Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1990 May 11;18(9):2715–2720. doi: 10.1093/nar/18.9.2715

Loss of DNA-binding and new transcriptional trans-activation function in polyomavirus large T-antigen with mutation of zinc finger motif.

A Bergqvist 1, M Nilsson 1, K Bondeson 1, G Magnusson 1
PMCID: PMC330756  PMID: 2160069

Abstract

A putative zinc finger in polyomavirus large T-antigen was investigated. We were unable to demonstrate unequivocally a requirement for zinc in specific DNA-binding using the chelating agent 1, 10-phenanthroline. An involvement of the putative zinc finger in specific DNA-binding was nevertheless suggested by the properties of a mutant protein with a cys----ser replacement in the finger motif. Probably as a result of the defective DNA-binding, the mutant protein had lost its activity in initiation of viral DNA-replication and in negative regulation of viral early transcription. However, the trans-activation of the viral late promoter was normal. The analysis also revealed a previously unrecognized activity of large T-antigen. The mutant protein trans-activated the viral early promoter. In the wild-type protein this activity is probably concealed by the separate, negative regulatory function.

Full text

PDF
2715

Images in this article

2717Image
on p.2717
2718Image
on p.2718
2718Image
on p.2718
2719Image
on p.2719

Selected References

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

  1. Arthur A. K., Höss A., Fanning E. Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids. J Virol. 1988 Jun;62(6):1999–2006. doi: 10.1128/jvi.62.6.1999-2006.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Auld D. S. Use of chelating agents to inhibit enzymes. Methods Enzymol. 1988;158:110–114. doi: 10.1016/0076-6879(88)58051-5. [DOI] [PubMed] [Google Scholar]
  3. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  4. Berg J. M. Proposed structure for the zinc-binding domains from transcription factor IIIA and related proteins. Proc Natl Acad Sci U S A. 1988 Jan;85(1):99–102. doi: 10.1073/pnas.85.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bourachot B., Yaniv M., Herbomel P. Control elements situated downstream of the major transcriptional start site are sufficient for highly efficient polyomavirus late transcription. J Virol. 1989 Jun;63(6):2567–2577. doi: 10.1128/jvi.63.6.2567-2577.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brady J., Loeken M. R., Khoury G. Interaction between two transcriptional control sequences required for tumor-antigen-mediated simian virus 40 late gene expression. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7299–7303. doi: 10.1073/pnas.82.21.7299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cogen B. Virus-specific early RNA in 3T6 cells infected by a tsA mutant of polyoma virus. Virology. 1978 Mar;85(1):222–230. doi: 10.1016/0042-6822(78)90426-9. [DOI] [PubMed] [Google Scholar]
  8. Cowie A., Kamen R. Multiple binding sites for polyomavirus large T antigen within regulatory sequences of polyomavirus DNA. J Virol. 1984 Dec;52(3):750–760. doi: 10.1128/jvi.52.3.750-760.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cowie A., de Villiers J., Kamen R. Immortalization of rat embryo fibroblasts by mutant polyomavirus large T antigens deficient in DNA binding. Mol Cell Biol. 1986 Dec;6(12):4344–4352. doi: 10.1128/mcb.6.12.4344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Evans R. M., Hollenberg S. M. Zinc fingers: gilt by association. Cell. 1988 Jan 15;52(1):1–3. doi: 10.1016/0092-8674(88)90522-3. [DOI] [PubMed] [Google Scholar]
  13. Farmerie W. G., Folk W. R. Regulation of polyomavirus transcription by large tumor antigen. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6919–6923. doi: 10.1073/pnas.81.22.6919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Francke B., Eckhart W. Polyoma gene function required for viral DNA synthesis. Virology. 1973 Sep;55(1):127–135. doi: 10.1016/s0042-6822(73)81014-1. [DOI] [PubMed] [Google Scholar]
  15. Gibson T. J., Postma J. P., Brown R. S., Argos P. A model for the tertiary structure of the 28 residue DNA-binding motif ('zinc finger') common to many eukaryotic transcriptional regulatory proteins. Protein Eng. 1988 Sep;2(3):209–218. doi: 10.1093/protein/2.3.209. [DOI] [PubMed] [Google Scholar]
  16. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hansen U., Tenen D. G., Livingston D. M., Sharp P. A. T antigen repression of SV40 early transcription from two promoters. Cell. 1981 Dec;27(3 Pt 2):603–613. doi: 10.1016/0092-8674(81)90402-5. [DOI] [PubMed] [Google Scholar]
  18. Katinka M., Yaniv M. Deletions of N-terminal sequences of polyoma virus T-antigens reduce but do not abolish transformation of rat fibroblasts. Mol Cell Biol. 1982 Oct;2(10):1238–1246. doi: 10.1128/mcb.2.10.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Keller J. M., Alwine J. C. Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication. Cell. 1984 Feb;36(2):381–389. doi: 10.1016/0092-8674(84)90231-9. [DOI] [PubMed] [Google Scholar]
  20. Kern F. G., Pellegrini S., Cowie A., Basilico C. Regulation of polyomavirus late promoter activity by viral early proteins. J Virol. 1986 Oct;60(1):275–285. doi: 10.1128/jvi.60.1.275-285.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Loeber G., Parsons R., Tegtmeyer P. The zinc finger region of simian virus 40 large T antigen. J Virol. 1989 Jan;63(1):94–100. doi: 10.1128/jvi.63.1.94-100.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lusky M., Botchan M. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature. 1981 Sep 3;293(5827):79–81. doi: 10.1038/293079a0. [DOI] [PubMed] [Google Scholar]
  24. Luthman H., Magnusson G. High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 1983 Mar 11;11(5):1295–1308. doi: 10.1093/nar/11.5.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Martens I., Nilsson S. A., Linder S., Magnusson G. Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation. J Virol. 1989 May;63(5):2126–2133. doi: 10.1128/jvi.63.5.2126-2133.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mazus B., Falchuk K. H., Vallee B. L. Inhibition of Euglena gracilis and wheat germ zinc RNA polymerases II by 1,10-phenanthroline acting as a chelating agent. Biochemistry. 1986 May 20;25(10):2941–2945. doi: 10.1021/bi00358a031. [DOI] [PubMed] [Google Scholar]
  27. McKay R. D. Binding of a simian virus 40 T antigen-related protein to DNA. J Mol Biol. 1981 Jan 25;145(3):471–488. doi: 10.1016/0022-2836(81)90540-4. [DOI] [PubMed] [Google Scholar]
  28. Miller J., McLachlan A. D., Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun;4(6):1609–1614. doi: 10.1002/j.1460-2075.1985.tb03825.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nilsson S. V., Magnusson G. Activities of polyomavirus large-T-antigen proteins expressed by mutant genes. J Virol. 1984 Sep;51(3):768–775. doi: 10.1128/jvi.51.3.768-775.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Pomerantz B. J., Mueller C. R., Hassell J. A. Polyomavirus large T antigen binds independently to multiple, unique regions on the viral genome. J Virol. 1983 Sep;47(3):600–610. doi: 10.1128/jvi.47.3.600-610.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rice W. C., Lorimer H. E., Prives C., Miller L. K. Expression of polyomavirus large T antigen by using a baculovirus vector. J Virol. 1987 May;61(5):1712–1716. doi: 10.1128/jvi.61.5.1712-1716.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rio D., Robbins A., Myers R., Tjian R. Regulation of simian virus 40 early transcription in vitro by a purified tumor antigen. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5706–5710. doi: 10.1073/pnas.77.10.5706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Stanssens P., Opsomer C., McKeown Y. M., Kramer W., Zabeau M., Fritz H. J. Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res. 1989 Jun 26;17(12):4441–4454. doi: 10.1093/nar/17.12.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tang W. J., Folk W. R. Asp-286----Asn-286 in polyomavirus large T antigen relaxes the specificity of binding to the polyomavirus origin. J Virol. 1989 Jan;63(1):242–249. doi: 10.1128/jvi.63.1.242-249.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Westin G., Schaffner W. Heavy metal ions in transcription factors from HeLa cells: Sp1, but not octamer transcription factor requires zinc for DNA binding and for activator function. Nucleic Acids Res. 1988 Jul 11;16(13):5771–5781. doi: 10.1093/nar/16.13.5771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Yamaguchi Y., Satake M., Ito Y. Two overlapping sequence motifs within the polyomavirus enhancer are independently the targets of stimulation by both the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the Ha-ras oncogene. J Virol. 1989 Mar;63(3):1040–1048. doi: 10.1128/jvi.63.3.1040-1048.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES