Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Feb;70(2):862–872. doi: 10.1128/jvi.70.2.862-872.1996

Human adenovirus type 9 E4 open reading frame 1 encodes a cytoplasmic transforming protein capable of increasing the oncogenicity of CREF cells.

R S Weiss 1, M J McArthur 1, R T Javier 1
PMCID: PMC189889  PMID: 8551625

Abstract

The induction of estrogen-dependent rat mammary tumors by human adenovirus type 9 (Ad9) requires the Ad9 E4 open reading frame 1 (9ORF1) protein, which alone can transform that rat embryo fibroblast cell line CREF in vitro. In the present study, independent pools of both 9ORF1-expressing and control CREF cells were generated by selection with G418 and compared with respect to transformed properties. Indirect immunofluorescence analyses revealed that more than 99% of the cells that made up the 9ORF1-transfected pools expressed 9ORF1 protein and, together with confocal laser scanning microscopy, indicated that this E4 protein was located predominantly within the cytoplasm of cells. With regard to transformation, cells of the 9ORF1-expressing pools differed from those of control pools by forming foci, displaying morphological alterations, growing more efficiently in soft agar, and reaching higher saturation densities. Following injection into immunocompetent syngeneic rats, the 9ORF1-expressing pool cells exhibited greatly enhanced oncogenicity compared with control pool cells. These results show that 9ORF1 protein (i) localizes predominantly within the cytoplasm, (ii) confers multiple general transformed characteristics to CREF cells in vitro, and (iii) increases the tumorigenic properties of these cells in vivo.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Ankerst J., Jonsson N. Adenovirus type 9-induced tumorigenesis in the rat mammary gland related to sex hormonal state. J Natl Cancer Inst. 1989 Feb 15;81(4):294–298. doi: 10.1093/jnci/81.4.294. [DOI] [PubMed] [Google Scholar]
  2. Ankerst J., Jonsson N., Kjellén L., Norrby E., Sjögren H. O. Induction of mammary fibroadenomas in rats by adenovirus type 9. Int J Cancer. 1974 Mar 15;13(3):286–290. doi: 10.1002/ijc.2910130303. [DOI] [PubMed] [Google Scholar]
  3. Babiss L. E., Liaw W. S., Zimmer S. G., Godman G. C., Ginsberg H. S., Fisher P. B. Mutations in the E1a gene of adenovirus type 5 alter the tumorigenic properties of transformed cloned rat embryo fibroblast cells. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2167–2171. doi: 10.1073/pnas.83.7.2167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bridge E., Ketner G. Redundant control of adenovirus late gene expression by early region 4. J Virol. 1989 Feb;63(2):631–638. doi: 10.1128/jvi.63.2.631-638.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bridge E., Medghalchi S., Ubol S., Leesong M., Ketner G. Adenovirus early region 4 and viral DNA synthesis. Virology. 1993 Apr;193(2):794–801. doi: 10.1006/viro.1993.1188. [DOI] [PubMed] [Google Scholar]
  6. Cutt J. R., Shenk T., Hearing P. Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells. J Virol. 1987 Feb;61(2):543–552. doi: 10.1128/jvi.61.2.543-552.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dix I., Leppard K. N. Expression of adenovirus type 5 E4 Orf2 protein during lytic infection. J Gen Virol. 1995 Apr;76(Pt 4):1051–1055. doi: 10.1099/0022-1317-76-4-1051. [DOI] [PubMed] [Google Scholar]
  8. Dix I., Leppard K. N. Regulated splicing of adenovirus type 5 E4 transcripts and regulated cytoplasmic accumulation of E4 mRNA. J Virol. 1993 Jun;67(6):3226–3231. doi: 10.1128/jvi.67.6.3226-3231.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duigou G. J., Babiss L. E., Liaw W. S., Zimmer S. G., Ginsberg H. S., Fisher P. B. Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells. J Cell Biochem. 1987 Feb;33(2):117–126. doi: 10.1002/jcb.240330206. [DOI] [PubMed] [Google Scholar]
  10. Evan G. I., Wyllie A. H., Gilbert C. S., Littlewood T. D., Land H., Brooks M., Waters C. M., Penn L. Z., Hancock D. C. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 1992 Apr 3;69(1):119–128. doi: 10.1016/0092-8674(92)90123-t. [DOI] [PubMed] [Google Scholar]
  11. Fisher P. B., Babiss L. E., Weinstein I. B., Ginsberg H. S. Analysis of type 5 adenovirus transformation with a cloned rat embryo cell line (CREF). Proc Natl Acad Sci U S A. 1982 Jun;79(11):3527–3531. doi: 10.1073/pnas.79.11.3527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guadagno T. M., Ohtsubo M., Roberts J. M., Assoian R. K. A link between cyclin A expression and adhesion-dependent cell cycle progression. Science. 1993 Dec 3;262(5139):1572–1575. doi: 10.1126/science.8248807. [DOI] [PubMed] [Google Scholar]
  13. Halbert D. N., Cutt J. R., Shenk T. Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff. J Virol. 1985 Oct;56(1):250–257. doi: 10.1128/jvi.56.1.250-257.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hinds P. W., Finlay C. A., Quartin R. S., Baker S. J., Fearon E. R., Vogelstein B., Levine A. J. Mutant p53 DNA clones from human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the "hot spot" mutant phenotypes. Cell Growth Differ. 1990 Dec;1(12):571–580. [PubMed] [Google Scholar]
  15. Hoang A. T., Cohen K. J., Barrett J. F., Bergstrom D. A., Dang C. V. Participation of cyclin A in Myc-induced apoptosis. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6875–6879. doi: 10.1073/pnas.91.15.6875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hogenkamp T., Esche H. Nucleotide sequence of the right 10% of adenovirus type 12 DNA encoding the entire region E4. Nucleic Acids Res. 1990 May 25;18(10):3065–3066. doi: 10.1093/nar/18.10.3065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huang M. M., Hearing P. Adenovirus early region 4 encodes two gene products with redundant effects in lytic infection. J Virol. 1989 Jun;63(6):2605–2615. doi: 10.1128/jvi.63.6.2605-2615.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hérissé J., Rigolet M., de Dinechin S. D., Galibert F. Nucleotide sequence of adenovirus 2 DNA fragment encoding for the carboxylic region of the fiber protein and the entire E4 region. Nucleic Acids Res. 1981 Aug 25;9(16):4023–4042. doi: 10.1093/nar/9.16.4023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ingram V. M. A side view of moving fibroblasts. Nature. 1969 May 17;222(5194):641–644. doi: 10.1038/222641a0. [DOI] [PubMed] [Google Scholar]
  20. Javier R. T. Adenovirus type 9 E4 open reading frame 1 encodes a transforming protein required for the production of mammary tumors in rats. J Virol. 1994 Jun;68(6):3917–3924. doi: 10.1128/jvi.68.6.3917-3924.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Javier R., Raska K., Jr, Macdonald G. J., Shenk T. Human adenovirus type 9-induced rat mammary tumors. J Virol. 1991 Jun;65(6):3192–3202. doi: 10.1128/jvi.65.6.3192-3202.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Javier R., Raska K., Jr, Shenk T. Requirement for the adenovirus type 9 E4 region in production of mammary tumors. Science. 1992 Aug 28;257(5074):1267–1271. doi: 10.1126/science.1519063. [DOI] [PubMed] [Google Scholar]
  23. Kiefer F., Courtneidge S. A., Wagner E. F. Oncogenic properties of the middle T antigens of polyomaviruses. Adv Cancer Res. 1994;64:125–157. doi: 10.1016/s0065-230x(08)60837-4. [DOI] [PubMed] [Google Scholar]
  24. Kleinberger T., Shenk T. Adenovirus E4orf4 protein binds to protein phosphatase 2A, and the complex down regulates E1A-enhanced junB transcription. J Virol. 1993 Dec;67(12):7556–7560. doi: 10.1128/jvi.67.12.7556-7560.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Meikrantz W., Gisselbrecht S., Tam S. W., Schlegel R. Activation of cyclin A-dependent protein kinases during apoptosis. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3754–3758. doi: 10.1073/pnas.91.9.3754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mosialos G., Birkenbach M., Yalamanchili R., VanArsdale T., Ware C., Kieff E. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell. 1995 Feb 10;80(3):389–399. doi: 10.1016/0092-8674(95)90489-1. [DOI] [PubMed] [Google Scholar]
  27. Nobes C. D., Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 1995 Apr 7;81(1):53–62. doi: 10.1016/0092-8674(95)90370-4. [DOI] [PubMed] [Google Scholar]
  28. Peters R. Fluorescence microphotolysis to measure nucleocytoplasmic transport and intracellular mobility. Biochim Biophys Acta. 1986 Dec 22;864(3-4):305–359. doi: 10.1016/0304-4157(86)90003-1. [DOI] [PubMed] [Google Scholar]
  29. Petti L., Nilson L. A., DiMaio D. Activation of the platelet-derived growth factor receptor by the bovine papillomavirus E5 transforming protein. EMBO J. 1991 Apr;10(4):845–855. doi: 10.1002/j.1460-2075.1991.tb08017.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  31. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  32. Sarnow P., Hearing P., Anderson C. W., Halbert D. N., Shenk T., Levine A. J. Adenovirus early region 1B 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells. J Virol. 1984 Mar;49(3):692–700. doi: 10.1128/jvi.49.3.692-700.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sarnow P., Hearing P., Anderson C. W., Reich N., Levine A. J. Identification and characterization of an immunologically conserved adenovirus early region 11,000 Mr protein and its association with the nuclear matrix. J Mol Biol. 1982 Dec 15;162(3):565–583. doi: 10.1016/0022-2836(82)90389-8. [DOI] [PubMed] [Google Scholar]
  34. Wilkinson D., de Vries R. R., Madrigal J. A., Lock C. B., Morgenstern J. P., Trowsdale J., Altmann D. M. Analysis of HLA-DR glycoproteins by DNA-mediated gene transfer. Definition of DR2 beta gene products and antigen presentation to T cell clones from leprosy patients. J Exp Med. 1988 Apr 1;167(4):1442–1458. doi: 10.1084/jem.167.4.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Xu G., Livingston D. M., Krek W. Multiple members of the E2F transcription factor family are the products of oncogenes. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1357–1361. doi: 10.1073/pnas.92.5.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES