholar]
  17. Haley K. P., Overhauser J., Babiss L. E., Ginsberg H. S., Jones N. C. Transformation properties of type 5 adenovirus mutants that differentially express the E1A gene products. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5734–5738. doi: 10.1073/pnas.81.18.5734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Harlow E., Crawford L. V., Pim D. C., Williamson N. M. Monoclonal antibodies specific for simian virus 40 tumor antigens. J Virol. 1981 Sep;39(3):861–869. doi: 10.1128/jvi.39.3.861-869.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Harlow E., Franza B. R., Jr, Schley C. Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products. J Virol. 1985 Sep;55(3):533–546. doi: 10.1128/jvi.55.3.533-546.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Hearing P., Shenk T. Sequence-independent autoregulation of the adenovirus type 5 E1A transcription unit. Mol Cell Biol. 1985 Nov;5(11):3214–3221. doi: 10.1128/mcb.5.11.3214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hen R., Borrelli E., Chambon P. Repression of the immunoglobulin heavy chain enhancer by the adenovirus-2 E1A products. Science. 1985 Dec 20;230(4732):1391–1394. doi: 10.1126/science.2999984. [DOI] [PubMed] [Google Scholar]
  23. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  24. Houweling A., van den Elsen P. J., van der Eb A. J. Partial transformation of primary rat cells by the leftmost 4.5% fragment of adenovirus 5 DNA. Virology. 1980 Sep;105(2):537–550. doi: 10.1016/0042-6822(80)90054-9. [DOI] [PubMed] [Google Scholar]
  25. Hurwitz D. R., Chinnadurai G. Evidence that a second tumor antigen coded by adenovirus early gene region E1a is required for efficient cell transformation. Proc Natl Acad Sci U S A. 1985 Jan;82(1):163–167. doi: 10.1073/pnas.82.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Imperiale M. J., Nevins J. R. Adenovirus 5 E2 transcription unit: an E1A-inducible promoter with an essential element that functions independently of position or orientation. Mol Cell Biol. 1984 May;4(5):875–882. doi: 10.1128/mcb.4.5.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Kao H. T., Nevins J. R. Transcriptional activation and subsequent control of the human heat shock gene during adenovirus infection. Mol Cell Biol. 1983 Nov;3(11):2058–2065. doi: 10.1128/mcb.3.11.2058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kitchingman G. R., Westphal H. The structure of adenovirus 2 early nuclear and cytoplasmic RNAs. J Mol Biol. 1980 Feb 15;137(1):23–48. doi: 10.1016/0022-2836(80)90155-2. [DOI] [PubMed] [Google Scholar]
  30. Krippl B., Ferguson B., Jones N., Rosenberg M., Westphal H. Mapping of functional domains in adenovirus E1A proteins. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7480–7484. doi: 10.1073/pnas.82.22.7480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lane E. B. Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization. J Cell Biol. 1982 Mar;92(3):665–673. doi: 10.1083/jcb.92.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Leff T., Elkaim R., Goding C. R., Jalinot P., Sassone-Corsi P., Perricaudet M., Kédinger C., Chambon P. Individual products of the adenovirus 12S and 13S EIa mRNAs stimulate viral EIIa and EIII expression at the transcriptional level. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4381–4385. doi: 10.1073/pnas.81.14.4381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moll R., Franke W. W., Schiller D. L., Geiger B., Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982 Nov;31(1):11–24. doi: 10.1016/0092-8674(82)90400-7. [DOI] [PubMed] [Google Scholar]
  34. Montell C., Courtois G., Eng C., Berk A. Complete transformation by adenovirus 2 requires both E1A proteins. Cell. 1984 Apr;36(4):951–961. doi: 10.1016/0092-8674(84)90045-x. [DOI] [PubMed] [Google Scholar]
  35. Montell C., Fisher E. F., Caruthers M. H., Berk A. J. Resolving the functions of overlapping viral genes by site-specific mutagenesis at a mRNA splice site. Nature. 1982 Feb 4;295(5848):380–384. doi: 10.1038/295380a0. [DOI] [PubMed] [Google Scholar]
  36. Moran E., Grodzicker T., Roberts R. J., Mathews M. B., Zerler B. Lytic and transforming functions of individual products of the adenovirus E1A gene. J Virol. 1986 Mar;57(3):765–775. doi: 10.1128/jvi.57.3.765-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Murray J. D., Bellett A. J., Braithwaite A. w., Waldron L. K., Taylor I. W. Altered cell cycle progression and aberrant mitosis in adenovirus-infected rodent cells. J Cell Physiol. 1982 Apr;111(1):89–96. doi: 10.1002/jcp.1041110114. [DOI] [PubMed] [Google Scholar]
  38. Murray J. D., Braithwaite A. W., Taylor I. W., Bellett A. J. Adenovirus-induced alterations of the cell growth cycle: effects of mutations in early regions E2A and E2B. J Virol. 1982 Dec;44(3):1072–1075. doi: 10.1128/jvi.44.3.1072-1075.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nevins J. R. Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product. Cell. 1982 Jul;29(3):913–919. doi: 10.1016/0092-8674(82)90453-6. [DOI] [PubMed] [Google Scholar]
  40. Nevins J. R. Mechanism of activation of early viral transcription by the adenovirus E1A gene product. Cell. 1981 Oct;26(2 Pt 2):213–220. doi: 10.1016/0092-8674(81)90304-4. [DOI] [PubMed] [Google Scholar]
  41. Perricaudet M., Akusjärvi G., Virtanen A., Pettersson U. Structure of two spliced mRNAs from the transforming region of human subgroup C adenoviruses. Nature. 1979 Oct 25;281(5733):694–696. doi: 10.1038/281694a0. [DOI] [PubMed] [Google Scholar]
  42. Pochron S., Rossini M., Darzynkiewicz Z., Traganos F., Baserga R. Failure of accumulation of cellular RNA in hamster cells stimulated to synthesize DNA by infection with adenovirus 2. J Biol Chem. 1980 May 25;255(10):4411–4413. [PubMed] [Google Scholar]
  43. Ricciardi R. P., Jones R. L., Cepko C. L., Sharp P. A., Roberts B. E. Expression of early adenovirus genes requires a viral encoded acidic polypeptide. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6121–6125. doi: 10.1073/pnas.78.10.6121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ross S. R., Levine A. J., Galos R. S., Williams J., Shenk T. Early viral proteins in HeLa cells infected with adenovirus type 5 host range mutants. Virology. 1980 Jun;103(2):475–492. doi: 10.1016/0042-6822(80)90205-6. [DOI] [PubMed] [Google Scholar]
  45. Ruben M., Bacchetti S., Graham F. L. Integration and expression of viral DNA in cells transformed by host range mutants of adenovirus type 5. J Virol. 1982 Feb;41(2):674–685. doi: 10.1128/jvi.41.2.674-685.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ruley H. E. Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature. 1983 Aug 18;304(5927):602–606. doi: 10.1038/304602a0. [DOI] [PubMed] [Google Scholar]
  47. Shenk T., Jones N., Colby W., Fowlkes D. Functional analysis of adenovirus-5 host-range deletion mutants defective for transformation of rat embryo cells. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):367–375. doi: 10.1101/sqb.1980.044.01.041. [DOI] [PubMed] [Google Scholar]
  48. Shimojo H., Yamashita T. Induction of DNA synthesis by adenoviruses in contact-inhibited hamster cells. Virology. 1968 Nov;36(3):422–433. doi: 10.1016/0042-6822(68)90167-0. [DOI] [PubMed] [Google Scholar]
  49. Sitch H. F., Yohn D. S. Viruses and chromosomes. Prog Med Virol. 1970;12:78–127. [PubMed] [Google Scholar]
  50. 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]
  51. Spindler K. R., Berk A. J. Rapid intracellular turnover of adenovirus 5 early region 1A proteins. J Virol. 1984 Nov;52(2):706–710. doi: 10.1128/jvi.52.2.706-710.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Spindler K. R., Eng C. Y., Berk A. J. An adenovirus early region 1A protein is required for maximal viral DNA replication in growth-arrested human cells. J Virol. 1985 Mar;53(3):742–750. doi: 10.1128/jvi.53.3.742-750.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stabel S., Argos P., Philipson L. The release of growth arrest by microinjection of adenovirus E1A DNA. EMBO J. 1985 Sep;4(9):2329–2336. doi: 10.1002/j.1460-2075.1985.tb03934.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Stein R., Ziff E. B. HeLa cell beta-tubulin gene transcription is stimulated by adenovirus 5 in parallel with viral early genes by an E1a-dependent mechanism. Mol Cell Biol. 1984 Dec;4(12):2792–2801. doi: 10.1128/mcb.4.12.2792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Strohl W. A. The response of BHK21 cells to infection with type 12 adenovirus. II. Relationship of virus-stimulated DNA synthesis to other viral functions. Virology. 1969 Dec;39(4):653–665. doi: 10.1016/0042-6822(69)90004-x. [DOI] [PubMed] [Google Scholar]
  56. Svensson C., Akusjärvi G. Adenovirus 2 early region 1A stimulates expression of both viral and cellular genes. EMBO J. 1984 Apr;3(4):789–794. doi: 10.1002/j.1460-2075.1984.tb01886.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Takahashi M., Ogino T., Baba K., Onaka M. Synthesis of deoxyribonucleic acid in human and hamster kidney cells infected with human adenovirus types 5 and 12. Virology. 1969 Apr;37(4):513–520. doi: 10.1016/0042-6822(69)90269-4. [DOI] [PubMed] [Google Scholar]
  58. Taub M., Chuman L., Saier M. H., Jr, Sato G. Growth of Madin-Darby canine kidney epithelial cell (MDCK) line in hormone-supplemented, serum-free medium. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3338–3342. doi: 10.1073/pnas.76.7.3338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Treisman R., Green M. R., Maniatis T. cis and trans activation of globin gene transcription in transient assays. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7428–7432. doi: 10.1073/pnas.80.24.7428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Van Doren K., Gluzman Y. Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants. Mol Cell Biol. 1984 Aug;4(8):1653–1656. doi: 10.1128/mcb.4.8.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Velcich A., Ziff E. Adenovirus E1a proteins repress transcription from the SV40 early promoter. Cell. 1985 Mar;40(3):705–716. doi: 10.1016/0092-8674(85)90219-3. [DOI] [PubMed] [Google Scholar]
  62. Weeks D. L., Jones N. C. E1A control of gene expression is mediated by sequences 5' to the transcriptional starts of the early viral genes. Mol Cell Biol. 1983 Jul;3(7):1222–1234. doi: 10.1128/mcb.3.7.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Younghusband H. B., Tyndall C., Bellett A. J. Replication and interaction of virus DNA and cellular DNA in mouse cells infected by a human adenovirus. J Gen Virol. 1979 Nov;45(2):455–467. doi: 10.1099/0022-1317-45-2-455. [DOI] [PubMed] [Google Scholar]
  64. Zerler B., Moran B., Maruyama K., Moomaw J., Grodzicker T., Ruley H. E. Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells. Mol Cell Biol. 1986 Mar;6(3):887–899. doi: 10.1128/mcb.6.3.887. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES