Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1982 Mar;41(3):1014–1024. doi: 10.1128/jvi.41.3.1014-1024.1982

Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation.

D Templeton, W Eckhart
PMCID: PMC256839  PMID: 6284958

Abstract

We used site-specific mutagenesis to introduce a termination codon, TGA, into the reading frame for the polyoma virus medium T antigen. We induced this mutation in a region of the polyoma genome in which the overlapping coding regions for the large and medium TE antigens are translated in different reading frames. Therefore, the mutation terminated translation of the medium T antigen, but it caused only a single amino acid substitution in the large T antigen and did not affect the small T antigen. Cells infected by the mutant virus produced normal-size small and large T antigens. The infected cells produced a 28,000-dalton fragment of the 48,000-dalton medium T antigen, whose size and tryptic peptide map were consistent with its being a truncated N-terminal fragment terminating at the new termination codon of the mutant. Immunoprecipitates of mutant-infected cell extracts did not show medium-T-antigen-associated protein kinase activity. The mutant virus replicated normally in mouse 3T6 cells and induced cellular DNA synthesis in resting mouse 3T3 cells, but it failed to transform rat or hamster cells, as judged by focus formation and growth in agar. The mutant complemented a tsA mutant which affects the large T antigen for transformation, implying that the mutant defect for transformation was in the medium T antigen. These results imply that the small T antigen and the large T antigen together are insufficient to cause transformation and support the conclusion that the medium T antigen is essential for cell transformation by polyoma virus.

Full text

PDF
1024

Images in this article

1018Image
on p.1018
1019Image
on p.1019
1020Image
on p.1020
1021Image
on p.1021
1022Image
on p.1022

Selected References

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

  1. Benjamin T. L. Host range mutants of polyoma virus. Proc Natl Acad Sci U S A. 1970 Sep;67(1):394–399. doi: 10.1073/pnas.67.1.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Deninger P. L., Esty A., LaPorte P., Hsu H., Friedmann T. The nucleotide sequence and restriction enzyme sites of the polyoma genome. Nucleic Acids Res. 1980 Feb 25;8(4):855–860. [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Freeman A. E., Igel H. J., Price P. J. I. In vitrol transformation of rat embryo cells: correlations with the known tumorigenic activities of chemicals in rodents. In Vitro. 1975 Mar-Apr;11(2):107–116. doi: 10.1007/BF02624083. [DOI] [PubMed] [Google Scholar]
  6. Friedmann T., LaPorte P., Esty A. Nucleotide sequence studies of polyoma DNA. The Hpa II 3/5 junction to the Hpa II 4/Hae III 18 junction, encoding the origin of DNA replication and the 5' end of the early region. J Biol Chem. 1978 Sep 25;253(18):6561–6567. [PubMed] [Google Scholar]
  7. Griffin B. E., Maddock C. New classes of viable deletion mutants in the early region of polyoma virus. J Virol. 1979 Sep;31(3):645–656. doi: 10.1128/jvi.31.3.645-656.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Hunter T., Hutchinson M. A., Eckhart W., Friedmann T., Esty A., LaPorte P., Deininger P. Regions of the polyoma genome coding for T antigens. Nucleic Acids Res. 1979 Dec 20;7(8):2275–2288. doi: 10.1093/nar/7.8.2275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Israel M. A., Simmons D. T., Hourihan S. L., Rowe W. P., Martin M. A. Interrupting the early region of polyoma virus DNA enhances tumorigenicity. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3713–3716. doi: 10.1073/pnas.76.8.3713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Kai K., Tsuruo T., Hayatsu H. The effect of bisulfite modification on the template activity of DNA for DNA polymerase I. Nucleic Acids Res. 1974 Jul;1(7):889–899. doi: 10.1093/nar/1.7.889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kimura G., Itagaki A., Summers J. Rat cell line 3y1 and its virogenic polyoma- and sv40- transformed derivatives. Int J Cancer. 1975 Apr 15;15(4):694–706. doi: 10.1002/ijc.2910150419. [DOI] [PubMed] [Google Scholar]
  15. Lania L., Gandini-Attardi D., Griffiths M., Cooke B., De Cicco D., Fried M. The polyoma virus 100K large T-antigen is not required for the maintenance of transformation. Virology. 1980 Feb;101(1):217–232. doi: 10.1016/0042-6822(80)90497-3. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Magnusson G., Berg P. Construction and analysis of viable deletion mutants of polyoma virus. J Virol. 1979 Nov;32(2):523–529. doi: 10.1128/jvi.32.2.523-529.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  19. Moore J. L., Chowdhury K., Martin M. A., Israel M. A. Polyoma large tumor antigen is not required for tumorigenesis mediated by viral DNA. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1336–1340. doi: 10.1073/pnas.77.3.1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Parker R. C., Watson R. M., Vinograd J. Mapping of closed circular DNAs by cleavage with restriction endonucleases and calibration by agarose gel electrophoresis. Proc Natl Acad Sci U S A. 1977 Mar;74(3):851–855. doi: 10.1073/pnas.74.3.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Shortle D., Nathans D. Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome. Proc Natl Acad Sci U S A. 1978 May;75(5):2170–2174. doi: 10.1073/pnas.75.5.2170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Smolar N., Griffin B. E. DNA sequences of polyoma virus early deletion mutants. J Virol. 1981 Jun;38(3):958–967. doi: 10.1128/jvi.38.3.958-967.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Türler H. The tumor antigens and the early functions of polyoma virus. Mol Cell Biochem. 1980 Sep 15;32(2):63–93. doi: 10.1007/BF00227801. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES