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. 1988 Sep;62(9):3109–3119. doi: 10.1128/jvi.62.9.3109-3119.1988

Expression and complex formation of simian virus 40 large T antigen and mouse p53 in insect cells.

D R O'Reilly 1, L K Miller 1
PMCID: PMC253427  PMID: 2457111

Abstract

Recombinant baculoviruses were constructed which express simian virus 40 large T antigen (SVT-Ag) or murine p53 to high levels in infected insect cells. Characterization of the expressed proteins revealed that they display many properties of the corresponding mammalian-derived proteins. Both proteins are of wild-type size, localize to the nucleus, are recognized by several SVT-Ag- or p53-specific monoclonal antibodies, and are phosphorylated in this system. Complexes are formed between baculovirus-derived SVT-Ag and p53 after coinfection of insect cells with both recombinant viruses. After infection of insect cells with either virus individually, each protein can self-associate to form a variety of oligomeric species. Pulse-chase experiments indicated that both SVT-Ag and p53 are highly stable in insect cells, even in the absence of complex formation.

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Selected References

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

  1. 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]
  2. Carroll R. B., Gurney E. G. Time-dependent maturation of the simian virus 40 large T antigen-p53 complex studied by using monoclonal antibodies. J Virol. 1982 Nov;44(2):565–573. doi: 10.1128/jvi.44.2.565-573.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  4. Dean F. B., Bullock P., Murakami Y., Wobbe C. R., Weissbach L., Hurwitz J. Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication. Proc Natl Acad Sci U S A. 1987 Jan;84(1):16–20. doi: 10.1073/pnas.84.1.16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eliyahu D., Raz A., Gruss P., Givol D., Oren M. Participation of p53 cellular tumour antigen in transformation of normal embryonic cells. Nature. 1984 Dec 13;312(5995):646–649. doi: 10.1038/312646a0. [DOI] [PubMed] [Google Scholar]
  6. Estes M. K., Crawford S. E., Penaranda M. E., Petrie B. L., Burns J. W., Chan W. K., Ericson B., Smith G. E., Summers M. D. Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system. J Virol. 1987 May;61(5):1488–1494. doi: 10.1128/jvi.61.5.1488-1494.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Greenfield C., Patel G., Clark S., Jones N., Waterfield M. D. Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector. EMBO J. 1988 Jan;7(1):139–146. doi: 10.1002/j.1460-2075.1988.tb02793.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grässer F. A., Mann K., Walter G. Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding. J Virol. 1987 Nov;61(11):3373–3380. doi: 10.1128/jvi.61.11.3373-3380.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gurney E. G., Harrison R. O., Fenno J. Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct sublcasses of large T antigen and for similarities among nonviral T antigens. J Virol. 1980 Jun;34(3):752–763. doi: 10.1128/jvi.34.3.752-763.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Hu S. I., Kosowski S. G., Schaaf K. F. Expression of envelope glycoproteins of human immunodeficiency virus by an insect virus vector. J Virol. 1987 Nov;61(11):3617–3620. doi: 10.1128/jvi.61.11.3617-3620.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jeang K. T., Giam C. Z., Nerenberg M., Khoury G. Abundant synthesis of functional human T-cell leukemia virus type I p40x protein in eucaryotic cells by using a baculovirus expression vector. J Virol. 1987 Mar;61(3):708–713. doi: 10.1128/jvi.61.3.708-713.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jeang K. T., Holmgren-Konig M., Khoury G. A baculovirus vector can express intron-containing genes. J Virol. 1987 May;61(5):1761–1764. doi: 10.1128/jvi.61.5.1761-1764.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jenkins J. R., Rudge K., Chumakov P., Currie G. A. The cellular oncogene p53 can be activated by mutagenesis. 1985 Oct 31-Nov 6Nature. 317(6040):816–818. doi: 10.1038/317816a0. [DOI] [PubMed] [Google Scholar]
  18. Jenkins J. R., Rudge K., Currie G. A. Cellular immortalization by a cDNA clone encoding the transformation-associated phosphoprotein p53. Nature. 1984 Dec 13;312(5995):651–654. doi: 10.1038/312651a0. [DOI] [PubMed] [Google Scholar]
  19. Kaczmarek L., Oren M., Baserga R. Co-operation between the p53 protein tumor antigen and platelet-poor plasma in the induction of cellular DNA synthesis. Exp Cell Res. 1986 Jan;162(1):268–272. doi: 10.1016/0014-4827(86)90445-3. [DOI] [PubMed] [Google Scholar]
  20. Kuroda K., Hauser C., Rott R., Klenk H. D., Doerfler W. Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector. EMBO J. 1986 Jun;5(6):1359–1365. doi: 10.1002/j.1460-2075.1986.tb04367.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Lee H. H., Miller L. K. Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus. J Virol. 1978 Sep;27(3):754–767. doi: 10.1128/jvi.27.3.754-767.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Madisen L., Travis B., Hu S. L., Purchio A. F. Expression of the human immunodeficiency virus gag gene in insect cells. Virology. 1987 May;158(1):248–250. doi: 10.1016/0042-6822(87)90262-5. [DOI] [PubMed] [Google Scholar]
  24. Maeda S., Kawai T., Obinata M., Fujiwara H., Horiuchi T., Saeki Y., Sato Y., Furusawa M. Production of human alpha-interferon in silkworm using a baculovirus vector. Nature. 1985 Jun 13;315(6020):592–594. doi: 10.1038/315592a0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Meek D. W., Eckhart W. Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells. Mol Cell Biol. 1988 Jan;8(1):461–465. doi: 10.1128/mcb.8.1.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Michalovitz D., Eliyahu D., Oren M. Overproduction of protein p53 contributes to simian virus 40-mediated transformation. Mol Cell Biol. 1986 Oct;6(10):3531–3536. doi: 10.1128/mcb.6.10.3531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Miller L. K. Baculoviruses as gene expression vectors. Annu Rev Microbiol. 1988;42:177–199. doi: 10.1146/annurev.mi.42.100188.001141. [DOI] [PubMed] [Google Scholar]
  29. Miyajima A., Schreurs J., Otsu K., Kondo A., Arai K., Maeda S. Use of the silkworm, Bombyx mori, and an insect baculovirus vector for high-level expression and secretion of biologically active mouse interleukin-3. Gene. 1987;58(2-3):273–281. doi: 10.1016/0378-1119(87)90382-9. [DOI] [PubMed] [Google Scholar]
  30. Miyamoto C., Smith G. E., Farrell-Towt J., Chizzonite R., Summers M. D., Ju G. Production of human c-myc protein in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1985 Oct;5(10):2860–2865. doi: 10.1128/mcb.5.10.2860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. O'Reilly D. R. p53 and transformation by SV40. Biol Cell. 1986;57(3):187–196. doi: 10.1111/j.1768-322x.1986.tb00475.x. [DOI] [PubMed] [Google Scholar]
  33. Ollo R., Maniatis T. Drosophila Krüppel gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to DNA. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5700–5704. doi: 10.1073/pnas.84.16.5700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Oren M., Maltzman W., Levine A. J. Post-translational regulation of the 54K cellular tumor antigen in normal and transformed cells. Mol Cell Biol. 1981 Feb;1(2):101–110. doi: 10.1128/mcb.1.2.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Parada L. F., Land H., Weinberg R. A., Wolf D., Rotter V. Cooperation between gene encoding p53 tumour antigen and ras in cellular transformation. Nature. 1984 Dec 13;312(5995):649–651. doi: 10.1038/312649a0. [DOI] [PubMed] [Google Scholar]
  36. Possee R. D. Cell-surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector. Virus Res. 1986 Jul;5(1):43–59. doi: 10.1016/0168-1702(86)90064-x. [DOI] [PubMed] [Google Scholar]
  37. Rice W. C., Lorimer H. E., Prives C., Miller L. K. Expression of polyomavirus large T antigen by using a baculovirus vector. J Virol. 1987 May;61(5):1712–1716. doi: 10.1128/jvi.61.5.1712-1716.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Samad A., Anderson C. W., Carroll R. B. Mapping of phosphomonoester and apparent phosphodiester bonds of the oncogene product p53 from simian virus 40-transformed 3T3 cells. Proc Natl Acad Sci U S A. 1986 Feb;83(4):897–901. doi: 10.1073/pnas.83.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Scheidtmann K. H., Kaiser A., Carbone A., Walter G. Phosphorylation of threonine in the proline-rich carboxy-terminal region of simian virus 40 large T antigen. J Virol. 1981 Apr;38(1):59–69. doi: 10.1128/jvi.38.1.59-69.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Scheidtmann K. H. Phosphorylation of simian virus 40 large T antigen: cytoplasmic and nuclear phophorylation sites differ in their metabolic stability. Virology. 1986 Apr 15;150(1):85–95. [PubMed] [Google Scholar]
  43. 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]
  44. Schmieg F. I., Simmons D. T. Intracellular location and kinetics of complex formation between simian virus 40 T antigen and cellular protein p53. J Virol. 1984 Nov;52(2):350–355. doi: 10.1128/jvi.52.2.350-355.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Smith G. E., Ju G., Ericson B. L., Moschera J., Lahm H. W., Chizzonite R., Summers M. D. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8404–8408. doi: 10.1073/pnas.82.24.8404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Smith G. E., Summers M. D., Fraser M. J. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1983 Dec;3(12):2156–2165. doi: 10.1128/mcb.3.12.2156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. St Angelo C., Smith G. E., Summers M. D., Krug R. M. Two of the three influenza viral polymerase proteins expressed by using baculovirus vectors form a complex in insect cells. J Virol. 1987 Feb;61(2):361–365. doi: 10.1128/jvi.61.2.361-365.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Tegtmeyer P., Rundell K., Collins J. K. Modification of simian virus 40 protein A. J Virol. 1977 Feb;21(2):647–657. doi: 10.1128/jvi.21.2.647-657.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Van Roy F., Fransen L., Fiers W. Phosphorylation patterns of tumour antigens in cells lytically infected or transformed by simian virus 40. J Virol. 1981 Oct;40(1):28–44. doi: 10.1128/jvi.40.1.28-44.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Vaughn J. L., Goodwin R. H., Tompkins G. J., McCawley P. The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro. 1977 Apr;13(4):213–217. doi: 10.1007/BF02615077. [DOI] [PubMed] [Google Scholar]
  54. Wade-Evans A., Jenkins J. R. Precise epitope mapping of the murine transformation-associated protein, p53. EMBO J. 1985 Mar;4(3):699–706. doi: 10.1002/j.1460-2075.1985.tb03686.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]

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