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. 1982 Oct;44(1):276–285. doi: 10.1128/jvi.44.1.276-285.1982

Enhanced expression of adenovirus transforming proteins.

R B Gaynor, A Tsukamoto, C Montell, A J Berk
PMCID: PMC256263  PMID: 7143568

Abstract

Proteins encoded in regions EIA and EIB of human adenoviruses cause transformation of rodent cells. One protein from EIA also stimulates transcription of other early regions at early times in a productive infection. In the past, direct analysis of these proteins synthesized in vivo has been difficult because of the low levels produced in both transformed cells and productively infected cells. We present a simple method which leads to expression of EIA and EIB mRNAs and proteins at 30-fold greater levels than those observed during the early phase of a standard productive infection. Under these conditions, these proteins are among the most prominent translation products of infected cells. This allowed direct visualization of EIA and EIB proteins on two-dimensional gels of pulse-labeled total cell protein. Experiments with EIA and EIB mutants confirm that the identified proteins are indeed encoded in these regions. Two EIA proteins are observed, one translated from each of the major early EIA mRNAs. Both of these EIA proteins are phosphorylated.

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Selected References

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  1. Aleström P., Akusjärvi G., Perricaudet M., Mathews M. B., Klessig D. F., Pettersson U. The gene for polypeptide IX of adenovirus type 2 and its unspliced messenger RNA. Cell. 1980 Mar;19(3):671–681. doi: 10.1016/s0092-8674(80)80044-4. [DOI] [PubMed] [Google Scholar]
  2. Anderson C. W., Lewis J. B. Amino-terminal sequence of adenovirus type 2 proteins: hexon, fiber, component IX, and early protein 1B-15K. Virology. 1980 Jul 15;104(1):27–41. doi: 10.1016/0042-6822(80)90363-3. [DOI] [PubMed] [Google Scholar]
  3. Baker C. C., Ziff E. B. Promoters and heterogeneous 5' termini of the messenger RNAs of adenovirus serotype 2. J Mol Biol. 1981 Jun 25;149(2):189–221. doi: 10.1016/0022-2836(81)90298-9. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Bos J. L., Polder L. J., Bernards R., Schrier P. I., van den Elsen P. J., van der Eb A. J., van Ormondt H. The 2.2 kb E1b mRNA of human Ad12 and Ad5 codes for two tumor antigens starting at different AUG triplets. Cell. 1981 Nov;27(1 Pt 2):121–131. doi: 10.1016/0092-8674(81)90366-4. [DOI] [PubMed] [Google Scholar]
  8. Brackmann K. H., Green M., Wold W. S., Cartas M., Matsuo T., Hashimoto S. Identification and peptide mapping of human adenovirus type 2-induced early polypeptides isolated by two-dimensional gel electrophoresis and immunoprecipitation. J Biol Chem. 1980 Jul 25;255(14):6772–6779. [PubMed] [Google Scholar]
  9. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [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. 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]
  12. Garrels J. I., Gibson W. Identification and characterization of multiple forms of actin. Cell. 1976 Dec;9(4 Pt 2):793–805. doi: 10.1016/0092-8674(76)90142-2. [DOI] [PubMed] [Google Scholar]
  13. 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]
  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. Green M., Wold W. S., Brackmann K. H., Cartas M. A. Identification of families of overlapping polypeptides coded by early "transforming" gene region 1 of human adenovirus type 2. Virology. 1979 Sep;97(2):275–286. doi: 10.1016/0042-6822(79)90339-8. [DOI] [PubMed] [Google Scholar]
  16. Halbert D. N., Raskas H. J. Tryptic and chymotryptic methionine peptide analysis of the in vitro translation products specified by the transforming region of adenovirus type 2. Virology. 1982 Jan 30;116(2):406–418. doi: 10.1016/0042-6822(82)90135-0. [DOI] [PubMed] [Google Scholar]
  17. Halbert D. N., Spector D. J., Raskas H. J. In vitro translation products specified by the transforming region of adenovirus type 2. J Virol. 1979 Sep;31(3):621–629. doi: 10.1128/jvi.31.3.621-629.1979. [DOI] [PMC free article] [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. Harter M. L., Lewis J. B. Adenovirus type 2 early proteins synthesized in vitro and in vivo: identification in infected cells of the 38,000- to 50,000- molecular-weight protein encoded by the left end of the adenovirus type 2 genome. J Virol. 1978 Jun;26(3):736–749. doi: 10.1128/jvi.26.3.736-749.1978. [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. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Levinson A. D., Postel E. H., Levine A. J. In vivo and in vitro phosphorylation of the adenovirus type 5 single strand-specific DNA-binding protein. Virology. 1977 Jun 1;79(1):144–159. doi: 10.1016/0042-6822(77)90341-5. [DOI] [PubMed] [Google Scholar]
  25. Lupker J. H., Davis A., Jochemsen H., van der Eb A. J. In vitro synthesis of adenovirus type 5 T antigens. I. Translation of early region 1-specific rna from lytically infected cells. J Virol. 1981 Jan;37(1):524–529. doi: 10.1128/jvi.37.1.524-529.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  29. Osborne T. F., Gaynor R. B., Berk A. J. The TATA homology and the mRNA 5' untranslated sequence are not required for expression of essential adenovirus E1A functions. Cell. 1982 May;29(1):139–148. doi: 10.1016/0092-8674(82)90098-8. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Perricaudet M., Le Moullec J. M., Pettersson U. Predicted structure of two adenovirus tumor antigens. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3778–3782. doi: 10.1073/pnas.77.7.3778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Schrier P. I., Van Den Elsen P. J., Hertoghs J. J., Van Der Eb A. J. Characterization of tumor antigens in cells transformed by fragments of adenovirus type 5 DNA. Virology. 1979 Dec;99(2):372–385. doi: 10.1016/0042-6822(79)90016-3. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Smart J. E., Lewis J. B., Mathews M. B., Harter M. L., Anderson C. W. Adenovirus type 2 early proteins: assignment of the early region 1A proteins synthesized in vivo and in vitro to specific mRNAs. Virology. 1981 Jul 30;112(2):703–713. doi: 10.1016/0042-6822(81)90315-9. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Vogt V. M. Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae. Eur J Biochem. 1973 Feb 15;33(1):192–200. doi: 10.1111/j.1432-1033.1973.tb02669.x. [DOI] [PubMed] [Google Scholar]
  40. Ziff E. B. Transcription and RNA processing by the DNA tumour viruses. Nature. 1980 Oct 9;287(5782):491–499. doi: 10.1038/287491a0. [DOI] [PubMed] [Google Scholar]
  41. 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]

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