Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Mar;71(3):1888–1896. doi: 10.1128/jvi.71.3.1888-1896.1997

Adding an Rb-binding site to an N-terminally truncated simian virus 40 T antigen restores growth to high cell density, and the T common region in trans provides anchorage-independent growth and rapid growth in low serum concentrations.

M J Tevethia 1, H A Lacko 1, T D Kierstead 1, D L Thompson 1
PMCID: PMC191260  PMID: 9032319

Abstract

The simian virus 40 large T antigen is sufficient to confer on cells multiple transformed cell growth characteristics, including growth to a high cell density, rapid growth in medium containing low serum concentrations, and anchorage-independent growth. We showed previously that distinct regions of the protein were involved in conferring these properties and that removal of the first 127 amino acids of T antigen abrogated all three activities. At least three large-T-antigen transformation-related activities have been localized to that region: binding of the tumor suppressor gene product Rb and two independent activities contained within the common region shared by large T and small t antigens. The experiments described here were directed toward determining whether these were the only activities from the N terminus that were needed. To do so we reintroduced an Rb-binding region into the N-terminally truncated T antigen (T128-708) and examined the growth properties of cells immortalized by it in the presence and absence of small t antigen, which can provide the T-common-region transformation-related activities in trans. We show that an Rb-binding region consisting of amino acids 101 to 118, when introduced into a heterologous site in T128-708, is capable of physically binding Rb and that binding is sufficient for cells expressing the protein to acquire the ability to grow to a high saturation density. However, in low-serum medium, the growth rate of the cells and maximal cell density are reduced relative to those of wild-type-T-antigen-expressing cells, and the cells cannot divide without anchorage. This result suggests that although Rb binding is sufficient in the context of T128-708 to confer growth to a high density, one or more other N-terminally located T-antigen activities are needed for cells to acquire the additional growth properties. Small t antigen in trans supplied those activities. These results indicate that the T-common-region activities and Rb binding are the only activities from the T-antigen N terminus needed to restore full transforming activity to the N-terminally truncated T antigen.

Full Text

The Full Text of this article is available as a PDF (635.4 KB).

Selected References

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

  1. 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]
  2. Berger L. C., Smith D. B., Davidson I., Hwang J. J., Fanning E., Wildeman A. G. Interaction between T antigen and TEA domain of the factor TEF-1 derepresses simian virus 40 late promoter in vitro: identification of T-antigen domains important for transcription control. J Virol. 1996 Feb;70(2):1203–1212. doi: 10.1128/jvi.70.2.1203-1212.1996. [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. Cavender J. F., Conn A., Epler M., Lacko H., Tevethia M. J. Simian virus 40 large T antigen contains two independent activities that cooperate with a ras oncogene to transform rat embryo fibroblasts. J Virol. 1995 Feb;69(2):923–934. doi: 10.1128/jvi.69.2.923-934.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Christensen J. B., Imperiale M. J. Inactivation of the retinoblastoma susceptibility protein is not sufficient for the transforming function of the conserved region 2-like domain of simian virus 40 large T antigen. J Virol. 1995 Jun;69(6):3945–3948. doi: 10.1128/jvi.69.6.3945-3948.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. DeCaprio J. A., Ludlow J. W., Lynch D., Furukawa Y., Griffin J., Piwnica-Worms H., Huang C. M., Livingston D. M. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell. 1989 Sep 22;58(6):1085–1095. doi: 10.1016/0092-8674(89)90507-2. [DOI] [PubMed] [Google Scholar]
  9. Dickmanns A., Zeitvogel A., Simmersbach F., Weber R., Arthur A. K., Dehde S., Wildeman A. G., Fanning E. The kinetics of simian virus 40-induced progression of quiescent cells into S phase depend on four independent functions of large T antigen. J Virol. 1994 Sep;68(9):5496–5508. doi: 10.1128/jvi.68.9.5496-5508.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dobbelstein M., Arthur A. K., Dehde S., van Zee K., Dickmanns A., Fanning E. Intracistronic complementation reveals a new function of SV40 T antigen that co-operates with Rb and p53 binding to stimulate DNA synthesis in quiescent cells. Oncogene. 1992 May;7(5):837–847. [PubMed] [Google Scholar]
  11. Dyson N., Bernards R., Friend S. H., Gooding L. R., Hassell J. A., Major E. O., Pipas J. M., Vandyke T., Harlow E. Large T antigens of many polyomaviruses are able to form complexes with the retinoblastoma protein. J Virol. 1990 Mar;64(3):1353–1356. doi: 10.1128/jvi.64.3.1353-1356.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  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. Fanning E., Knippers R. Structure and function of simian virus 40 large tumor antigen. Annu Rev Biochem. 1992;61:55–85. doi: 10.1146/annurev.bi.61.070192.000415. [DOI] [PubMed] [Google Scholar]
  15. Figge J., Webster T., Smith T. F., Paucha E. Prediction of similar transforming regions in simian virus 40 large T, adenovirus E1A, and myc oncoproteins. J Virol. 1988 May;62(5):1814–1818. doi: 10.1128/jvi.62.5.1814-1818.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fu T. M., Bonneau R. H., Tevethia M. J., Tevethia S. S. Simian virus 40 T antigen as a carrier for the expression of cytotoxic T-lymphocyte recognition epitopes. J Virol. 1993 Nov;67(11):6866–6871. doi: 10.1128/jvi.67.11.6866-6871.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Graña X., Reddy E. P. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene. 1995 Jul 20;11(2):211–219. [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. Johnston S. D., Yu X. M., Mertz J. E. The major transcriptional transactivation domain of simian virus 40 large T antigen associates nonconcurrently with multiple components of the transcriptional preinitiation complex. J Virol. 1996 Feb;70(2):1191–1202. doi: 10.1128/jvi.70.2.1191-1202.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kalderon D., Richardson W. D., Markham A. F., Smith A. E. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature. 1984 Sep 6;311(5981):33–38. doi: 10.1038/311033a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kierstead T. D., Tevethia M. J. Association of p53 binding and immortalization of primary C57BL/6 mouse embryo fibroblasts by using simian virus 40 T-antigen mutants bearing internal overlapping deletion mutations. J Virol. 1993 Apr;67(4):1817–1829. doi: 10.1128/jvi.67.4.1817-1829.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kohrman D. C., Imperiale M. J. Simian virus 40 large T antigen stably complexes with a 185-kilodalton host protein. J Virol. 1992 Mar;66(3):1752–1760. doi: 10.1128/jvi.66.3.1752-1760.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Loeken M. R. Multiple, distinct trans-activation functions are encoded by the simian virus 40 large T and small t antigens, only some of which require the 82-residue amino-terminal common domain. J Virol. 1993 Dec;67(12):7684–7689. doi: 10.1128/jvi.67.12.7684-7689.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Loeken M. R. Simian virus 40 small t antigen trans activates the adenovirus E2A promoter by using mechanisms distinct from those used by adenovirus E1A. J Virol. 1992 Apr;66(4):2551–2555. doi: 10.1128/jvi.66.4.2551-2555.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Loeken M., Bikel I., Livingston D. M., Brady J. trans-activation of RNA polymerase II and III promoters by SV40 small t antigen. Cell. 1988 Dec 23;55(6):1171–1177. doi: 10.1016/0092-8674(88)90261-9. [DOI] [PubMed] [Google Scholar]
  27. Ludlow J. W., Shon J., Pipas J. M., Livingston D. M., DeCaprio J. A. The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T. Cell. 1990 Feb 9;60(3):387–396. doi: 10.1016/0092-8674(90)90590-b. [DOI] [PubMed] [Google Scholar]
  28. Ludlow J. W., Skuse G. R. Viral oncoprotein binding to pRB, p107, p130, and p300. Virus Res. 1995 Feb;35(2):113–121. doi: 10.1016/0168-1702(94)00094-s. [DOI] [PubMed] [Google Scholar]
  29. Manfredi J. J., Prives C. The transforming activity of simian virus 40 large tumor antigen. Biochim Biophys Acta. 1994 May 27;1198(1):65–83. doi: 10.1016/0304-419x(94)90006-x. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Montano X., Millikan R., Milhaven J. M., Newsom D. A., Ludlow J. W., Arthur A. K., Fanning E., Bikel I., Livingston D. M. Simian virus 40 small tumor antigen and an amino-terminal domain of large tumor antigen share a common transforming function. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7448–7452. doi: 10.1073/pnas.87.19.7448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moran E. A region of SV40 large T antigen can substitute for a transforming domain of the adenovirus E1A products. Nature. 1988 Jul 14;334(6178):168–170. doi: 10.1038/334168a0. [DOI] [PubMed] [Google Scholar]
  33. Peden K. W., Pipas J. M. Simian virus 40 mutants with amino-acid substitutions near the amino terminus of large T antigen. Virus Genes. 1992 Apr;6(2):107–118. doi: 10.1007/BF01703060. [DOI] [PubMed] [Google Scholar]
  34. Peden K. W., Spence S. L., Tack L. C., Cartwright C. A., Srinivasan A., Pipas J. M. A DNA replication-positive mutant of simian virus 40 that is defective for transformation and the production of infectious virions. J Virol. 1990 Jun;64(6):2912–2921. doi: 10.1128/jvi.64.6.2912-2921.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Pipas J. M., Peden K. W., Nathans D. Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene. Mol Cell Biol. 1983 Feb;3(2):203–213. doi: 10.1128/mcb.3.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Porrás A., Bennett J., Howe A., Tokos K., Bouck N., Henglein B., Sathyamangalam S., Thimmapaya B., Rundell K. A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays. J Virol. 1996 Oct;70(10):6902–6908. doi: 10.1128/jvi.70.10.6902-6908.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rutila J. E., Imperiale M. J., Brockman W. W. Replication and transformation functions of in vitro-generated simian virus 40 large T antigen mutants. J Virol. 1986 May;58(2):526–535. doi: 10.1128/jvi.58.2.526-535.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sawai E. T., Rasmussen G., Butel J. S. Construction of SV40 deletion mutants and delimitation of the binding domain for heat shock protein to the amino terminus of large T-antigen. Virus Res. 1994 Mar;31(3):367–378. doi: 10.1016/0168-1702(94)90029-9. [DOI] [PubMed] [Google Scholar]
  40. Schulze A., Zerfass-Thome K., Bergès J., Middendorp S., Jansen-Dürr P., Henglein B. Anchorage-dependent transcription of the cyclin A gene. Mol Cell Biol. 1996 Sep;16(9):4632–4638. doi: 10.1128/mcb.16.9.4632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sompayrac L., Danna K. J. Simian virus 40 deletion mutants that transform with reduced efficiency. Mol Cell Biol. 1983 Mar;3(3):484–489. doi: 10.1128/mcb.3.3.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Tavis J. E., Trowbridge P. W., Frisque R. J. Converting the JCV T antigen Rb binding domain to that of SV40 does not alter JCV's limited transforming activity but does eliminate viral viability. Virology. 1994 Mar;199(2):384–392. doi: 10.1006/viro.1994.1136. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Wang W. B., Bikel I., Marsilio E., Newsome D., Livingston D. M. Transrepression of RNA polymerase II promoters by the simian virus 40 small t antigen. J Virol. 1994 Oct;68(10):6180–6187. doi: 10.1128/jvi.68.10.6180-6187.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zalvide J., DeCaprio J. A. Role of pRb-related proteins in simian virus 40 large-T-antigen-mediated transformation. Mol Cell Biol. 1995 Oct;15(10):5800–5810. doi: 10.1128/mcb.15.10.5800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zerrahn J., Deppert W. Analysis of simian virus 40 small t antigen-induced progression of rat F111 cells minimally transformed by large T antigen. J Virol. 1993 Mar;67(3):1555–1563. doi: 10.1128/jvi.67.3.1555-1563.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zerrahn J., Knippschild U., Winkler T., Deppert W. Independent expression of the transforming amino-terminal domain of SV40 large I antigen from an alternatively spliced third SV40 early mRNA. EMBO J. 1993 Dec;12(12):4739–4746. doi: 10.1002/j.1460-2075.1993.tb06162.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES