Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1992 May;66(5):2780–2791. doi: 10.1128/jvi.66.5.2780-2791.1992

Transformation of a continuous rat embryo fibroblast cell line requires three separate domains of simian virus 40 large T antigen.

J Zhu 1, P W Rice 1, L Gorsch 1, M Abate 1, C N Cole 1
PMCID: PMC241034  PMID: 1313902

Abstract

Mouse C3H 10T1/2 cells and the established rat embryo fibroblast cell line REF-52 are two cell lines widely used in studies of viral transformation. Studies have shown that transformation of 10T1/2 cells requires only the amino-terminal 121 amino acids of simian virus 40 (SV40) large T antigen, while transformation of REF-52 cells requires considerably more of large T antigen, extending from near the N terminus to beyond residue 600. The ability of a large set of linker insertion, small deletion, and point mutants of SV40 T antigen to transform these two cell lines and to bind p105Rb was determined. Transformation of 10T1/2 cells was greatly reduced by mutations within the first exon of the gene for large T antigen but was only modestly affected by mutations affecting the p105Rb binding site or the p53 binding region. All mutants defective for transformation of 10T1/2 cells were also defective for transformation of REF-52 cells. In addition, mutants whose T antigens had alterations in the Rb binding site showed a substantial reduction in transformation of REF-52 cells, and the degree of this reduction could be correlated with the ability of the mutant T antigens to bind p105Rb. There was a tight correlation between the ability of mutants to transform REF-52 cells and the ability of their T antigens to bind p53. These results demonstrate that multiple regions of large T antigen are required for full transformation by SV40.

Full text

PDF
2780

Images in this article

2784Image
on p.2784
2786Image
on p.2786
2786Image
on p.2786

Selected References

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

  1. Alwine J. C. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins. Mol Cell Biol. 1985 May;5(5):1034–1042. doi: 10.1128/mcb.5.5.1034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arthur A. K., Höss A., Fanning E. Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids. J Virol. 1988 Jun;62(6):1999–2006. doi: 10.1128/jvi.62.6.1999-2006.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bikel I., Montano X., Agha M. E., Brown M., McCormack M., Boltax J., Livingston D. M. SV40 small t antigen enhances the transformation activity of limiting concentrations of SV40 large T antigen. Cell. 1987 Jan 30;48(2):321–330. doi: 10.1016/0092-8674(87)90435-1. [DOI] [PubMed] [Google Scholar]
  4. Borowiec J. A., Dean F. B., Bullock P. A., Hurwitz J. Binding and unwinding--how T antigen engages the SV40 origin of DNA replication. Cell. 1990 Jan 26;60(2):181–184. doi: 10.1016/0092-8674(90)90730-3. [DOI] [PubMed] [Google Scholar]
  5. Brady J., Bolen J. B., Radonovich M., Salzman N., Khoury G. Stimulation of simian virus 40 late gene expression by simian virus 40 tumor antigen. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2040–2044. doi: 10.1073/pnas.81.7.2040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen S., Paucha E. Identification of a region of simian virus 40 large T antigen required for cell transformation. J Virol. 1990 Jul;64(7):3350–3357. doi: 10.1128/jvi.64.7.3350-3357.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cherington V., Brown M., Paucha E., St Louis J., Spiegelman B. M., Roberts T. M. Separation of simian virus 40 large-T-antigen-transforming and origin-binding functions from the ability to block differentiation. Mol Cell Biol. 1988 Mar;8(3):1380–1384. doi: 10.1128/mcb.8.3.1380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cole C. N., Tornow J., Clark R., Tjian R. Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A. J Virol. 1986 Feb;57(2):539–546. doi: 10.1128/jvi.57.2.539-546.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DeCaprio J. A., Ludlow J. W., Figge J., Shew J. Y., Huang C. M., Lee W. H., Marsilio E., Paucha E., Livingston D. M. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell. 1988 Jul 15;54(2):275–283. doi: 10.1016/0092-8674(88)90559-4. [DOI] [PubMed] [Google Scholar]
  10. Dornreiter I., Höss A., Arthur A. K., Fanning E. SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha. EMBO J. 1990 Oct;9(10):3329–3336. doi: 10.1002/j.1460-2075.1990.tb07533.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dyson N., Buchkovich K., Whyte P., Harlow E. The cellular 107K protein that binds to adenovirus E1A also associates with the large T antigens of SV40 and JC virus. Cell. 1989 Jul 28;58(2):249–255. doi: 10.1016/0092-8674(89)90839-8. [DOI] [PubMed] [Google Scholar]
  12. Dyson N., Howley P. M., Münger K., Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 1989 Feb 17;243(4893):934–937. doi: 10.1126/science.2537532. [DOI] [PubMed] [Google Scholar]
  13. Ewen M. E., Ludlow J. W., Marsilio E., DeCaprio J. A., Millikan R. C., Cheng S. H., Paucha E., Livingston D. M. An N-terminal transformation-governing sequence of SV40 large T antigen contributes to the binding of both p110Rb and a second cellular protein, p120. Cell. 1989 Jul 28;58(2):257–267. doi: 10.1016/0092-8674(89)90840-4. [DOI] [PubMed] [Google Scholar]
  14. Franza B. R., Jr, Maruyama K., Garrels J. I., Ruley H. E. In vitro establishment is not a sufficient prerequisite for transformation by activated ras oncogenes. Cell. 1986 Feb 14;44(3):409–418. doi: 10.1016/0092-8674(86)90462-9. [DOI] [PubMed] [Google Scholar]
  15. Green M., Loewenstein P. M., Pusztai R., Symington J. S. An adenovirus E1A protein domain activates transcription in vivo and in vitro in the absence of protein synthesis. Cell. 1988 Jun 17;53(6):921–926. doi: 10.1016/s0092-8674(88)90429-1. [DOI] [PubMed] [Google Scholar]
  16. Hirakawa T., Ruley H. E. Rescue of cells from ras oncogene-induced growth arrest by a second, complementing, oncogene. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1519–1523. doi: 10.1073/pnas.85.5.1519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hollstein M., Sidransky D., Vogelstein B., Harris C. C. p53 mutations in human cancers. Science. 1991 Jul 5;253(5015):49–53. doi: 10.1126/science.1905840. [DOI] [PubMed] [Google Scholar]
  18. Hu Q. J., Dyson N., Harlow E. The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations. EMBO J. 1990 Apr;9(4):1147–1155. doi: 10.1002/j.1460-2075.1990.tb08221.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hunter T. Cooperation between oncogenes. Cell. 1991 Jan 25;64(2):249–270. doi: 10.1016/0092-8674(91)90637-e. [DOI] [PubMed] [Google Scholar]
  20. JENSEN F. C., GIRARDI A. J., GILDEN R. V., KOPROWSKI H. INFECTION OF HUMAN AND SIMIAN TISSUE CULTURES WITH ROUS SARCOMA VIRUS. Proc Natl Acad Sci U S A. 1964 Jul;52:53–59. doi: 10.1073/pnas.52.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
  22. Kalderon D., Smith A. E. In vitro mutagenesis of a putative DNA binding domain of SV40 large-T. Virology. 1984 Nov;139(1):109–137. doi: 10.1016/0042-6822(84)90334-9. [DOI] [PubMed] [Google Scholar]
  23. Keller J. M., Alwine J. C. Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication. Cell. 1984 Feb;36(2):381–389. doi: 10.1016/0092-8674(84)90231-9. [DOI] [PubMed] [Google Scholar]
  24. Kohl N. E., Ruley H. E. Role of c-myc in the transformation of REF52 cells by viral and cellular oncogenes. Oncogene. 1987;2(1):41–48. [PubMed] [Google Scholar]
  25. Land H., Parada L. F., Weinberg R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature. 1983 Aug 18;304(5927):596–602. doi: 10.1038/304596a0. [DOI] [PubMed] [Google Scholar]
  26. Lanford R. E., Butel J. S. Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell. 1984 Jul;37(3):801–813. doi: 10.1016/0092-8674(84)90415-x. [DOI] [PubMed] [Google Scholar]
  27. Lanford R. E., Wong C., Butel J. S. Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells. Mol Cell Biol. 1985 May;5(5):1043–1050. doi: 10.1128/mcb.5.5.1043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lillie J. W., Green M. R. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. doi: 10.1038/338039a0. [DOI] [PubMed] [Google Scholar]
  29. Lillie J. W., Loewenstein P. M., Green M. R., Green M. Functional domains of adenovirus type 5 E1a proteins. Cell. 1987 Sep 25;50(7):1091–1100. doi: 10.1016/0092-8674(87)90175-9. [DOI] [PubMed] [Google Scholar]
  30. Lin J. Y., Simmons D. T. Stable T-p53 complexes are not required for replication of simian virus 40 in culture or for enhanced phosphorylation of T antigen and p53. J Virol. 1991 Apr;65(4):2066–2072. doi: 10.1128/jvi.65.4.2066-2072.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu F., Green M. R. A specific member of the ATF transcription factor family can mediate transcription activation by the adenovirus E1a protein. Cell. 1990 Jun 29;61(7):1217–1224. doi: 10.1016/0092-8674(90)90686-9. [DOI] [PubMed] [Google Scholar]
  32. Loeber G., Parsons R., Tegtmeyer P. The zinc finger region of simian virus 40 large T antigen. J Virol. 1989 Jan;63(1):94–100. doi: 10.1128/jvi.63.1.94-100.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Logan J., Nicolas J. C., Topp W. C., Girard M., Shenk T., Levine A. J. Transformation by adenovirus early region 2A temperature-sensitive mutants and their revertants. Virology. 1981 Dec;115(2):419–422. doi: 10.1016/0042-6822(81)90126-4. [DOI] [PubMed] [Google Scholar]
  34. Ludlow J. W., DeCaprio J. A., Huang C. M., Lee W. H., Paucha E., Livingston D. M. SV40 large T antigen binds preferentially to an underphosphorylated member of the retinoblastoma susceptibility gene product family. Cell. 1989 Jan 13;56(1):57–65. doi: 10.1016/0092-8674(89)90983-5. [DOI] [PubMed] [Google Scholar]
  35. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  36. Manos M. M., Gluzman Y. Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants. J Virol. 1985 Jan;53(1):120–127. doi: 10.1128/jvi.53.1.120-127.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Manos M. M., Gluzman Y. Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation. Mol Cell Biol. 1984 Jun;4(6):1125–1133. doi: 10.1128/mcb.4.6.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Marsilio E., Cheng S. H., Schaffhausen B., Paucha E., Livingston D. M. The T/t common region of simian virus 40 large T antigen contains a distinct transformation-governing sequence. J Virol. 1991 Oct;65(10):5647–5652. doi: 10.1128/jvi.65.10.5647-5652.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Michalovitz D., Fischer-Fantuzzi L., Vesco C., Pipas J. M., Oren M. Activated Ha-ras can cooperate with defective simian virus 40 in the transformation of nonestablished rat embryo fibroblasts. J Virol. 1987 Aug;61(8):2648–2654. doi: 10.1128/jvi.61.8.2648-2654.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Moran E., Mathews M. B. Multiple functional domains in the adenovirus E1A gene. Cell. 1987 Jan 30;48(2):177–178. doi: 10.1016/0092-8674(87)90418-1. [DOI] [PubMed] [Google Scholar]
  41. Münger K., Werness B. A., Dyson N., Phelps W. C., Harlow E., Howley P. M. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 1989 Dec 20;8(13):4099–4105. doi: 10.1002/j.1460-2075.1989.tb08594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Peden K. W., Srinivasan A., Farber J. M., Pipas J. M. Mutants with changes within or near a hydrophobic region of simian virus 40 large tumor antigen are defective for binding cellular protein p53. Virology. 1989 Jan;168(1):13–21. doi: 10.1016/0042-6822(89)90398-x. [DOI] [PubMed] [Google Scholar]
  43. Prives C. The replication functions of SV40 T antigen are regulated by phosphorylation. Cell. 1990 Jun 1;61(5):735–738. doi: 10.1016/0092-8674(90)90179-i. [DOI] [PubMed] [Google Scholar]
  44. Reznikoff C. A., Brankow D. W., Heidelberger C. Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division. Cancer Res. 1973 Dec;33(12):3231–3238. [PubMed] [Google Scholar]
  45. Robbins P. D., Rio D. C., Botchan M. R. trans Activation of the simian virus 40 enhancer. Mol Cell Biol. 1986 Apr;6(4):1283–1295. doi: 10.1128/mcb.6.4.1283. [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. Schneider J. F., Fisher F., Goding C. R., Jones N. C. Mutational analysis of the adenovirus E1a gene: the role of transcriptional regulation in transformation. EMBO J. 1987 Jul;6(7):2053–2060. doi: 10.1002/j.1460-2075.1987.tb02470.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sompayrac L., Danna K. J. The amino-terminal 147 amino acids of SV40 large T antigen transform secondary rat embryo fibroblasts. Virology. 1991 Mar;181(1):412–415. doi: 10.1016/0042-6822(91)90516-e. [DOI] [PubMed] [Google Scholar]
  49. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  50. Srinivasan A., Peden K. W., Pipas J. M. The large tumor antigen of simian virus 40 encodes at least two distinct transforming functions. J Virol. 1989 Dec;63(12):5459–5463. doi: 10.1128/jvi.63.12.5459-5463.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tack L. C., Cartwright C. A., Wright J. H., Eckhart W., Peden K. W., Srinivasan A., Pipas J. M. Properties of a simian virus 40 mutant T antigen substituted in the hydrophobic region: defective ATPase and oligomerization activities and altered phosphorylation accompany an inability to complex with cellular p53. J Virol. 1989 Aug;63(8):3362–3367. doi: 10.1128/jvi.63.8.3362-3367.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tevethia M. J., Pipas J. M., Kierstead T., Cole C. Requirements for immortalization of primary mouse embryo fibroblasts probed with mutants bearing deletions in the 3' end of SV40 gene A. Virology. 1988 Jan;162(1):76–89. doi: 10.1016/0042-6822(88)90396-0. [DOI] [PubMed] [Google Scholar]
  53. Thompson D. L., Kalderon D., Smith A. E., Tevethia M. J. Dissociation of Rb-binding and anchorage-independent growth from immortalization and tumorigenicity using SV40 mutants producing N-terminally truncated large T antigens. Virology. 1990 Sep;178(1):15–34. doi: 10.1016/0042-6822(90)90375-2. [DOI] [PubMed] [Google Scholar]
  54. Tornow J., Cole C. N. Intracistronic complementation in the simian virus 40 A gene. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6312–6316. doi: 10.1073/pnas.80.20.6312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Whyte P., Buchkovich K. J., Horowitz J. M., Friend S. H., Raybuck M., Weinberg R. A., Harlow E. Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature. 1988 Jul 14;334(6178):124–129. doi: 10.1038/334124a0. [DOI] [PubMed] [Google Scholar]
  56. Whyte P., Ruley H. E., Harlow E. Two regions of the adenovirus early region 1A proteins are required for transformation. J Virol. 1988 Jan;62(1):257–265. doi: 10.1128/jvi.62.1.257-265.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Whyte P., Williamson N. M., Harlow E. Cellular targets for transformation by the adenovirus E1A proteins. Cell. 1989 Jan 13;56(1):67–75. doi: 10.1016/0092-8674(89)90984-7. [DOI] [PubMed] [Google Scholar]
  58. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yaciuk P., Carter M. C., Pipas J. M., Moran E. Simian virus 40 large-T antigen expresses a biological activity complementary to the p300-associated transforming function of the adenovirus E1A gene products. Mol Cell Biol. 1991 Apr;11(4):2116–2124. doi: 10.1128/mcb.11.4.2116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. 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]
  61. Zhu J. Y., Abate M., Rice P. W., Cole C. N. The ability of simian virus 40 large T antigen to immortalize primary mouse embryo fibroblasts cosegregates with its ability to bind to p53. J Virol. 1991 Dec;65(12):6872–6880. doi: 10.1128/jvi.65.12.6872-6880.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zhu J. Y., Cole C. N. Linker insertion mutants of simian virus 40 large T antigen that show trans-dominant interference with wild-type large T antigen map to multiple sites within the T-antigen gene. J Virol. 1989 Nov;63(11):4777–4786. doi: 10.1128/jvi.63.11.4777-4786.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zhu J. Y., Rice P. W., Chamberlain M., Cole C. N. Mapping the transcriptional transactivation function of simian virus 40 large T antigen. J Virol. 1991 Jun;65(6):2778–2790. doi: 10.1128/jvi.65.6.2778-2790.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES