Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1985 Jul;55(1):1–9. doi: 10.1128/jvi.55.1.1-9.1985

Site-directed mutagenesis of the simian virus 40 large T-antigen gene: replication-defective amino acid substitution mutants that retain the ability to induce morphological transformation.

K W Peden, J M Pipas
PMCID: PMC254890  PMID: 2989548

Abstract

We used a heteroduplex deletion loop mutagenesis procedure for directing sodium bisulfite-induced mutations to specific sites on viral or plasmid DNA to generate a series of SV40 large T-antigen point mutants. The mutations were directed to a region of the T-antigen gene, 0.5 map units, that is thought to be important for interaction of the protein with the viral origin of DNA replication. Of the 16 mutants reported here, 10 had lost the ability to replicate their DNA, and 3 others showed a reduced level of replication compared to wild type. All of the mutants tested were capable of transforming rat cells in culture by the dense focus assay. We conclude that the sequences of the early region around 0.5 map units are critical for the replication of viral DNA but not for the transformation function of T antigen.

Full text

PDF
1

Images in this article

7Image
on p.7

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. Ariga H., Sugano S. Initiation of simian virus 40 DNA replication in vitro. J Virol. 1983 Nov;48(2):481–491. doi: 10.1128/jvi.48.2.481-491.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brockman W. W., Lee T. N., Nathans D. The evolution of new species of viral DNA during serial passage of simian virus 40 at high multiplicity. Virology. 1973 Aug;54(2):384–397. doi: 10.1016/0042-6822(73)90151-7. [DOI] [PubMed] [Google Scholar]
  4. Brockman W. W., Nathans D. The isolation of simian virus 40 variants with specifically altered genomes. Proc Natl Acad Sci U S A. 1974 Mar;71(3):942–946. doi: 10.1073/pnas.71.3.942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brockman W. W. Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: temperature sensitivity of the transformed phenotype and retransofrmation by wild-type virus. J Virol. 1978 Mar;25(3):860–870. doi: 10.1128/jvi.25.3.860-870.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brugge J. S., Butel J. S. Role of simian virus 40 gene A function in maintenance of transformation. J Virol. 1975 Mar;15(3):619–635. doi: 10.1128/jvi.15.3.619-635.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chou J. Y., Martin R. G. DNA infectivity and the induction of host DNA synthesis with temperature-sensitive mutants of simian virus 40. J Virol. 1975 Jan;15(1):145–150. doi: 10.1128/jvi.15.1.145-150.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clark R., Lane D. P., Tjian R. Use of monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity. J Biol Chem. 1981 Nov 25;256(22):11854–11858. [PubMed] [Google Scholar]
  9. Clark R., Peden K., Pipas J. M., Nathans D., Tjian R. Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants. Mol Cell Biol. 1983 Feb;3(2):220–228. doi: 10.1128/mcb.3.2.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clertant P., Seif I. A common function for polyoma virus large-T and papillomavirus E1 proteins? Nature. 1984 Sep 20;311(5983):276–279. doi: 10.1038/311276a0. [DOI] [PubMed] [Google Scholar]
  11. Cosman D. J., Tevethia M. J. Characterization of a temperature-sensitive, DNA-positive, nontransforming mutant of simian virus 40. Virology. 1981 Jul 30;112(2):605–624. doi: 10.1016/0042-6822(81)90306-8. [DOI] [PubMed] [Google Scholar]
  12. Cowan K., Tegtmeyer P., Anthony D. D. Relationship of replication and transcription of Simian Virus 40 DNA. Proc Natl Acad Sci U S A. 1973 Jul;70(7):1927–1930. doi: 10.1073/pnas.70.7.1927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Friedmann T., Esty A., LaPorte P., Deininger P. The nucleotide sequence and genome organization of the polyoma early region: extensive nucleotide and amino acid homology with SV40. Cell. 1979 Jul;17(3):715–724. doi: 10.1016/0092-8674(79)90278-2. [DOI] [PubMed] [Google Scholar]
  14. Frost E., Williams J. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology. 1978 Nov;91(1):39–50. doi: 10.1016/0042-6822(78)90353-7. [DOI] [PubMed] [Google Scholar]
  15. Galanti N., Jonak G. J., Soprano K. J., Floros J., Kaczmarek L., Weissman S., Reddy V. B., Tilghman S. M., Baserga R. Characterization and biological activity of cloned simian virus 40 DNA fragments. J Biol Chem. 1981 Jun 25;256(12):6469–6474. [PubMed] [Google Scholar]
  16. Giacherio D., Hager L. P. A poly(dT)-stimulated ATPase activity associated with simian virus 40 large T antigen. J Biol Chem. 1979 Sep 10;254(17):8113–8116. [PubMed] [Google Scholar]
  17. Giza P. E., Schmit D. M., Murr B. L. Region- and strand-specific mutagensis of a recombinant plasmid. Gene. 1981 Dec;15(4):331–342. doi: 10.1016/0378-1119(81)90176-1. [DOI] [PubMed] [Google Scholar]
  18. Gluzman Y., Ahrens B. SV40 early mutants that are defective for viral DNA synthesis but competent for transformation of cultured rat and simian cells. Virology. 1982 Nov;123(1):78–92. doi: 10.1016/0042-6822(82)90296-3. [DOI] [PubMed] [Google Scholar]
  19. Gluzman Y., Davison J., Oren M., Winocour E. Properties of permissive monkey cells transformed by UV-irradiated simian virus 40. J Virol. 1977 May;22(2):256–266. doi: 10.1128/jvi.22.2.256-266.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  21. Hiscott J. B., Defendi V. Simian virus 40 gene A regulation of cellular DNA synthesis. I. In permissive cells. J Virol. 1979 May;30(2):590–599. doi: 10.1128/jvi.30.2.590-599.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hiscott J. B., Defendi V. Simian virus 40 gene A regulation of cellular DNA synthesis. II. In nonpermissive cells. J Virol. 1981 Feb;37(2):802–812. doi: 10.1128/jvi.37.2.802-812.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  24. Kalderon D., Smith A. E. In vitro mutagenesis of a putative DNA binding domain of SV40 large-T. Virology. 1984 Nov;139(1):109–137. doi: 10.1016/0042-6822(84)90334-9. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Kimura G., Dulbecco R. A temperature-sensitive mutant of simian virus 40 affecting transforming ability. Virology. 1973 Apr;52(2):529–534. doi: 10.1016/0042-6822(73)90348-6. [DOI] [PubMed] [Google Scholar]
  28. Kimura G., Itagaki A. Initiation and maintenance of cell transformation by simian virus 40: a viral genetic property. Proc Natl Acad Sci U S A. 1975 Feb;72(2):673–677. doi: 10.1073/pnas.72.2.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lai C. J., Nathans D. A map of temperature-sensitive mutants of simian virus 40. Virology. 1975 Jul;66(1):70–81. doi: 10.1016/0042-6822(75)90179-8. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Lederberg E. M., Cohen S. N. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. doi: 10.1128/jb.119.3.1072-1074.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6973–6977. doi: 10.1073/pnas.81.22.6973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Manos M. M., Gluzman Y. Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants. J Virol. 1985 Jan;53(1):120–127. doi: 10.1128/jvi.53.1.120-127.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Manos M. M., Gluzman Y. Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation. Mol Cell Biol. 1984 Jun;4(6):1125–1133. doi: 10.1128/mcb.4.6.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Margolskee R. F., Nathans D. Simian virus 40 mutant T antigens with relaxed specificity for the nucleotide sequence at the viral DNA origin of replication. J Virol. 1984 Feb;49(2):386–393. doi: 10.1128/jvi.49.2.386-393.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Martin R. G., Chou J. Y. Simian virus 40 functions required for the establishment and maintenance of malignant transformation. J Virol. 1975 Mar;15(3):599–612. doi: 10.1128/jvi.15.3.599-612.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. McCutchan J. H., Pagano J. S. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed] [Google Scholar]
  41. 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]
  42. McKay R., DiMaio D. Binding of an SV40 T antigen-related protein to the DNA of SV40 regulatory mutants. Nature. 1981 Feb 26;289(5800):810–813. doi: 10.1038/289810a0. [DOI] [PubMed] [Google Scholar]
  43. Meselson M., Yuan R. DNA restriction enzyme from E. coli. Nature. 1968 Mar 23;217(5134):1110–1114. doi: 10.1038/2171110a0. [DOI] [PubMed] [Google Scholar]
  44. Osborn M., Weber K. Simian virus 40 gene A function and maintenance of transformation. J Virol. 1975 Mar;15(3):636–644. doi: 10.1128/jvi.15.3.636-644.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. POPE J. H., ROWE W. P. DETECTION OF SPECIFIC ANTIGEN IN SV40-TRANSFORMED CELLS BY IMMUNOFLUORESCENCE. J Exp Med. 1964 Aug 1;120:121–128. doi: 10.1084/jem.120.2.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Peden K. W., Nathans D. Local mutagenesis within deletion loops of DNA heteroduplexes. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7214–7217. doi: 10.1073/pnas.79.23.7214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Peden K. W., Pipas J. M., Pearson-White S., Nathans D. Isolation of mutants of an animal virus in bacteria. Science. 1980 Sep 19;209(4463):1392–1396. doi: 10.1126/science.6251547. [DOI] [PubMed] [Google Scholar]
  49. Peden K., Mounts P., Hayward G. S. Homology between mammalian cell DNA sequences and human herpesvirus genomes detected by a hybridization procedure with high-complexity probe. Cell. 1982 Nov;31(1):71–80. doi: 10.1016/0092-8674(82)90406-8. [DOI] [PubMed] [Google Scholar]
  50. Pipas J. M., Peden K. W., Nathans D. Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene. Mol Cell Biol. 1983 Feb;3(2):203–213. doi: 10.1128/mcb.3.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Prives C., Covey L., Scheller A., Gluzman Y. DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication. Mol Cell Biol. 1983 Nov;3(11):1958–1966. doi: 10.1128/mcb.3.11.1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Reed S. I., Ferguson J., Davis R. W., Stark G. R. T antigen binds to simian virus 40 DNA at the origin of replication. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1605–1609. doi: 10.1073/pnas.72.4.1605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Reed S. I., Stark G. R., Alwine J. C. Autoregulation of simian virus 40 gene A by T antigen. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3083–3087. doi: 10.1073/pnas.73.9.3083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  55. Rosenthal L. J., Brown M. The control of SV40 transcription during a lytic infection: late RNA synthesis in the presence of inhibitors of DNA replication. Nucleic Acids Res. 1977 Mar;4(3):551–565. doi: 10.1093/nar/4.3.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sanger F., Coulson A. R. The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 1978 Mar 1;87(1):107–110. doi: 10.1016/0014-5793(78)80145-8. [DOI] [PubMed] [Google Scholar]
  57. Scott W. A., Brockman W. W., Nathans D. Biological activities of deletion mutants of simian virus 40. Virology. 1976 Dec;75(2):319–334. doi: 10.1016/0042-6822(76)90031-3. [DOI] [PubMed] [Google Scholar]
  58. Shalloway D., Kleinberger T., Livingston D. M. Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III digestion. Cell. 1980 Jun;20(2):411–422. doi: 10.1016/0092-8674(80)90627-3. [DOI] [PubMed] [Google Scholar]
  59. Shiroki K., Shimojo H. Transformation of green monkey kidney cells by SV40 genome: the establishment of transformed cell lines and the replication of human adenoviruses and SV40 in transformed cells. Virology. 1971 Jul;45(1):163–171. doi: 10.1016/0042-6822(71)90123-1. [DOI] [PubMed] [Google Scholar]
  60. Shortle D. R., Margolskee R. F., Nathans D. Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6128–6131. doi: 10.1073/pnas.76.12.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. 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]
  62. Shortle D., Nathans D. Regulatory mutants of simian virus 40: constructed mutants with base substitutions at the origin of DNA replication. J Mol Biol. 1979 Jul 15;131(4):801–817. doi: 10.1016/0022-2836(79)90202-x. [DOI] [PubMed] [Google Scholar]
  63. Smith H. O. Recovery of DNA from gels. Methods Enzymol. 1980;65(1):371–380. doi: 10.1016/s0076-6879(80)65048-4. [DOI] [PubMed] [Google Scholar]
  64. Soprano K. J., Dev V. G., Croce C. M., Baserga R. Reactivation of silent rRNA genes by simian virus 40 in human-mouse hybrid cells. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3885–3889. doi: 10.1073/pnas.76.8.3885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Soprano K. J., Galanti N., Jonak G. J., McKercher S., Pipas J. M., Peden K. W., Baserga R. Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T-antigen gene. Mol Cell Biol. 1983 Feb;3(2):214–219. doi: 10.1128/mcb.3.2.214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Soprano K. J., Rossini M., Croce C., Baserga R. The role of large T antigen in simian virus 40-induced reactivation of silent rRNA genes in human-mouse hybrid cells. Virology. 1980 Apr 30;102(2):317–326. doi: 10.1016/0042-6822(80)90099-9. [DOI] [PubMed] [Google Scholar]
  67. 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]
  68. Steinberg B., Pollack R., Topp W., Botchan M. Isolation and characterization of T antigen-negative revertants from a line of transformed rat cells containing one copy of the SV40 genome. Cell. 1978 Jan;13(1):19–32. doi: 10.1016/0092-8674(78)90134-4. [DOI] [PubMed] [Google Scholar]
  69. Stringer J. R. Mutant of simian virus 40 large T-antigen that is defective for viral DNA synthesis, but competent for transformation of cultured rat cells. J Virol. 1982 Jun;42(3):854–864. doi: 10.1128/jvi.42.3.854-864.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Suarez H. G., Bourali M. F., Wicker R., Estrade S., Cassingena R. Properties of an SV40-transformed African green monkey kidney (BSC-1) cell line. Int J Cancer. 1972 Mar 15;9(2):324–333. doi: 10.1002/ijc.2910090210. [DOI] [PubMed] [Google Scholar]
  71. Tegtmeyer P. Function of simian virus 40 gene A in transforming infection. J Virol. 1975 Mar;15(3):613–618. doi: 10.1128/jvi.15.3.613-618.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Tegtmeyer P., Schwartz M., Collins J. K., Rundell K. Regulation of tumor antigen synthesis by simain virus 40 gene A. J Virol. 1975 Jul;16(1):168–178. doi: 10.1128/jvi.16.1.168-178.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Tegtmeyer P. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J Virol. 1972 Oct;10(4):591–598. doi: 10.1128/jvi.10.4.591-598.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Tjian R., Fey G., Graessmann A. Biological activity of purified simian virus 40 T antigen proteins. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1279–1283. doi: 10.1073/pnas.75.3.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Tjian R., Robbins A. Enzymatic activities associated with a purified simian virus 40 T antigen-related protein. Proc Natl Acad Sci U S A. 1979 Feb;76(2):610–614. doi: 10.1073/pnas.76.2.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Tjian R. The binding site on SV40 DNA for a T antigen-related protein. Cell. 1978 Jan;13(1):165–179. doi: 10.1016/0092-8674(78)90147-2. [DOI] [PubMed] [Google Scholar]
  77. Topp W. C. Normal rat cell lines deficient in nuclear thymidine kinase. Virology. 1981 Aug;113(1):408–411. doi: 10.1016/0042-6822(81)90168-9. [DOI] [PubMed] [Google Scholar]
  78. Tordaro G. J., Green H. An assay for cellular transformation by SV40. Virology. 1964 May;23(1):117–119. doi: 10.1016/s0042-6822(64)80018-0. [DOI] [PubMed] [Google Scholar]
  79. van Roy F., Fransen L., Fiers W. Improved localization of phosphorylation sites in simian virus 40 large T antigen. J Virol. 1983 Jan;45(1):315–331. doi: 10.1128/jvi.45.1.315-331.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES