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. 1985 Sep;55(3):660–669. doi: 10.1128/jvi.55.3.660-669.1985

Construction and biological analysis of deletion mutants of Fujinami sarcoma virus: 5'-fps sequence has a role in the transforming activity.

K Ariizumi, M Shibuya
PMCID: PMC255035  PMID: 2991588

Abstract

Fujinami sarcoma virus (FSV) genome codes for the gag-fps fusion protein FSV-P130. The amino acid sequence of the 3' one-third portion in v-fps is partially homologous to the 3' half of pp60src, or the kinase domain, but the sequence of the 5' portion is unique to v-fps. To identify a possible domain structure in the v-fps sequence responsible for cell transformation, we constructed various deletion mutants of FSV with molecularly cloned viral DNA. Their transforming activities were assayed by measuring focus formation on chicken embryo fibroblasts and rat 3Y1 cells and tumor formation in chickens. The mutants carrying a deletion at the 3' portion in v-fps, the kinase domain, lost transforming activity. The mutants carrying an approximately 1-kilobase deletion within the 5' portion of the v-fps sequence retained focus-forming activity and tumorigenicity in the chicken system, but the efficiency of focus formation was about 10 times lower than that of the wild type. The morphology of these transformed cells was distinct from that observed in cells infected with wild-type FSV. Furthermore, these mutants could not transform rat 3Y1 cells, although wild-type FSV DNA transformed rat 3Y1 cells at a high frequency. The mutants carrying a larger deletion in the 5' portion of fps completely lacked the transforming activity. These results suggest that the 3' portion of the v-fps sequence is necessary but not sufficient for cell transformation and that the 5' portion of v-fps has a role in the transforming activity.

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Selected References

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  1. Balduzzi P. C., Notter M. F., Morgan H. R., Shibuya M. Some biological properties of two new avian sarcoma viruses. J Virol. 1981 Oct;40(1):268–275. doi: 10.1128/jvi.40.1.268-275.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Breitman M. L., Hirano A., Wong T., Vogt P. K. Characteristics of avian sarcoma virus strain PRCIV and comparison with strain PRCII-p. Virology. 1981 Oct 30;114(2):451–462. doi: 10.1016/0042-6822(81)90225-7. [DOI] [PubMed] [Google Scholar]
  3. Breitman M. L., Neil J. C., Moscovici C., Vogt P. K. The pathogenicity and defectiveness of PRCII: a new type of avian sarcoma virus. Virology. 1981 Jan 15;108(1):1–12. doi: 10.1016/0042-6822(81)90522-5. [DOI] [PubMed] [Google Scholar]
  4. Bryant D., Parsons J. T. Site-directed mutagenesis of the src gene of Rous sarcoma virus: construction and characterization of a deletion mutant temperature sensitive for transformation. J Virol. 1982 Nov;44(2):683–691. doi: 10.1128/jvi.44.2.683-691.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. CARR J. G., CAMPBELL J. G. Three new virus-induced fowl sarcomata. Br J Cancer. 1958 Dec;12(4):631–635. doi: 10.1038/bjc.1958.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carlberg K., Chamberlin M. E., Beemon K. The avian sarcoma virus PRCII lacks 1020 nucleotides of the fps transforming gene. Virology. 1984 May;135(1):157–167. doi: 10.1016/0042-6822(84)90126-0. [DOI] [PubMed] [Google Scholar]
  7. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooper G. M., Okenquist S. Mechanism of transfection of chicken embryo fibroblasts by Rous sarcoma virus DNA. J Virol. 1978 Oct;28(1):45–52. doi: 10.1128/jvi.28.1.45-52.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cross F. R., Garber E. A., Pellman D., Hanafusa H. A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation. Mol Cell Biol. 1984 Sep;4(9):1834–1842. doi: 10.1128/mcb.4.9.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duesberg P. H., Phares W., Lee W. H. The low tumorigenic potential of PRCII, among viruses of the Fujinami sarcoma virus subgroup, corresponds to an internal (fps) deletion of the transforming gene. Virology. 1983 Nov;131(1):144–158. doi: 10.1016/0042-6822(83)90541-x. [DOI] [PubMed] [Google Scholar]
  11. Dugaiczyk A., Boyer H. W., Goodman H. M. Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures. J Mol Biol. 1975 Jul 25;96(1):171–184. doi: 10.1016/0022-2836(75)90189-8. [DOI] [PubMed] [Google Scholar]
  12. Feldman R. A., Hanafusa T., Hanafusa H. Characterization of protein kinase activity associated with the transforming gene product of Fujinami sarcoma virus. Cell. 1980 Dec;22(3):757–765. doi: 10.1016/0092-8674(80)90552-8. [DOI] [PubMed] [Google Scholar]
  13. Feldman R. A., Wang E., Hanafusa H. Cytoplasmic localization of the transforming protein of Fujinami sarcoma virus: salt-sensitive association with subcellular components. J Virol. 1983 Feb;45(2):782–791. doi: 10.1128/jvi.45.2.782-791.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Foster D. A., Hanafusa H. A fps gene without gag gene sequences transforms cells in culture and induces tumors in chickens. J Virol. 1983 Dec;48(3):744–751. doi: 10.1128/jvi.48.3.744-751.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guyden J. C., Martin G. S. Transformation parameters of chick embryo fibroblasts transformed by Fujinami, PRCII, PRCII-p, and Y73 avian sarcoma viruses. Virology. 1982 Oct 15;122(1):71–83. doi: 10.1016/0042-6822(82)90378-6. [DOI] [PubMed] [Google Scholar]
  16. Hanafusa H. Rapid transformation of cells by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1969 Jun;63(2):318–325. doi: 10.1073/pnas.63.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hanafusa T., Mathey-Prevot B., Feldman R. A., Hanafusa H. Mutants of Fujinami sarcoma virus which are temperature sensitive for cellular transformation and protein kinase activity. J Virol. 1981 Apr;38(1):347–355. doi: 10.1128/jvi.38.1.347-355.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hanafusa T., Wang L. H., Anderson S. M., Karess R. E., Hayward W. S., Hanafusa H. Characterization of the transforming gene of Fujinami sarcoma virus. Proc Natl Acad Sci U S A. 1980 May;77(5):3009–3013. doi: 10.1073/pnas.77.5.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Huang C. C., Hammond C., Bishop J. M. Nucleotide sequence of v-fps in the PRCII strain of avian sarcoma virus. J Virol. 1984 Apr;50(1):125–131. doi: 10.1128/jvi.50.1.125-131.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kawai S., Nishizawa M. New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol Cell Biol. 1984 Jun;4(6):1172–1174. doi: 10.1128/mcb.4.6.1172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kitamura N., Yoshida M. Small deletion in src of Rous sarcoma virus modifying transformation phenotypes: identification of 207-nucleotide deletion and its smaller product with protein kinase activity. J Virol. 1983 Jun;46(3):985–992. doi: 10.1128/jvi.46.3.985-992.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koyama T., Harada F., Kawai S. Characterization of a Rous sarcoma virus mutant defective in packaging its own genomic RNA: biochemical properties of mutant TK15 and mutant-induced transformants. J Virol. 1984 Jul;51(1):154–162. doi: 10.1128/jvi.51.1.154-162.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krueger J. G., Garber E. A., Goldberg A. R., Hanafusa H. Changes in amino-terminal sequences of pp60src lead to decreased membrane association and decreased in vivo tumorigenicity. Cell. 1982 Apr;28(4):889–896. doi: 10.1016/0092-8674(82)90068-x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Lee W. H., Bister K., Pawson A., Robins T., Moscovici C., Duesberg P. H. Fujinami sarcoma virus: an avian RNA tumor virus with a unique transforming gene. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2018–2022. doi: 10.1073/pnas.77.4.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lee W. H., Phares W., Duesberg P. H. Structural relationship between the chicken DNA locus, proto-fps, and the transforming gene of Fujinami sarcoma virus, delta gag-fps. Virology. 1983 Aug;129(1):79–93. doi: 10.1016/0042-6822(83)90397-5. [DOI] [PubMed] [Google Scholar]
  28. Levinson A. D., Courtneidge S. A., Bishop J. M. Structural and functional domains of the Rous sarcoma virus transforming protein (pp60src). Proc Natl Acad Sci U S A. 1981 Mar;78(3):1624–1628. doi: 10.1073/pnas.78.3.1624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Levitt M. A simplified representation of protein conformations for rapid simulation of protein folding. J Mol Biol. 1976 Jun 14;104(1):59–107. doi: 10.1016/0022-2836(76)90004-8. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Marinus M. G., Morris N. R. Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J Bacteriol. 1973 Jun;114(3):1143–1150. doi: 10.1128/jb.114.3.1143-1150.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mathey-Prevot B., Hanafusa H., Kawai S. A cellular protein is immunologically crossreactive with and functionally homologous to the Fujinami sarcoma virus transforming protein. Cell. 1982 Apr;28(4):897–906. doi: 10.1016/0092-8674(82)90069-1. [DOI] [PubMed] [Google Scholar]
  33. Neckameyer W. S., Wang L. H. Molecular cloning and characterization of avian sarcoma virus UR2 and comparison of its transforming sequence with those of other avian sarcoma viruses. J Virol. 1984 Jun;50(3):914–921. doi: 10.1128/jvi.50.3.914-921.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Neel B. G., Wang L. H., Mathey-Prevot B., Hanafusa T., Hanafusa H., Hayward W. S. Isolation of 16L virus: a rapidly transforming sarcoma virus from an avian leukosis virus-induced sarcoma. Proc Natl Acad Sci U S A. 1982 Aug;79(16):5088–5092. doi: 10.1073/pnas.79.16.5088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Neil J. C. Defective avian sarcoma viruses. Curr Top Microbiol Immunol. 1983;103:51–74. doi: 10.1007/978-3-642-68943-7_3. [DOI] [PubMed] [Google Scholar]
  36. Pawson T., Guyden J., Kung T. H., Radke K., Gilmore T., Martin G. S. A strain of Fujinami sarcoma virus which is temperature-sensitive in protein phosphorylation and cellular transformation. Cell. 1980 Dec;22(3):767–775. doi: 10.1016/0092-8674(80)90553-x. [DOI] [PubMed] [Google Scholar]
  37. Pawson T., Kung T. H., Martin G. S. Structure and phosphorylation of the Fujinami sarcoma virus gene product. J Virol. 1981 Dec;40(3):665–672. doi: 10.1128/jvi.40.3.665-672.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Prywes R., Foulkes J. G., Rosenberg N., Baltimore D. Sequences of the A-MuLV protein needed for fibroblast and lymphoid cell transformation. Cell. 1983 Sep;34(2):569–579. doi: 10.1016/0092-8674(83)90389-6. [DOI] [PubMed] [Google Scholar]
  39. Pugatsch T., Stacey D. W. Identification of a sequence likely to be required for avian retroviral packaging. Virology. 1983 Jul 30;128(2):505–511. doi: 10.1016/0042-6822(83)90279-9. [DOI] [PubMed] [Google Scholar]
  40. Segawa K., Yamaguchi N., Oda K. Simian virus 40 gene A regulates the association between a highly phosphorylated protein and chromatin and ribosomes in simian virus 40-transformed cells. J Virol. 1977 Jun;22(3):679–693. doi: 10.1128/jvi.22.3.679-693.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shibuya M., Hanafusa H. Nucleotide sequence of Fujinami sarcoma virus: evolutionary relationship of its transforming gene with transforming genes of other sarcoma viruses. Cell. 1982 Oct;30(3):787–795. doi: 10.1016/0092-8674(82)90283-5. [DOI] [PubMed] [Google Scholar]
  42. Shibuya M., Hanafusa T., Hanafusa H., Stephenson J. R. Homology exists among the transforming sequences of avian and feline sarcoma viruses. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6536–6540. doi: 10.1073/pnas.77.11.6536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shibuya M., Wang L. H., Hanafusa H. Molecular cloning of the Fujinami sarcoma virus genome and its comparison with sequences of other related transforming viruses. J Virol. 1982 Jun;42(3):1007–1016. doi: 10.1128/jvi.42.3.1007-1016.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Soberon X., Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene. 1980 May;9(3-4):287–305. doi: 10.1016/0378-1119(90)90328-o. [DOI] [PubMed] [Google Scholar]
  45. Srinivasan A., Dunn C. Y., Yuasa Y., Devare S. G., Reddy E. P., Aaronson S. A. Abelson murine leukemia virus: structural requirements for transforming gene function. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5508–5512. doi: 10.1073/pnas.79.18.5508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stone J. C., Atkinson T., Smith M., Pawson T. Identification of functional regions in the transforming protein of Fujinami sarcoma virus by in-phase insertion mutagenesis. Cell. 1984 Jun;37(2):549–558. doi: 10.1016/0092-8674(84)90385-4. [DOI] [PubMed] [Google Scholar]
  47. Walseth T. F., Johnson R. A. The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP. Biochim Biophys Acta. 1979 Mar 28;562(1):11–31. doi: 10.1016/0005-2787(79)90122-9. [DOI] [PubMed] [Google Scholar]
  48. Wang L. H., Feldman R., Shibuya M., Hanafusa H., Notter M. F., Balduzzi P. C. Genetic structure, transforming sequence, and gene product of avian sarcoma virus UR1. J Virol. 1981 Oct;40(1):258–267. doi: 10.1128/jvi.40.1.258-267.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Weinmaster G., Hinze E., Pawson T. Mapping of multiple phosphorylation sites within the structural and catalytic domains of the Fujinami avian sarcoma virus transforming protein. J Virol. 1983 Apr;46(1):29–41. doi: 10.1128/jvi.46.1.29-41.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weinmaster G., Zoller M. J., Smith M., Hinze E., Pawson T. Mutagenesis of Fujinami sarcoma virus: evidence that tyrosine phosphorylation of P130gag-fps modulates its biological activity. Cell. 1984 Jun;37(2):559–568. doi: 10.1016/0092-8674(84)90386-6. [DOI] [PubMed] [Google Scholar]
  51. Wong T. C., Hirano A., Lai M. M., Vogt P. K. Genome structure of the defective avian sarcoma virus PRCIV. Virology. 1982 Feb;117(1):156–164. doi: 10.1016/0042-6822(82)90515-3. [DOI] [PubMed] [Google Scholar]
  52. Woolford J., Beemon K. Transforming proteins of fujinami and PRCII avian sarcoma viruses have different subcellular locations. Virology. 1984 May;135(1):168–180. doi: 10.1016/0042-6822(84)90127-2. [DOI] [PubMed] [Google Scholar]
  53. Yamamoto T., Nishida T., Miyajima N., Kawai S., Ooi T., Toyoshima K. The erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell. 1983 Nov;35(1):71–78. doi: 10.1016/0092-8674(83)90209-x. [DOI] [PubMed] [Google Scholar]
  54. Yoshida M., Kawai S., Toyoshima K. Genome structure of avian sarcoma virus Y73 and unique sequence coding for polyprotein p90. J Virol. 1981 May;38(2):430–437. doi: 10.1128/jvi.38.2.430-437.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

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