Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1983 Jun;46(3):1003–1013. doi: 10.1128/jvi.46.3.1003-1013.1983

Identification of human adenovirus early region 1 products by using antisera against synthetic peptides corresponding to the predicted carboxy termini.

S P Yee, D T Rowe, M L Tremblay, M McDermott, P E Branton
PMCID: PMC256575  PMID: 6343626

Abstract

Synthetic peptides were prepared which corresponded to the carboxy termini of the human adenovirus type 5 early region 1B (E1B) 58,000-molecular-weight (58K) protein (Tyr-Ser-Asp-Glu-Asp-Thr-Asp) and of the E1A gene products (Tyr-Gly-Lys-Arg-Pro-Arg-Pro). Antisera raised against these peptides precipitated polypeptides from adenovirus type 5-infected KB cells; serum raised against the 58K carboxy terminus was active against the E1B 58K phosphoprotein, whereas serum raised against the E1A peptide immunoprecipitated four major and at least two minor polypeptides. These latter proteins migrated with apparent molecular weights of 52K, 50K, 48.5K, 45K, 37.5K, and 35K, and all were phosphoproteins. By using tryptic phosphopeptide analysis, the four major species (52K, 50K, 48.5K, and 45K) were found to be related, as would be expected if all were products of the E1A region. The ability of the antipeptide sera to precipitate these viral proteins thus confirmed that the previously proposed sequence of E1 DNA and mRNA and the reading frame of the mRNA are correct. Immunofluorescent-antibody staining with the antipeptide sera indicated that the 58K E1B protein was localized both in the nucleus and in the cytoplasm, especially in the perinuclear region. The E1A-specific serum also stained both discrete patches in the nucleus and diffuse areas of the cytoplasm. These data suggest that both the 58K protein and the E1A proteins may function in or around the nucleus. These highly specific antipeptide sera should allow for a more complete identification and characterization of these important viral proteins.

Full text

PDF
1013

Images in this article

1006Image
on p.1006
1006Image
on p.1006
1007Image
on p.1007
1008Image
on p.1008
1009Image
on p.1009
1009Image
on p.1009
1010Image
on p.1010
1011Image
on p.1011

Selected References

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

  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. 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]
  3. Berget S. M., Moore C., Sharp P. A. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3171–3175. doi: 10.1073/pnas.74.8.3171. [DOI] [PMC free article] [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. 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]
  6. 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]
  7. 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]
  8. Branton P. E., Lassam N. J., Downey J. F., Yee S. P., Graham F. L., Mak S., Bayley S. T. Protein kinase activity immunoprecipitated from adenovirus infected cells by sera from tumor-bearing hamsters. J Virol. 1981 Feb;37(2):601–608. doi: 10.1128/jvi.37.2.601-608.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  11. Dijkema R., Dekker B. M., van Ormondt H. Gene organization of the transforming region of adenovirus type 7 DNA. Gene. 1982 May;18(2):143–156. doi: 10.1016/0378-1119(82)90112-3. [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. 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]
  15. Gaynor R. B., Tsukamoto A., Montell C., Berk A. J. Enhanced expression of adenovirus transforming proteins. J Virol. 1982 Oct;44(1):276–285. doi: 10.1128/jvi.44.1.276-285.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Graham F. L., van der Eb A. J., Heijneker H. L. Size and location of the transforming region in human adenovirus type 5 DNA. Nature. 1974 Oct 25;251(5477):687–691. doi: 10.1038/251687a0. [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. 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]
  19. Johansson K., Persson H., Lewis A. M., Pettersson U., Tibbetts C., Philipson L. Viral DNA sequences and gene products in hamster cells transformed by adenovirus type 2. J Virol. 1978 Sep;27(3):628–639. doi: 10.1128/jvi.27.3.628-639.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. Levinson A., Levine A. J. The isolation and identification of the adenovirus group C tumor antigens. Virology. 1977 Jan;76(1):1–11. doi: 10.1016/0042-6822(77)90275-6. [DOI] [PubMed] [Google Scholar]
  24. Lewis J. B., Atkins J. F., Baum P. R., Solem R., Gesteland R. F., Anderson C. W. Location and identification of the genes for adenovirus type 2 early polypeptides. Cell. 1976 Jan;7(1):141–151. doi: 10.1016/0092-8674(76)90264-6. [DOI] [PubMed] [Google Scholar]
  25. Lewis J. B., Esche H., Smart J. E., Stillman B. W., Harter M. L., Mathews M. B. Organization and expression of the left third of the genome of adenovirus. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):493–508. doi: 10.1101/sqb.1980.044.01.052. [DOI] [PubMed] [Google Scholar]
  26. Maat J., Van Ormondt H. The nucleotide sequence of the transforming HindIII-G fragment of adenovirus type 5 DNA. The region between map positions 4.5 (HpaI site) and 8.0 (HindIII site). Gene. 1979 May;6(1):75–90. doi: 10.1016/0378-1119(79)90086-6. [DOI] [PubMed] [Google Scholar]
  27. Maat J., van Beveren C. P., van Ormondt H. The nucleotide sequence of adenovirus type 5 early region E1: the region between map positions 8.0 (HindIII site) and 11.8 (SmaI site). Gene. 1980 Jun;10(1):27–38. doi: 10.1016/0378-1119(80)90140-7. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Persson H., Monstein H. J., Akusjärvi G., Philipson L. Adenovirus early gene products may control viral mRNA accumulation and translation in vivo. Cell. 1981 Feb;23(2):485–496. doi: 10.1016/0092-8674(81)90144-6. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Scheidtmann K. H., Kaiser A., Carbone A., Walter G. Phosphorylation of threonine in the proline-rich carboxy-terminal region of simian virus 40 large T antigen. J Virol. 1981 Apr;38(1):59–69. doi: 10.1128/jvi.38.1.59-69.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Spector D. J., McGrogan M., Raskas H. J. Regulation of the appearance of cytoplasmic RNAs from region 1 of the adenovirus 2 genome. J Mol Biol. 1978 Dec 15;126(3):395–414. doi: 10.1016/0022-2836(78)90048-7. [DOI] [PubMed] [Google Scholar]
  35. Sutcliffe J. G., Shinnick T. M., Green N., Liu F. T., Niman H. L., Lerner R. A. Chemical synthesis of a polypeptide predicted from nucleotide sequence allows detection of a new retroviral gene product. Nature. 1980 Oct 30;287(5785):801–805. doi: 10.1038/287801a0. [DOI] [PubMed] [Google Scholar]
  36. Van der Eb A. J., Mulder C., Graham F. L., Houweling A. Transformation with specific fragments of adenovirus DNAs. I. Isolation of specific fragments with transforming activity of adenovirus 2 and 5 DNA. Gene. 1977;2(3-4):115–132. doi: 10.1016/0378-1119(77)90012-9. [DOI] [PubMed] [Google Scholar]
  37. Walter G., Scheidtmann K. H., Carbone A., Laudano A. P., Doolittle R. F. Antibodies specific for the carboxy- and amino-terminal regions of simian virus 40 large tumor antigen. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5197–5200. doi: 10.1073/pnas.77.9.5197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Williams J. F. Oncogenic transformation of hamster embryo cells in vitro by adenovirus type 5. Nature. 1973 May 18;243(5403):162–163. doi: 10.1038/243162a0. [DOI] [PubMed] [Google Scholar]
  39. van Ormondt H., Maat J., van Beveren C. P. The nucleotide sequence of the transforming early region E1 of adenovirus type 5 DNA. Gene. 1980 Nov;11(3-4):299–309. doi: 10.1016/0378-1119(80)90070-0. [DOI] [PubMed] [Google Scholar]
  40. 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