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. 1987 Jul;61(7):2076–2083. doi: 10.1128/jvi.61.7.2076-2083.1987

Oligomerization and origin DNA-binding activity of simian virus 40 large T antigen.

R Runzler, S Thompson, E Fanning
PMCID: PMC254227  PMID: 3035209

Abstract

Simian virus 40 (SV40) large tumor antigen (T antigen) exists in multiple molecular forms, some of which are separable by zone velocity sedimentation of soluble extracts from infected monkey cells. Three subclasses of this antigen from SV40-infected monkey cells have been separated and characterized: the 5S, 7S, and 14S forms. Newly synthesized T antigen occurs primarily in the 5S form. Chemical cross-linking provided evidence that the 14S form is primarily a tetramer, whereas the 5S and 7S forms could not be cross-linked into oligomers. The DNA-binding properties of each subclass were investigated after immunopurification. The affinities of the three forms for SV40 DNA and for a synthetic 19-base-pair sequence from binding site I are very similar (equilibrium dissociation constant [KD], 0.3 to 0.4 nM). The specific activity of DNA binding was greatest for the 5S and 7S subclasses and least for the 14S subclass. Moreover, the specific activity of the 5S and 7S subclasses increased sharply at about 40 h after infection, whereas the activity of the 14S subclass was maintained at a constant low level throughout infection. A model relating oligomerization and DNA binding of T antigen in infected cells is presented.

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Selected References

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

  1. Adams J. C. Heavy metal intensification of DAB-based HRP reaction product. J Histochem Cytochem. 1981 Jun;29(6):775–775. doi: 10.1177/29.6.7252134. [DOI] [PubMed] [Google Scholar]
  2. Baumann E. A., Hand R. Phosphorylation and dephosphorylation alter the structure of D2 hybrid T antigen. J Virol. 1982 Oct;44(1):78–87. doi: 10.1128/jvi.44.1.78-87.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baumgartner I., Kuhn C., Fanning E. Identification and characterization of fast-sedimenting SV40 nucleoprotein complexes. Virology. 1979 Jul 15;96(1):54–63. doi: 10.1016/0042-6822(79)90172-7. [DOI] [PubMed] [Google Scholar]
  4. Bradley M. K., Griffin J. D., Livingston D. M. Relationship of oligomerization to enzymatic and DNA-binding properties of the SV40 large T antigen. Cell. 1982 Jan;28(1):125–134. doi: 10.1016/0092-8674(82)90382-8. [DOI] [PubMed] [Google Scholar]
  5. Bradley M. K., Hudson J., Villanueva M. S., Livingston D. M. Specific in vitro adenylylation of the simian virus 40 large tumor antigen. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6574–6578. doi: 10.1073/pnas.81.21.6574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Brady J., Khoury G. trans Activation of the simian virus 40 late transcription unit by T-antigen. Mol Cell Biol. 1985 Jun;5(6):1391–1399. doi: 10.1128/mcb.5.6.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Covey L., Choi Y., Prives C. Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells. Mol Cell Biol. 1984 Jul;4(7):1384–1392. doi: 10.1128/mcb.4.7.1384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. DiMaio D., Nathans D. Regulatory mutants of simian virus 40. Effect of mutations at a T antigen binding site on DNA replication and expression of viral genes. J Mol Biol. 1982 Apr 15;156(3):531–548. doi: 10.1016/0022-2836(82)90265-0. [DOI] [PubMed] [Google Scholar]
  11. Dixon R. A., Nathans D. Purification of simian virus 40 large T antigen by immunoaffinity chromatography. J Virol. 1985 Mar;53(3):1001–1004. doi: 10.1128/jvi.53.3.1001-1004.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dorn A., Brauer D., Otto B., Fanning E., Knippers R. Subclasses of simian-virus-40 large tumor antigen. Partial purification and DNA-binding properties of two subclasses of tumor antigen from productively infected cells. Eur J Biochem. 1982 Nov;128(1):53–62. [PubMed] [Google Scholar]
  13. Fanning E., Nowak B., Burger C. Detection and characterization of multiple forms of simian virus 40 large T antigen. J Virol. 1981 Jan;37(1):92–102. doi: 10.1128/jvi.37.1.92-102.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fanning E., Westphal K. H., Brauer D., Cörlin D. Subclasses of simian virus 40 large T antigen: differential binding of two subclasses of T antigen from productively infected cells to viral and cellular DNA. EMBO J. 1982;1(9):1023–1028. doi: 10.1002/j.1460-2075.1982.tb01290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gidoni D., Scheller A., Barnet B., Hantzopoulos P., Oren M., Prives C. Different forms of simian virus 40 large tumor antigen varying in their affinities for DNA. J Virol. 1982 May;42(2):456–466. doi: 10.1128/jvi.42.2.456-466.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goldman N., Brown M., Khoury G. Modification of SV40 T antigen by poly ADP-ribosylation. Cell. 1981 May;24(2):567–572. doi: 10.1016/0092-8674(81)90347-0. [DOI] [PubMed] [Google Scholar]
  17. Greenspan D. S., Carroll R. B. Complex of simian virus 40 large tumor antigen and 48,000-dalton host tumor antigen. Proc Natl Acad Sci U S A. 1981 Jan;78(1):105–109. doi: 10.1073/pnas.78.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gurney E. G., Tamowski S., Deppert W. Antigenic binding sites of monoclonal antibodies specific for simian virus 40 large T antigen. J Virol. 1986 Mar;57(3):1168–1172. doi: 10.1128/jvi.57.3.1168-1172.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hansen U., Tenen D. G., Livingston D. M., Sharp P. A. T antigen repression of SV40 early transcription from two promoters. Cell. 1981 Dec;27(3 Pt 2):603–613. doi: 10.1016/0092-8674(81)90402-5. [DOI] [PubMed] [Google Scholar]
  20. Hinzpeter M., Fanning E., Deppert W. A new sensitive target-bound DNA binding assay for SV40 large T antigen. Virology. 1986 Jan 15;148(1):159–167. doi: 10.1016/0042-6822(86)90411-3. [DOI] [PubMed] [Google Scholar]
  21. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  22. Huber B., Vakalopoulou E., Burger C., Fanning E. Identification and biochemical analysis of DNA replication-defective large T antigens from SV40-transformed cells. Virology. 1985 Oct 30;146(2):188–202. doi: 10.1016/0042-6822(85)90003-0. [DOI] [PubMed] [Google Scholar]
  23. Jones K. A., Tjian R. Essential contact residues within SV40 large T antigen binding sites I and II identified by alkylation-interference. Cell. 1984 Jan;36(1):155–162. doi: 10.1016/0092-8674(84)90084-9. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Keller J. M., Alwine J. C. Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation. Mol Cell Biol. 1985 Aug;5(8):1859–1869. doi: 10.1128/mcb.5.8.1859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klockmann U., Deppert W. Acylated simian virus 40 large T-antigen: a new subclass associated with a detergent-resistant lamina of the plasma membrane. EMBO J. 1983;2(7):1151–1157. doi: 10.1002/j.1460-2075.1983.tb01560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Margolskee R. F., Nathans D. Simian virus 40 mutant T antigens with relaxed specificity for the nucleotide sequence at the viral DNA origin of replication. J Virol. 1984 Feb;49(2):386–393. doi: 10.1128/jvi.49.2.386-393.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mastrangelo I. A., Hough P. V., Wilson V. G., Wall J. S., Hainfeld J. F., Tegtmeyer P. Monomers through trimers of large tumor antigen bind in region I and monomers through tetramers bind in region II of simian virus 40 origin of replication DNA as stable structures in solution. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3626–3630. doi: 10.1073/pnas.82.11.3626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McKay R. D. Binding of a simian virus 40 T antigen-related protein to DNA. J Mol Biol. 1981 Jan 25;145(3):471–488. doi: 10.1016/0022-2836(81)90540-4. [DOI] [PubMed] [Google Scholar]
  31. Montenarh M., Henning R. Disaggregation and reconstitution of oligomeric complexes of simian virus 40 large T-antigen. J Gen Virol. 1983 Jan;64(Pt 1):241–246. doi: 10.1099/0022-1317-64-1-241. [DOI] [PubMed] [Google Scholar]
  32. Myers R. M., Williams R. C., Tjian R. Oligomeric structure of a simian virus 40 T antigen in free form and bound to DNA. J Mol Biol. 1981 Jun 5;148(4):347–353. doi: 10.1016/0022-2836(81)90180-7. [DOI] [PubMed] [Google Scholar]
  33. Oren M., Winocour E., Prives C. Differential affinities of simian virus 40 large tumor antigen for DNA. Proc Natl Acad Sci U S A. 1980 Jan;77(1):220–224. doi: 10.1073/pnas.77.1.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Putney S. D., Sauer R. T., Schimmel P. R. Purification and properties of alanine tRNA synthetase from Escherichia coli A tetramer of identical subunits. J Biol Chem. 1981 Jan 10;256(1):198–204. [PubMed] [Google Scholar]
  35. Rio D., Robbins A., Myers R., Tjian R. Regulation of simian virus 40 early transcription in vitro by a purified tumor antigen. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5706–5710. doi: 10.1073/pnas.77.10.5706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Robb J. A., Huebner K. Effect of cell chromosome number on simian virus 40 replication. Exp Cell Res. 1973 Sep;81(1):120–126. doi: 10.1016/0014-4827(73)90118-3. [DOI] [PubMed] [Google Scholar]
  37. Ryder K., Vakalopoulou E., Mertz R., Mastrangelo I., Hough P., Tegtmeyer P., Fanning E. Seventeen base pairs of region I encode a novel tripartite binding signal for SV40 T antigen. Cell. 1985 Sep;42(2):539–548. doi: 10.1016/0092-8674(85)90111-4. [DOI] [PubMed] [Google Scholar]
  38. Scheidtmann K. H., Echle B., Walter G. Simian virus 40 large T antigen is phosphorylated at multiple sites clustered in two separate regions. J Virol. 1982 Oct;44(1):116–133. doi: 10.1128/jvi.44.1.116-133.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Scheidtmann K. H., Hardung M., Echle B., Walter G. DNA-binding activity of simian virus 40 large T antigen correlates with a distinct phosphorylation state. J Virol. 1984 Apr;50(1):1–12. doi: 10.1128/jvi.50.1.1-12.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Scheller A., Covey L., Barnet B., Prives C. A small subclass of SV40 T antigen binds to the viral origin of replication. Cell. 1982 Jun;29(2):375–383. doi: 10.1016/0092-8674(82)90154-4. [DOI] [PubMed] [Google Scholar]
  41. Shortle D. R., Margolskee R. F., Nathans D. Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6128–6131. doi: 10.1073/pnas.76.12.6128. [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. Staufenbiel M., Deppert W. Different structural systems of the nucleus are targets for SV40 large T antigen. Cell. 1983 May;33(1):173–181. doi: 10.1016/0092-8674(83)90346-x. [DOI] [PubMed] [Google Scholar]
  44. Twigg A. J., Sherratt D. Trans-complementable copy-number mutants of plasmid ColE1. Nature. 1980 Jan 10;283(5743):216–218. doi: 10.1038/283216a0. [DOI] [PubMed] [Google Scholar]
  45. Vogt B., Vakalopoulou E., Fanning E. Allosteric control of simian virus 40 T-antigen binding to viral origin DNA. J Virol. 1986 Jun;58(3):765–772. doi: 10.1128/jvi.58.3.765-772.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wachter M., Riedle G., Henning R. Functional implications of oligomerization of simian virus 40 large T antigen during lytic virus infection. J Virol. 1985 Nov;56(2):520–526. doi: 10.1128/jvi.56.2.520-526.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  48. van Roy F., Fransen L., Fiers W. Improved localization of phosphorylation sites in simian virus 40 large T antigen. J Virol. 1983 Jan;45(1):315–331. doi: 10.1128/jvi.45.1.315-331.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

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