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. 1984 Dec;4(12):2774–2783. doi: 10.1128/mcb.4.12.2774

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.

T J Liang, G G Carmichael, T L Benjamin
PMCID: PMC369288  PMID: 6098822

Abstract

The hr-t gene of polyoma virus encodes both the small and middle T (tumor) antigens and exerts pleiotropic effects on cells. By mutating the 3' splice site for middle T mRNA, we have constructed a virus mutant, Py808A, which fails to express middle T but encodes normal small and large T proteins. The mutant failed to induce morphological transformation or growth in soft agar, but did stimulate postconfluent growth of normal cells. Cells infected by Py808A became fully agglutinable by lectins while retaining normal actin cable architecture and normal levels of extracellular fibronectin. These properties of Py808A demonstrated the separability of structural changes at the cell surface from those in the cytoskeleton and extracellular matrix, parameters which have heretofore been linked in the action of the hr-t and other viral oncogenes.

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Selected References

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  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. Ballmer K., Mannino R. J., Burger M. M. Modulation of the cell cycle during reversible growth inhibition of 3T3 and SV40-3T3 cells with succinylated concanavalin A. Exp Cell Res. 1980 Apr;126(2):311–319. doi: 10.1016/0014-4827(80)90269-4. [DOI] [PubMed] [Google Scholar]
  4. Bendig M. M., Thomas T., Folk W. R. Viable deletion mutant in the medium and large T-antigen-coding sequences of the polyoma virus genome. J Virol. 1980 Mar;33(3):1215–1220. doi: 10.1128/jvi.33.3.1215-1220.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benjamin T. L., Burger M. M. Absence of a cell membrane alteration function in non-transforming mutants of polyoma virus. Proc Natl Acad Sci U S A. 1970 Oct;67(2):929–934. doi: 10.1073/pnas.67.2.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Benjamin T. L. The hr-t gene of polyoma virus. Biochim Biophys Acta. 1982 Dec 21;695(2):69–95. doi: 10.1016/0304-419x(82)90018-x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Birchmeier C., Kreis T. E., Eppenberger H. M., Winterhalter K. H., Birchmeier W. Corrugated attachment membrane in WI-38 fibroblasts: alternating fibronectin fibers and actin-containing focal contacts. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4108–4112. doi: 10.1073/pnas.77.7.4108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burger M. M., Noonan K. D. Restoration of normal growth by covering of agglutinin sites on tumour cell surface. Nature. 1970 Nov 7;228(5271):512–515. doi: 10.1038/228512a0. [DOI] [PubMed] [Google Scholar]
  10. Burger M. M. Proteolytic enzymes initiating cell division and escape from contact inhibition of growth. Nature. 1970 Jul 11;227(5254):170–171. doi: 10.1038/227170a0. [DOI] [PubMed] [Google Scholar]
  11. Calos M. P., Lebkowski J. S., Botchan M. R. High mutation frequency in DNA transfected into mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3015–3019. doi: 10.1073/pnas.80.10.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Carmichael G., Schaffhausen B. S., Mandel G., Liang T. J., Benjamin T. L. Transformation by polyoma virus is drastically reduced by substitution of phenylalanine for tyrosine at residue 315 of middle-sized tumor antigen. Proc Natl Acad Sci U S A. 1984 Feb;81(3):679–683. doi: 10.1073/pnas.81.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chen L. B., Gallimore P. H., McDougall J. K. Correlation between tumor induction and the large external transformation sensitive protein on the cell surface. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3570–3574. doi: 10.1073/pnas.73.10.3570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chen W. T., Singer S. J. Fibronectin is not present in the focal adhesions formed between normal cultured fibroblasts and their substrata. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7318–7322. doi: 10.1073/pnas.77.12.7318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Dulbecco R. Behavior of tissue culture cells infected with polyoma virus. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1214–1220. doi: 10.1073/pnas.67.3.1214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Eckhart W., Weber M. Uptake of 2-deoxyglucose by BALB-3T3 cells: changes after polyoma infection. Virology. 1974 Sep;61(1):223–228. doi: 10.1016/0042-6822(74)90256-6. [DOI] [PubMed] [Google Scholar]
  18. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  19. Felber B. K., Orkin S. H., Hamer D. H. Abnormal RNA splicing causes one form of alpha thalassemia. Cell. 1982 Jul;29(3):895–902. doi: 10.1016/0092-8674(82)90451-2. [DOI] [PubMed] [Google Scholar]
  20. Feunteun J., Benjamin T. L. Isolation of transformation-defective host-range mutants of polyoma virus on normal mouse cells. Virology. 1982 Jun;119(2):310–316. doi: 10.1016/0042-6822(82)90091-5. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Foster D. O., Pardee A. B. Transport of amino acids by confluent and nonconfluent 3T3 and polyoma virus-transformed 3T3 cells growing on glass cover slips. J Biol Chem. 1969 May 25;244(10):2675–2681. [PubMed] [Google Scholar]
  23. Garcea R. L., Benjamin T. L. Host range transforming gene of polyoma virus plays a role in virus assembly. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3613–3617. doi: 10.1073/pnas.80.12.3613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Graham F. L., Abrahams P. J., Mulder C., Heijneker H. L., Warnaar S. O., De Vries F. A., Fiers W., Van Der Eb A. J. Studies on in vitro transformation by DNA and DNA fragments of human adenoviruses and simian virus 40. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):637–650. doi: 10.1101/sqb.1974.039.01.077. [DOI] [PubMed] [Google Scholar]
  26. Hatanaka M., Augl C., Gilden R. V. Evidence for a functional change in the plasma membrane of murine sarcoma virus-infected mouse embryo cells. Transport and transport-associated phosphorylation of 14C-2-deoxy-D-glucose. J Biol Chem. 1970 Feb 25;245(4):714–717. [PubMed] [Google Scholar]
  27. Heggeness M. H., Ash J. F., Singer S. J. Transmembrane linkage of fibronectin to intracellular actin-containing filaments in cultured human fibroblasts. Ann N Y Acad Sci. 1978 Jun 20;312:414–417. doi: 10.1111/j.1749-6632.1978.tb16822.x. [DOI] [PubMed] [Google Scholar]
  28. Hirschhorn R., Beratis N., Rosen F. S. Characterization of residual enzyme activity in fibroblasts from patients with adenosine deaminase deficiency and combined immunodeficiency: evidence for a mutant enzyme. Proc Natl Acad Sci U S A. 1976 Jan;73(1):213–217. doi: 10.1073/pnas.73.1.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hogg N. M. A comparison of membrane proteins of normal and transformed cells by lactoperoxidase labeling. Proc Natl Acad Sci U S A. 1974 Feb;71(2):489–492. doi: 10.1073/pnas.71.2.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hunter T. Protein phosphorylated by the RSV transforming function. Cell. 1980 Dec;22(3):647–648. doi: 10.1016/0092-8674(80)90539-5. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Hynes R. O. Alteration of cell-surface proteins by viral transformation and by proteolysis. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3170–3174. doi: 10.1073/pnas.70.11.3170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hynes R. O., Destree A. T. Relationships between fibronectin (LETS protein) and actin. Cell. 1978 Nov;15(3):875–886. doi: 10.1016/0092-8674(78)90272-6. [DOI] [PubMed] [Google Scholar]
  34. Hynes R. O., Yamada K. M. Fibronectins: multifunctional modular glycoproteins. J Cell Biol. 1982 Nov;95(2 Pt 1):369–377. doi: 10.1083/jcb.95.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hynes R. Phosphorylation of vinculin by pp60src: what might it mean? Cell. 1982 Mar;28(3):437–438. doi: 10.1016/0092-8674(82)90196-9. [DOI] [PubMed] [Google Scholar]
  36. Isselbacher K. J. Increased uptake of amino acids and 2-deoxy-D-glucose by virus-transformed cells in culture. Proc Natl Acad Sci U S A. 1972 Mar;69(3):585–589. doi: 10.1073/pnas.69.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Izzard C. S., Lochner L. R. Cell-to-substrate contacts in living fibroblasts: an interference reflexion study with an evaluation of the technique. J Cell Sci. 1976 Jun;21(1):129–159. doi: 10.1242/jcs.21.1.129. [DOI] [PubMed] [Google Scholar]
  38. Kamen R., Favaloro J., Parker J., Treisman R., Lania L., Fried M., Mellor A. Comparison of polyoma virus transcription in productively infected mouse cells and transformed rodent cell lines. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):63–75. doi: 10.1101/sqb.1980.044.01.009. [DOI] [PubMed] [Google Scholar]
  39. Kawai S., Hanafusa H. The effects of reciprocal changes in temperature on the transformed state of cells infected with a rous sarcoma virus mutant. Virology. 1971 Nov;46(2):470–479. doi: 10.1016/0042-6822(71)90047-x. [DOI] [PubMed] [Google Scholar]
  40. Lewin B. Alternatives for splicing: recognizing the ends of introns. Cell. 1980 Nov;22(2 Pt 2):324–326. doi: 10.1016/0092-8674(80)90340-2. [DOI] [PubMed] [Google Scholar]
  41. Loyter A., Scangos G. A., Ruddle F. H. Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc Natl Acad Sci U S A. 1982 Jan;79(2):422–426. doi: 10.1073/pnas.79.2.422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Montell C., Fisher E. F., Caruthers M. H., Berk A. J. Resolving the functions of overlapping viral genes by site-specific mutagenesis at a mRNA splice site. Nature. 1982 Feb 4;295(5848):380–384. doi: 10.1038/295380a0. [DOI] [PubMed] [Google Scholar]
  44. Nigg E. A., Sefton B. M., Hunter T., Walter G., Singer S. J. Immunofluorescent localization of the transforming protein of Rous sarcoma virus with antibodies against a synthetic src peptide. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5322–5326. doi: 10.1073/pnas.79.17.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Nilsson S. V., Magnusson G. T-antigen expression by polyoma mutants with modified RNA splicing. EMBO J. 1983;2(12):2095–2101. doi: 10.1002/j.1460-2075.1983.tb01708.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Oostra B. A., Harvey R., Ely B. K., Markham A. F., Smith A. E. Transforming activity of polyoma virus middle-T antigen probed by site-directed mutagenesis. Nature. 1983 Aug 4;304(5925):456–459. doi: 10.1038/304456a0. [DOI] [PubMed] [Google Scholar]
  47. Pollack R., Osborn M., Weber K. Patterns of organization of actin and myosin in normal and transformed cultured cells. Proc Natl Acad Sci U S A. 1975 Mar;72(3):994–998. doi: 10.1073/pnas.72.3.994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Razzaque A., Mizusawa H., Seidman M. M. Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3010–3014. doi: 10.1073/pnas.80.10.3010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rubin H., Figge J., Bladon M. T., Chen L. B., Ellman M., Bikel I., Farrell M., Livingston D. M. Role of small t antigen in the acute transforming activity of SV40. Cell. 1982 Sep;30(2):469–480. doi: 10.1016/0092-8674(82)90244-6. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Schaffhausen B. Transforming genes and gene products of polyoma and SV40. CRC Crit Rev Biochem. 1982;13(3):215–286. doi: 10.3109/10409238209114230. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. 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]
  54. Sharp P. A. Speculations on RNA splicing. Cell. 1981 Mar;23(3):643–646. doi: 10.1016/0092-8674(81)90425-6. [DOI] [PubMed] [Google Scholar]
  55. Silver J., Schaffhausen B., Benjamin T. Tumor antigens induced by nontransforming mutants of polyoma virus. Cell. 1978 Oct;15(2):485–496. doi: 10.1016/0092-8674(78)90018-1. [DOI] [PubMed] [Google Scholar]
  56. Singer I. I. Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts. J Cell Biol. 1982 Feb;92(2):398–408. doi: 10.1083/jcb.92.2.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Singer I. I., Paradiso P. R. A transmembrane relationship between fibronectin and vinculin (130 kd protein): serum modulation in normal and transformed hamster fibroblasts. Cell. 1981 May;24(2):481–492. doi: 10.1016/0092-8674(81)90339-1. [DOI] [PubMed] [Google Scholar]
  58. Singer I. I., Paradiso P. R. A transmembrane relationship between fibronectin and vinculin (130 kd protein): serum modulation in normal and transformed hamster fibroblasts. Cell. 1981 May;24(2):481–492. doi: 10.1016/0092-8674(81)90339-1. [DOI] [PubMed] [Google Scholar]
  59. Singer I. I., Paradiso P. R. A transmembrane relationship between fibronectin and vinculin (130 kd protein): serum modulation in normal and transformed hamster fibroblasts. Cell. 1981 May;24(2):481–492. doi: 10.1016/0092-8674(81)90339-1. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  62. Stoker M., Dulbecco R. Abortive transformation by the Tsa mutant of polyoma virus. Nature. 1969 Jul 26;223(5204):397–398. doi: 10.1038/223397a0. [DOI] [PubMed] [Google Scholar]
  63. Templeton D., Eckhart W. Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation. J Virol. 1982 Mar;41(3):1014–1024. doi: 10.1128/jvi.41.3.1014-1024.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Templeton D., Voronova A., Eckhart W. Construction and expression of a recombinant DNA gene encoding a polyomavirus middle-size tumor antigen with the carboxyl terminus of the vesicular stomatitis virus glycoprotein G. Mol Cell Biol. 1984 Feb;4(2):282–289. doi: 10.1128/mcb.4.2.282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. 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]
  66. Treisman R., Proudfoot N. J., Shander M., Maniatis T. A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing. Cell. 1982 Jul;29(3):903–911. doi: 10.1016/0092-8674(82)90452-4. [DOI] [PubMed] [Google Scholar]
  67. WINOCOUR E. Purification of polyoma virus. Virology. 1963 Feb;19:158–168. doi: 10.1016/0042-6822(63)90005-9. [DOI] [PubMed] [Google Scholar]
  68. Wallace R. B., Johnson M. J., Hirose T., Miyake T., Kawashima E. H., Itakura K. The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA. Nucleic Acids Res. 1981 Feb 25;9(4):879–894. doi: 10.1093/nar/9.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Wehland J., Osborn M., Weber K. Cell-to-substratum contacts in living cells: a direct correlation between interference-reflexion and indirect-immunofluorescence microscopy using antibodies against actin and alpha-actinin. J Cell Sci. 1979 Jun;37:257–273. doi: 10.1242/jcs.37.1.257. [DOI] [PubMed] [Google Scholar]
  70. Wieringa B., Meyer F., Reiser J., Weissmann C. Unusual splice sites revealed by mutagenic inactivation of an authentic splice site of the rabbit beta-globin gene. Nature. 1983 Jan 6;301(5895):38–43. doi: 10.1038/301038a0. [DOI] [PubMed] [Google Scholar]

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