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. 1986 Oct;60(1):105–113. doi: 10.1128/jvi.60.1.105-113.1986

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen.

T Noda, M Satake, T Robins, Y Ito
PMCID: PMC253907  PMID: 3018277

Abstract

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into a retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of psi 2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10(6) cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.

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Selected References

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

  1. Asselin C., Gelinas C., Bastin M. Role of the three polyoma virus early proteins in tumorigenesis. Mol Cell Biol. 1983 Aug;3(8):1451–1459. doi: 10.1128/mcb.3.8.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asselin C., Gélinas C., Branton P. E., Bastin M. Polyoma middle T antigen requires cooperation from another gene to express the malignant phenotype in vivo. Mol Cell Biol. 1984 Apr;4(4):755–760. doi: 10.1128/mcb.4.4.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bikel I., Roberts T. M., Bladon M. T., Green R., Amann E., Livingston D. M. Purification of biologically active simian virus 40 small tumor antigen. Proc Natl Acad Sci U S A. 1983 Feb;80(4):906–910. doi: 10.1073/pnas.80.4.906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolen J. B., Thiele C. J., Israel M. A., Yonemoto W., Lipsich L. A., Brugge J. S. Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation. Cell. 1984 Oct;38(3):767–777. doi: 10.1016/0092-8674(84)90272-1. [DOI] [PubMed] [Google Scholar]
  5. Bossert A., Mulgaonkar P., Rundell K. Interaction of simian virus 40 small-T antigen produced in bacteria with 56K and 32K proteins of animal cells. J Virol. 1985 Oct;56(1):325–327. doi: 10.1128/jvi.56.1.325-327.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Colbère-Garapin F., Horodniceanu F., Kourilsky P., Garapin A. C. A new dominant hybrid selective marker for higher eukaryotic cells. J Mol Biol. 1981 Jul 25;150(1):1–14. doi: 10.1016/0022-2836(81)90321-1. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Dilworth S. M., Griffin B. E. Monoclonal antibodies against polyoma virus tumor antigens. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1059–1063. doi: 10.1073/pnas.79.4.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eckhart W., Hutchinson M. A., Hunter T. An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell. 1979 Dec;18(4):925–933. doi: 10.1016/0092-8674(79)90205-8. [DOI] [PubMed] [Google Scholar]
  10. Ellman M., Bikel I., Figge J., Roberts T., Schlossman R., Livingston D. M. Localization of the simian virus 40 small t antigen in the nucleus and cytoplasm of monkey and mouse cells. J Virol. 1984 May;50(2):623–628. doi: 10.1128/jvi.50.2.623-628.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Emerman M., Temin H. M. Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell. 1984 Dec;39(3 Pt 2):449–467. [PubMed] [Google Scholar]
  12. Gaudray P., Tyndall C., Kamen R., Cuzin F. The high affinity binding site on polyoma virus DNA for the viral large-T protein. Nucleic Acids Res. 1981 Nov 11;9(21):5697–5710. doi: 10.1093/nar/9.21.5697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Graessmann A., Graessmann M., Tjian R., Topp W. C. Simian virus 40 small-t protein is required for loss of actin cable networks in rat cells. J Virol. 1980 Mar;33(3):1182–1191. doi: 10.1128/jvi.33.3.1182-1191.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ito Y., Brocklehurst J. R., Dulbecco R. Virus-specific proteins in the plasma membrane of cells lytically infected or transformed by pol-oma virus. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4666–4670. doi: 10.1073/pnas.74.10.4666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ito Y., Hamagishi Y., Segawa K., Dalianis T., Appella E., Willingham M. Antibodies against a nonapeptide of polyomavirus middle T antigen: cross-reaction with a cellular protein(s). J Virol. 1983 Dec;48(3):709–720. doi: 10.1128/jvi.48.3.709-720.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Ito Y., Spurr N., Griffin B. E. Middle T antigen as primary inducer of full expression of the phenotype of transformation by polyoma virus. J Virol. 1980 Jul;35(1):219–232. doi: 10.1128/jvi.35.1.219-232.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lania L., Griffiths M., Cooke B., Ito Y., Fried M. Untransformed rat cells containing free and integrated DNA of a polyoma nontransforming (Hr-t) mutant. Cell. 1979 Nov;18(3):793–802. doi: 10.1016/0092-8674(79)90132-6. [DOI] [PubMed] [Google Scholar]
  20. Liang T. J., Carmichael G. G., Benjamin T. L. A polyoma mutant that encodes small T antigen but not middle T antigen demonstrates uncoupling of cell surface and cytoskeletal changes associated with cell transformation. Mol Cell Biol. 1984 Dec;4(12):2774–2783. doi: 10.1128/mcb.4.12.2774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. Richardson W. D., Roberts B. L., Smith A. E. Nuclear location signals in polyoma virus large-T. Cell. 1986 Jan 17;44(1):77–85. doi: 10.1016/0092-8674(86)90486-1. [DOI] [PubMed] [Google Scholar]
  26. Rundell K., Major E. O., Lampert M. Association of cellular 56,000- and 32,000-molecular-weight protein with BK virus and polyoma virus t-antigens. J Virol. 1981 Mar;37(3):1090–1093. doi: 10.1128/jvi.37.3.1090-1093.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schaffhausen B. S., Benjamin T. L. Phosphorylation of polyoma T antigens. Cell. 1979 Dec;18(4):935–946. doi: 10.1016/0092-8674(79)90206-x. [DOI] [PubMed] [Google Scholar]
  28. Scher C. D., Shepard R. C., Antoniades H. N., Stiles C. D. Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle. Biochim Biophys Acta. 1979 Aug 10;560(2):217–241. doi: 10.1016/0304-419x(79)90020-9. [DOI] [PubMed] [Google Scholar]
  29. Schlegel R., Benjamin T. L. Cellular alterations dependent upon the polyoma virus Hr-t function: separation of mitogenic from transforming capacities. Cell. 1978 Jul;14(3):587–599. doi: 10.1016/0092-8674(78)90244-1. [DOI] [PubMed] [Google Scholar]
  30. Segawa K., Ito Y. Differential subcellular localization of in vivo-phosphorylated and nonphosphorylated middle-sized tumor antigen of polyoma virus and its relationship to middle-sized tumor antigen phosphorylating activity in vitro. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6812–6816. doi: 10.1073/pnas.79.22.6812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Smart J. E., Ito Y. Three species of polyoma virus tumor antigens share common peptides probably near the amino termini of the proteins. Cell. 1978 Dec;15(4):1427–1437. doi: 10.1016/0092-8674(78)90066-1. [DOI] [PubMed] [Google Scholar]
  32. Smith A. E., Smith R., Griffin B., Fried M. Protein kinase activity associated with polyoma virus middle T antigen in vitro. Cell. 1979 Dec;18(4):915–924. doi: 10.1016/0092-8674(79)90204-6. [DOI] [PubMed] [Google Scholar]
  33. Soeda E., Arrand J. R., Smolar N., Walsh J. E., Griffin B. E. Coding potential and regulatory signals of the polyoma virus genome. Nature. 1980 Jan 31;283(5746):445–453. doi: 10.1038/283445a0. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Tyndall C., La Mantia G., Thacker C. M., Favaloro J., Kamen R. A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication. Nucleic Acids Res. 1981 Dec 11;9(23):6231–6250. doi: 10.1093/nar/9.23.6231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  38. Wulf E., Deboben A., Bautz F. A., Faulstich H., Wieland T. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4498–4502. doi: 10.1073/pnas.76.9.4498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yang Y. C., Hearing P., Rundell K. Cellular proteins associated with simian virus 40 early gene products in newly infected cells. J Virol. 1979 Oct;32(1):147–154. doi: 10.1128/jvi.32.1.147-154.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhu Z. Y., Veldman G. M., Cowie A., Carr A., Schaffhausen B., Kamen R. Construction and functional characterization of polyomavirus genomes that separately encode the three early proteins. J Virol. 1984 Jul;51(1):170–180. doi: 10.1128/jvi.51.1.170-180.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. de Larco J. E., Todaro G. J. Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci U S A. 1978 Aug;75(8):4001–4005. doi: 10.1073/pnas.75.8.4001. [DOI] [PMC free article] [PubMed] [Google Scholar]

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