Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1980 May;34(2):315–329. doi: 10.1128/jvi.34.2.315-329.1980

Simian virus 40 T-antigen: identification of tryptic peptides in the C-terminal region and definition of the reading frame.

D T Denhardt, L V Crawford
PMCID: PMC288708  PMID: 6246267

Abstract

T-antigen (the simian virus 40 A cistron protein) was purified by immunoprecipitation and electrophoresis on polyacrylamide gels from monkey kidney CV-1 cells infected with simian virus S (SV-S), dl1263, or dl1265 and digested with trypsin. The tryptic peptides, labeled with [35S]methionine, [35S]cysteine, or [3H]proline, were fractionated either by chromatography on Chromobead-P resin or by two-dimensional electrophoresis and chromatography on cellulose thin layers. The T-antigen of SV-S was shown to give rise to a proline-rich (approximately 6 mol of proline) tryptic peptide which was absent in dl1265 T-antigen and hence, on the basis of DNA sequence data, must originate from the C-terminus of the SV-S protein. T-antigen from dl1265, but not SV-S, yielded a cysteine-rich terminal tryptic peptide. The presence of these cysteines caused the protein to be retarded during electrophoresis under the usual conditions in polyacrylamide gels. The T-antigen of dl1263 possessed the proline-rich tryptic peptide; the data are consistent with there being only one peptide altered by the deletion. Both deletion mutants produced a T-antigen that had a higher electrophoretic mobility than SV-S T-antigen but still a larger apparent molecular weight than was predicted by the DNA sequence. The major form of T-antigen found in several lines of 3T3 cells transformed by these mutants was indistinguishable from the T-antigen found in infected cells, and in addition seemed to associate normally with the host-coded 53,000-dalton protein. Except for a minor form of T-antigen with a slightly lower mobility in gels but the same C-terminus, no other polypeptides were detected among the extracted and immunoprecipitated proteins whose electrophoretic mobility was affected by either deletion.

Full text

PDF
329

Images in this article

321Image
on p.321
322Image
on p.322
323Image
on p.323
324Image
on p.324
325Image
on p.325
326Image
on p.326
327Image
on p.327

Selected References

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

  1. Alwine J. C., Reed S. I., Stark G. R. Characterization of the autoregulation of simian virus 40 gene A. J Virol. 1977 Oct;24(1):22–27. doi: 10.1128/jvi.24.1.22-27.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson J. L., Martin R. G., Chang C., Mora P. T., Livingston D. M. Nuclear preparations of SV40-transformed cells contain tumor-specific transplantation antigen activity. Virology. 1977 Jan;76(1):420–425. doi: 10.1016/0042-6822(77)90314-2. [DOI] [PubMed] [Google Scholar]
  3. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Chang C., Martin R. G., Livingston D. M., Luborsky S. W., Hu C. P., Mora P. T. Relationship between T-antigen and tumor-specific transplantation antigen in simian virus 40-transformed cells. J Virol. 1979 Jan;29(1):69–75. doi: 10.1128/jvi.29.1.69-75.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cole C. N., Crawford L. V., Berg P. Simian virus 40 mutants with deletions at the 3' end of the early region are defective in adenovirus helper function. J Virol. 1979 Jun;30(3):683–691. doi: 10.1128/jvi.30.3.683-691.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cole C. N., Landers T., Goff S. P., Manteuil-Brutlag S., Berg P. Physical and genetic characterization of deletion mutants of simian virus 40 constructed in vitro. J Virol. 1977 Oct;24(1):277–294. doi: 10.1128/jvi.24.1.277-294.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crawford L. V., Cole C. N., Smith A. E., Paucha E., Tegtmeyer P., Rundell K., Berg P. Organization and expression of early genes of simian virus 40. Proc Natl Acad Sci U S A. 1978 Jan;75(1):117–121. doi: 10.1073/pnas.75.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Crawford L. V., O'Farrell P. Z. Effect of alkylation on the physical properties of simian virus 40 T-antigen species. J Virol. 1979 Feb;29(2):587–596. doi: 10.1128/jvi.29.2.587-596.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deppert W., Walter G. Simian virus to (SV40) tumor-specific proteins in nucleus and plasma membrane of HeLa cells infected by adenovirus 2-SV40 hybrid virus Ad2+ND2. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2505–2509. doi: 10.1073/pnas.73.7.2505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feunteun J., Kress M., Gardes M., Monier R. Viable deletion mutants in the simian virus 40 early region. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4455–4459. doi: 10.1073/pnas.75.9.4455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fey G., Lewis J. B., Grodzicker T., Bothwell A. Characterization of a fused protein specified by the adenovirus type 2-simian virus 40 hybrid Ad2+ND1 dp2. J Virol. 1979 Apr;30(1):201–217. doi: 10.1128/jvi.30.1.201-217.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fiers W., Contreras R., Haegemann G., Rogiers R., Van de Voorde A., Van Heuverswyn H., Van Herreweghe J., Volckaert G., Ysebaert M. Complete nucleotide sequence of SV40 DNA. Nature. 1978 May 11;273(5658):113–120. doi: 10.1038/273113a0. [DOI] [PubMed] [Google Scholar]
  14. Gaudray P., Rassoulzadegan M., Cuzin F. Expression of simian virus 40 early genes in transformed rat cells is correlated with maintenance of the transformed phenotype. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4987–4991. doi: 10.1073/pnas.75.10.4987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Griffin J. D., Light S., Livingston D. M. Measurements of the molecular size of the simian virus 40 large T antigen. J Virol. 1978 Jul;27(1):218–226. doi: 10.1128/jvi.27.1.218-226.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hedrick J. L., Smith A. J. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys. 1968 Jul;126(1):155–164. doi: 10.1016/0003-9861(68)90569-9. [DOI] [PubMed] [Google Scholar]
  17. Kelly T. J., Jr, Nathans D. The genome of simian virus 40. Adv Virus Res. 1977;21:85–173. doi: 10.1016/s0065-3527(08)60762-9. [DOI] [PubMed] [Google Scholar]
  18. Khoury G., May E. Regulation of early and late simian virus 40 transcription: overproduction of early viral RNA in the absence of a functional T-antigen. J Virol. 1977 Jul;23(1):167–176. doi: 10.1128/jvi.23.1.167-176.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kuchino T., Yamaguchi N. Characterization of T antigen in cells infected with a temperature-sensitive mutant of simian virus 40. J Virol. 1975 Jun;15(6):1302–1307. doi: 10.1128/jvi.15.6.1302-1307.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Lane D. P., Robbins A. K. An immunochemical investigation of SV40 T antigens. 1. Production properties and specificity of rabbit antibody to purified simian virus 40 large-T antigen. Virology. 1978 Jun 1;87(1):182–193. doi: 10.1016/0042-6822(78)90170-8. [DOI] [PubMed] [Google Scholar]
  23. Lebowitz P., Kelly T. J., Jr, Nathans D., Lee T. N., Lewis A. M., Jr A colinear map relating the simian virus 40 (SV40) DNA segments of six adenovirus-SV40 hybrids to the DNA fragments produced by restriction endonuclease cleavage of SV40 DNA. Proc Natl Acad Sci U S A. 1974 Feb;71(2):441–445. doi: 10.1073/pnas.71.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Linke H. K., Hunter T., Walter G. Structural relationship between the 100,000- and 17,000- molecular-weight T antigens of simian virus 40 (SV40) as deduced by comparison with the SV40-specific proteins coded by the nondefective adenovirus type 2-SV40 hybrid viruses. J Virol. 1979 Jan;29(1):390–394. doi: 10.1128/jvi.29.1.390-394.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Martin R. G., Setlow V. P., Edwards C. A. Roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells: studies with simian virus 40 double mutants. J Virol. 1979 Sep;31(3):596–607. doi: 10.1128/jvi.31.3.596-607.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. May E., Kress M., May P. Characterization of two SV40 early mRNAs and evidence for a nuclear "prespliced" RNA species. Nucleic Acids Res. 1978 Sep;5(9):3083–3099. doi: 10.1093/nar/5.9.3083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Oppenheim A., Martin R. G. Initiation points for DNA replication in nontransformed and simian virus 40-transformed BALB/c 3T3 cells. J Virol. 1978 Jan;25(1):450–452. doi: 10.1128/jvi.25.1.450-452.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Paucha E., Smith A. E. The sequences between 0.59 and 0.54 map units on SV40 DNA code for the unique region of small t antigen. Cell. 1978 Nov;15(3):1011–1020. doi: 10.1016/0092-8674(78)90285-4. [DOI] [PubMed] [Google Scholar]
  32. Prives C., Gilboa E., Revel M., Winocour E. Cell-free translation of simian virus 40 early messenger RNA coding for viral T-antigen. Proc Natl Acad Sci U S A. 1977 Feb;74(2):457–461. doi: 10.1073/pnas.74.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rassoulzadegan M., Seif R., Cuzin F. Conditions leading to the establishment of the N (a gene dependent) and A (a gene independent) transformed states after polyoma virus infection of rat fibroblasts. J Virol. 1978 Nov;28(2):421–426. doi: 10.1128/jvi.28.2.421-426.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reddy V. B., Ghosh P. K., Lebowitz P., Piatak M., Weissman S. M. Simian virus 40 early mRNA's. I. Genomic localization of 3' and 5' termini and two major splices in mRNA from transformed and lytically infected cells. J Virol. 1979 Apr;30(1):279–296. doi: 10.1128/jvi.30.1.279-296.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Reddy V. B., Thimmappaya B., Dhar R., Subramanian K. N., Zain B. S., Pan J., Ghosh P. K., Celma M. L., Weissman S. M. The genome of simian virus 40. Science. 1978 May 5;200(4341):494–502. doi: 10.1126/science.205947. [DOI] [PubMed] [Google Scholar]
  36. Rundell K., Collins J. K., Tegtmeyer P., Ozer H. L., Lai C. J., Nathans D. Identification of simian virus 40 protein A. J Virol. 1977 Feb;21(2):636–646. doi: 10.1128/jvi.21.2.636-646.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Soule H. R., Butel J. S. Subcellular Localization of simian virus 40 large tumor antigen. J Virol. 1979 May;30(2):523–532. doi: 10.1128/jvi.30.2.523-532.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Titani K., Koide A., Hermann J., Ericsson L. H., Kumar S., Wade R. D., Walsh K. A., Neurath H., Fischer E. H. Complete amino acid sequence of rabbit muscle glycogen phosphorylase. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4762–4766. doi: 10.1073/pnas.74.11.4762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Van Heuverswyn H., Cole C., Berg P., Fiers W. Nucleotide sequence analysis of two simian virus 40 mutants with deletions in the region coding for the carboxyl terminus of the T antigen. J Virol. 1979 Jun;30(3):936–941. doi: 10.1128/jvi.30.3.936-941.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES