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. 1986 Apr;6(4):1204–1217. doi: 10.1128/mcb.6.4.1204

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

P S Jat, C L Cepko, R C Mulligan, P A Sharp
PMCID: PMC367632  PMID: 3023876

Abstract

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.

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Selected References

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

  1. Bastin M., Bourgaux-Ramoisy D., Bourgaux P. Biological properties of polyoma DNA fragments cloned in plasmid pBR322. J Gen Virol. 1980 Sep;50(1):179–184. doi: 10.1099/0022-1317-50-1-179. [DOI] [PubMed] [Google Scholar]
  2. Bouck N., Beales N., Shenk T., Berg P., di Mayorca G. New region of the simian virus 40 genome required for efficient viral transformation. Proc Natl Acad Sci U S A. 1978 May;75(5):2473–2477. doi: 10.1073/pnas.75.5.2473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brockman W. W. Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: temperature sensitivity of the transformed phenotype and retransofrmation by wild-type virus. J Virol. 1978 Mar;25(3):860–870. doi: 10.1128/jvi.25.3.860-870.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  6. Cepko C. L., Changelian P. S., Sharp P. A. Immunoprecipitation with two-dimensional pools as a hybridoma screening technique: production and characterization of monoclonal antibodies against adenovirus 2 proteins. Virology. 1981 Apr 30;110(2):385–401. doi: 10.1016/0042-6822(81)90069-6. [DOI] [PubMed] [Google Scholar]
  7. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
  8. Courtneidge S. A., Smith A. E. Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature. 1983 Jun 2;303(5916):435–439. doi: 10.1038/303435a0. [DOI] [PubMed] [Google Scholar]
  9. Davies J., Jimenez A. A new selective agent for eukaryotic cloning vectors. Am J Trop Med Hyg. 1980 Sep;29(5 Suppl):1089–1092. doi: 10.4269/ajtmh.1980.29.1089. [DOI] [PubMed] [Google Scholar]
  10. Di Mayorca G., Callender J., Marin G., Giordano R. Temperature-sensitive mutants of polyoma virus. Virology. 1969 May;38(1):126–133. doi: 10.1016/0042-6822(69)90134-2. [DOI] [PubMed] [Google Scholar]
  11. Donoghue D. J., Anderson C., Hunter T., Kaplan P. L. Transmission of the polyoma virus middle T gene as the oncogene of a murine retrovirus. Nature. 1984 Apr 19;308(5961):748–750. doi: 10.1038/308748a0. [DOI] [PubMed] [Google Scholar]
  12. Eckhart W. Complementation and transformation by temperature-sensitive mutants of polyoma virus. Virology. 1969 May;38(1):120–125. doi: 10.1016/0042-6822(69)90133-0. [DOI] [PubMed] [Google Scholar]
  13. Fluck M. M., Benjamin T. L. Comparisons of two early gene functions essential for transformation in polyoma virus and SV-40. Virology. 1979 Jul 15;96(1):205–228. doi: 10.1016/0042-6822(79)90185-5. [DOI] [PubMed] [Google Scholar]
  14. Gelinas C., Chartrand P., Bastin M. Polyoma virus mutant with normal transforming ability but impaired tumorigenic potential. J Virol. 1982 Sep;43(3):1072–1081. doi: 10.1128/jvi.43.3.1072-1081.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  16. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hassell J. A., Topp W. C., Rifkin D. B., Moreau P. E. Transformation of rat embryo fibroblasts by cloned polyoma virus DNA fragments containing only part of the early region. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3978–3982. doi: 10.1073/pnas.77.7.3978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Horowitz M., Cepko C. L., Sharp P. A. Expression of chimeric genes in the early region of SV40. J Mol Appl Genet. 1983;2(2):147–159. [PubMed] [Google Scholar]
  20. Hunter T., Hutchinson M. A., Eckhart W. Translation of polyoma virus T antigens in vitro. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5917–5921. doi: 10.1073/pnas.75.12.5917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hutchinson M. A., Hunter T., Eckhart W. Characterization of T antigens in polyoma-infected and transformed cells. Cell. 1978 Sep;15(1):65–77. doi: 10.1016/0092-8674(78)90083-1. [DOI] [PubMed] [Google Scholar]
  22. Imbert J., Clertant P., de Bovis B., Planche J., Birg F. Stabilization of the large T protein in temperature-independent (type A) FR 3T3 rat cells transformed with the simian virus 40 tsA30 mutant. J Virol. 1983 Sep;47(3):442–451. doi: 10.1128/jvi.47.3.442-451.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Imbert J., Lawrence J. J., Birg F. Simian virus 40 T antigen is detected only in cells in the G2 phase of the cell cycle in one group of rat transformants. Virology. 1983 Apr 30;126(2):711–716. doi: 10.1016/s0042-6822(83)80028-2. [DOI] [PubMed] [Google Scholar]
  24. Ito Y. Polyoma virus-specific 55K protein isolated from plasma membrane of productively infected cells is virus-coded and important for cell transformation. Virology. 1979 Oct 15;98(1):261–266. doi: 10.1016/0042-6822(79)90545-2. [DOI] [PubMed] [Google Scholar]
  25. Ito Y., Spurr N., Dulbecco R. Characterization of polyoma virus T antigen. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1259–1263. doi: 10.1073/pnas.74.3.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Keath E. J., Caimi P. G., Cole M. D. Fibroblast lines expressing activated c-myc oncogenes are tumorigenic in nude mice and syngeneic animals. Cell. 1984 Dec;39(2 Pt 1):339–348. doi: 10.1016/0092-8674(84)90012-6. [DOI] [PubMed] [Google Scholar]
  28. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  29. 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]
  30. Kriegler M., Perez C. F., Hardy C., Botchan M. Transformation mediated by the SV40 T antigens: separation of the overlapping SV40 early genes with a retroviral vector. Cell. 1984 Sep;38(2):483–491. doi: 10.1016/0092-8674(84)90503-8. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Lane D. P., Crawford L. V. T antigen is bound to a host protein in SV40-transformed cells. Nature. 1979 Mar 15;278(5701):261–263. doi: 10.1038/278261a0. [DOI] [PubMed] [Google Scholar]
  34. Linzer D. I., Levine A. J. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell. 1979 May;17(1):43–52. doi: 10.1016/0092-8674(79)90293-9. [DOI] [PubMed] [Google Scholar]
  35. MACPHERSON I., MONTAGNIER L. AGAR SUSPENSION CULTURE FOR THE SELECTIVE ASSAY OF CELLS TRANSFORMED BY POLYOMA VIRUS. Virology. 1964 Jun;23:291–294. doi: 10.1016/0042-6822(64)90301-0. [DOI] [PubMed] [Google Scholar]
  36. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Martin R. G., Setlow V. P., Edwards C. A., Vembu D. The roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells. Cell. 1979 Jul;17(3):635–643. doi: 10.1016/0092-8674(79)90271-x. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. McCoy M. S., Bargmann C. I., Weinberg R. A. Human colon carcinoma Ki-ras2 oncogene and its corresponding proto-oncogene. Mol Cell Biol. 1984 Aug;4(8):1577–1582. doi: 10.1128/mcb.4.8.1577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Novak U., Dilworth S. M., Griffin B. E. Coding capacity of a 35 percent fragment of the polyoma virus genome is sufficient to initiate and maintain cellular transformation. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3278–3282. doi: 10.1073/pnas.77.6.3278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Otsuka H., Dubbs D. R., Kit S. Variant lines of mouse kidney cells transformed by an SV40tsA mutant with growth properties of wild-type transformed cells at nonpermissive temperature. J Gen Virol. 1979 Feb;42(2):373–386. doi: 10.1099/0022-1317-42-2-373. [DOI] [PubMed] [Google Scholar]
  45. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Parker R. C., Varmus H. E., Bishop J. M. Expression of v-src and chicken c-src in rat cells demonstrates qualitative differences between pp60v-src and pp60c-src. Cell. 1984 May;37(1):131–139. doi: 10.1016/0092-8674(84)90308-8. [DOI] [PubMed] [Google Scholar]
  47. Paucha E., Mellor A., Harvey R., Smith A. E., Hewick R. M., Waterfield M. D. Large and small tumor antigens from simian virus 40 have identical amino termini mapping at 0.65 map units. Proc Natl Acad Sci U S A. 1978 May;75(5):2165–2169. doi: 10.1073/pnas.75.5.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Petit C. A., Gardes M., Feunteun J. Immortalization of rodent embryo fibroblasts by SV40 is maintained by the A gene. Virology. 1983 May;127(1):74–82. doi: 10.1016/0042-6822(83)90372-0. [DOI] [PubMed] [Google Scholar]
  49. Pollack R., Risser R., Conlon S., Rifkin D. Plasminogen activator production accompanies loss of anchorage regulation in transformation of primary rat embryo cells by simian virus 40. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4792–4796. doi: 10.1073/pnas.71.12.4792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Prives C., Gluzman Y., Winocour E. Cellular and cell-free synthesis of simian virus 40 T-antigens in permissive and transformed cells. J Virol. 1978 Feb;25(2):587–595. doi: 10.1128/jvi.25.2.587-595.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Rassoulzadegan M., Cowie A., Carr A., Glaichenhaus N., Kamen R., Cuzin F. The roles of individual polyoma virus early proteins in oncogenic transformation. Nature. 1982 Dec 23;300(5894):713–718. doi: 10.1038/300713a0. [DOI] [PubMed] [Google Scholar]
  52. Rassoulzadegan M., Cuzin F. Transformation of rat fibroblast cells with early mutants of polyoma (tsa) and simian virus 40 (tsA30): occurrence of either A or N transformants depends on the multiplicity of infection. J Virol. 1980 Feb;33(2):909–911. doi: 10.1128/jvi.33.2.909-911.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Rassoulzadegan M., Naghashfar Z., Cowie A., Carr A., Grisoni M., Kamen R., Cuzin F. Expression of the large T protein of polyoma virus promotes the establishment in culture of "normal" rodent fibroblast cell lines. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4354–4358. doi: 10.1073/pnas.80.14.4354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  55. Risser R., Pollack R. A nonselective analysis of SV40 transformation of mouse 3T3 cells. Virology. 1974 Jun;59(2):477–489. doi: 10.1016/0042-6822(74)90457-7. [DOI] [PubMed] [Google Scholar]
  56. Schaffhausen B. S., Silver J. E., Benjamin T. L. Tumor antigen(s) in cell productively infected by wild-type polyoma virus and mutant NG-18. Proc Natl Acad Sci U S A. 1978 Jan;75(1):79–83. doi: 10.1073/pnas.75.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Seif R., Martin R. G. Growth state of the cell early after infection with simian virus 40 determines whether the maintenance of transformation will be A-gene dependent or independent. J Virol. 1979 Aug;31(2):350–359. doi: 10.1128/jvi.31.2.350-359.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Seif R., Martin R. G. Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells. J Virol. 1979 Dec;32(3):979–988. doi: 10.1128/jvi.32.3.979-988.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Shenk T. E., Carbon J., Berg P. Construction and analysis of viable deletion mutants of simian virus 40. J Virol. 1976 May;18(2):664–671. doi: 10.1128/jvi.18.2.664-671.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Shih C., Weinberg R. A. Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell. 1982 May;29(1):161–169. doi: 10.1016/0092-8674(82)90100-3. [DOI] [PubMed] [Google Scholar]
  61. Shimotohno K., Temin H. M. Loss of intervening sequences in genomic mouse alpha-globin DNA inserted in an infectious retrovirus vector. Nature. 1982 Sep 16;299(5880):265–268. doi: 10.1038/299265a0. [DOI] [PubMed] [Google Scholar]
  62. Shin S. I., Freedman V. H., Risser R., Pollack R. Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4435–4439. doi: 10.1073/pnas.72.11.4435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Sleigh M. J., Topp W. C., Hanich R., Sambrook J. F. Mutants of SV40 with an altered small t protein are reduced in their ability to transform cells. Cell. 1978 May;14(1):79–88. doi: 10.1016/0092-8674(78)90303-3. [DOI] [PubMed] [Google Scholar]
  64. Sorge J., Hughes S. H. Splicing of intervening sequences introduced into an infectious retroviral vector. J Mol Appl Genet. 1982;1(6):547–559. [PubMed] [Google Scholar]
  65. 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]
  66. Treisman R., Novak U., Favaloro J., Kamen R. Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature. 1981 Aug 13;292(5824):595–600. doi: 10.1038/292595a0. [DOI] [PubMed] [Google Scholar]

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