Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1991 Apr;65(4):2066–2072. doi: 10.1128/jvi.65.4.2066-2072.1991

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 Y Lin 1, D T Simmons 1
PMCID: PMC240060  PMID: 1705996

Abstract

We generated a number of simian virus 40 (SV40) mutants with single amino acid substitutions in T antigen between residues 388 and 411. All but one mutant (398LV) replicated like wild-type SV40 and gave rise to normal-size plaques. Three different mutations at residue 402 (Asp to Glu, Asn, or His) totally prevented the formation of stable complexes with the cellular protein p53 in monkey cells but had no effect on virus replication. Only one other mutation in this region, involving residue 401 (Met to Thr), slightly inhibited the formation of T-monkey p53 complexes. The three mutant T antigens with substitutions at residue 402 also formed no stable complexes with human p53 but generated low levels of complexes with mouse p53. These results indicate that residue 402 is critical for binding to monkey and human p53 proteins and is important for binding to mouse p53. We suggest that it is one of several points of contact. In cells infected with any one of the three residue 402 mutant viruses. T antigen and p53 became increasingly phosphorylated, as they were in cells infected with wild-type virus. Our data therefore show that stable T-p53 complexes are not required for replication of SV40 in culture or for enhanced phosphorylation of either protein.

Full text

PDF
2066

Images in this article

2068Image
on p.2068
2069Image
on p.2069
2069Image
on p.2069
2070Image
on p.2070

Selected References

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

  1. Aaronson S. A., Todaro G. J. Development of 3T3-like lines from Balb-c mouse embryo cultures: transformation susceptibility to SV40. J Cell Physiol. 1968 Oct;72(2):141–148. doi: 10.1002/jcp.1040720208. [DOI] [PubMed] [Google Scholar]
  2. Bischoff J. R., Friedman P. N., Marshak D. R., Prives C., Beach D. Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4766–4770. doi: 10.1073/pnas.87.12.4766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borowiec J. A., Hurwitz J. Localized melting and structural changes in the SV40 origin of replication induced by T-antigen. EMBO J. 1988 Oct;7(10):3149–3158. doi: 10.1002/j.1460-2075.1988.tb03182.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Braithwaite A. W., Sturzbecher H. W., Addison C., Palmer C., Rudge K., Jenkins J. R. Mouse p53 inhibits SV40 origin-dependent DNA replication. Nature. 1987 Oct 1;329(6138):458–460. doi: 10.1038/329458a0. [DOI] [PubMed] [Google Scholar]
  5. Brugge J. S., Butel J. S. Role of simian virus 40 gene A function in maintenance of transformation. J Virol. 1975 Mar;15(3):619–635. doi: 10.1128/jvi.15.3.619-635.1975. [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. Clark R., Lane D. P., Tjian R. Use of monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity. J Biol Chem. 1981 Nov 25;256(22):11854–11858. [PubMed] [Google Scholar]
  8. 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]
  9. Deppert W., Haug M., Steinmayer T. Modulation of p53 protein expression during cellular transformation with simian virus 40. Mol Cell Biol. 1987 Dec;7(12):4453–4463. doi: 10.1128/mcb.7.12.4453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DiMaio D., Nathans D. Cold-sensitive regulatory mutants of simian virus 40. J Mol Biol. 1980 Jun 15;140(1):129–142. doi: 10.1016/0022-2836(80)90359-9. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Gannon J. V., Lane D. P. p53 and DNA polymerase alpha compete for binding to SV40 T antigen. Nature. 1987 Oct 1;329(6138):456–458. doi: 10.1038/329456a0. [DOI] [PubMed] [Google Scholar]
  13. Giacherio D., Hager L. P. A poly(dT)-stimulated ATPase activity associated with simian virus 40 large T antigen. J Biol Chem. 1979 Sep 10;254(17):8113–8116. [PubMed] [Google Scholar]
  14. 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]
  15. Harlow E., Pim D. C., Crawford L. V. Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells. J Virol. 1981 Feb;37(2):564–573. doi: 10.1128/jvi.37.2.564-573.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kimura G., Itagaki A. Initiation and maintenance of cell transformation by simian virus 40: a viral genetic property. Proc Natl Acad Sci U S A. 1975 Feb;72(2):673–677. doi: 10.1073/pnas.72.2.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Levitsky K., Chandrasekaran K., Mora P. T., Simmons D. T. Immunoaffinity chromatography of a cellular tumor antigen from mouse neuroblastoma cells. Int J Cancer. 1983 Nov 15;32(5):597–602. doi: 10.1002/ijc.2910320513. [DOI] [PubMed] [Google Scholar]
  19. Lin J. Y., Simmons D. T. Transformation by simian virus 40 does not involve the mutational activation of p53 to an oncogenic form. Virology. 1990 May;176(1):302–305. doi: 10.1016/0042-6822(90)90258-s. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Martin R. G., Chou J. Y. Simian virus 40 functions required for the establishment and maintenance of malignant transformation. J Virol. 1975 Mar;15(3):599–612. doi: 10.1128/jvi.15.3.599-612.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McCormick F., Harlow E. Association of a murine 53,000-dalton phosphoprotein with simian virus 40 large-T antigen in transformed cells. J Virol. 1980 Apr;34(1):213–224. doi: 10.1128/jvi.34.1.213-224.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McCutchan J. H., Pagano J. S. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed] [Google Scholar]
  24. McVey D., Brizuela L., Mohr I., Marshak D. R., Gluzman Y., Beach D. Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication. Nature. 1989 Oct 12;341(6242):503–507. doi: 10.1038/341503a0. [DOI] [PubMed] [Google Scholar]
  25. Milner J., Gamble J., Cook A. p53 is associated with a 35 kD protein in cells transformed by simian virus 40. Oncogene. 1989 May;4(5):665–668. [PubMed] [Google Scholar]
  26. Mohr I. J., Stillman B., Gluzman Y. Regulation of SV40 DNA replication by phosphorylation of T antigen. EMBO J. 1987 Jan;6(1):153–160. doi: 10.1002/j.1460-2075.1987.tb04733.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Montenarh M., Vesco C., Kemmerling G., Müller D., Henning R. Regions of SV40 large T antigen necessary for oligomerization and complex formation with the cellular oncoprotein p53. FEBS Lett. 1986 Aug 11;204(1):51–55. doi: 10.1016/0014-5793(86)81386-2. [DOI] [PubMed] [Google Scholar]
  28. Myers R. M., Tjian R. Construction and analysis of simian virus 40 origins defective in tumor antigen binding and DNA replication. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6491–6495. doi: 10.1073/pnas.77.11.6491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Osborn M., Weber K. Simian virus 40 gene A function and maintenance of transformation. J Virol. 1975 Mar;15(3):636–644. doi: 10.1128/jvi.15.3.636-644.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Scheidtmann K. H., Haber A. Simian virus 40 large T antigen induces or activates a protein kinase which phosphorylates the transformation-associated protein p53. J Virol. 1990 Feb;64(2):672–679. doi: 10.1128/jvi.64.2.672-679.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schmieg F. I., Simmons D. T. Characterization of the in vitro interaction between SV40 T antigen and p53: mapping the p53 binding site. Virology. 1988 May;164(1):132–140. doi: 10.1016/0042-6822(88)90628-9. [DOI] [PubMed] [Google Scholar]
  34. Shalloway D., Kleinberger T., Livingston D. M. Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III digestion. Cell. 1980 Jun;20(2):411–422. doi: 10.1016/0092-8674(80)90627-3. [DOI] [PubMed] [Google Scholar]
  35. Simmons D. T., Chou W., Rodgers K. Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen. J Virol. 1986 Dec;60(3):888–894. doi: 10.1128/jvi.60.3.888-894.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Simmons D. T. DNA-binding region of the simian virus 40 tumor antigen. J Virol. 1986 Mar;57(3):776–785. doi: 10.1128/jvi.57.3.776-785.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Simmons D. T., Loeber G., Tegtmeyer P. Four major sequence elements of simian virus 40 large T antigen coordinate its specific and nonspecific DNA binding. J Virol. 1990 May;64(5):1973–1983. doi: 10.1128/jvi.64.5.1973-1983.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simmons D. T. Stepwise phosphorylation of the NH2-terminal region of the simian virus 40 large T antigen. J Biol Chem. 1984 Jul 10;259(13):8633–8640. [PubMed] [Google Scholar]
  39. Simmons D. T., Wun-Kim K., Young W. Identification of simian virus 40 T-antigen residues important for specific and nonspecific binding to DNA and for helicase activity. J Virol. 1990 Oct;64(10):4858–4865. doi: 10.1128/jvi.64.10.4858-4865.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Smale S. T., Tjian R. T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication. Mol Cell Biol. 1986 Nov;6(11):4077–4087. doi: 10.1128/mcb.6.11.4077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sompayrac L. M., Gurney E. G., Danna K. J. Stabilization of the 53,000-dalton nonviral tumor antigen is not required for transformation by simian virus 40. Mol Cell Biol. 1983 Feb;3(2):290–296. doi: 10.1128/mcb.3.2.290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stürzbecher H. W., Montenarh M., Henning R. Enhanced protein phosphorylation in SV40-transformed and -infected cells. Virology. 1987 Oct;160(2):445–455. doi: 10.1016/0042-6822(87)90016-x. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Tack L. C., Wright J. H., Deb S. P., Tegtmeyer P. The p53 complex from monkey cells modulates the biochemical activities of simian virus 40 large T antigen. J Virol. 1989 Mar;63(3):1310–1317. doi: 10.1128/jvi.63.3.1310-1317.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tegtmeyer P. Function of simian virus 40 gene A in transforming infection. J Virol. 1975 Mar;15(3):613–618. doi: 10.1128/jvi.15.3.613-618.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tegtmeyer P. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J Virol. 1972 Oct;10(4):591–598. doi: 10.1128/jvi.10.4.591-598.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Tjian R., Robbins A. Enzymatic activities associated with a purified simian virus 40 T antigen-related protein. Proc Natl Acad Sci U S A. 1979 Feb;76(2):610–614. doi: 10.1073/pnas.76.2.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tjian R. The binding site on SV40 DNA for a T antigen-related protein. Cell. 1978 Jan;13(1):165–179. doi: 10.1016/0092-8674(78)90147-2. [DOI] [PubMed] [Google Scholar]
  51. Virshup D. M., Kauffman M. G., Kelly T. J. Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A. EMBO J. 1989 Dec 1;8(12):3891–3898. doi: 10.1002/j.1460-2075.1989.tb08568.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wang E. H., Friedman P. N., Prives C. The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen. Cell. 1989 May 5;57(3):379–392. doi: 10.1016/0092-8674(89)90913-6. [DOI] [PubMed] [Google Scholar]
  53. Yewdell J. W., Gannon J. V., Lane D. P. Monoclonal antibody analysis of p53 expression in normal and transformed cells. J Virol. 1986 Aug;59(2):444–452. doi: 10.1128/jvi.59.2.444-452.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES