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. 1983 May;46(2):594–605. doi: 10.1128/jvi.46.2.594-605.1983

Construction and isolation of a transforming murine retrovirus containing the src gene of Rous sarcoma virus.

S M Anderson, E M Scolnick
PMCID: PMC255162  PMID: 6302322

Abstract

Recombinant murine retroviruses containing the src gene of the avian retrovirus Rous sarcoma virus were isolated. Such viruses were isolated from cells after transfection with DNAs in which the src gene was inserted into the genome of the amphotropic murine retrovirus 4070A. The isolated viruses had functional gag and pol genes, but they were all env defective since the src gene was inserted in the middle of the env gene coding region. Infectious transforming virus could be isolated only from cells transfected with DNA constructions in which the src gene was in the same polarity as that of a long terminal repeat of the amphotropic viral genome. These recombinant viruses encoded a pp60src protein with a molecular weight similar to that of the Schmidt-Ruppin strain of Rous sarcoma virus. In addition, the src protein(s) of these recombinant viruses was as active as protein kinases in the immune complex protein kinase assay. Intravenous injection of helper-independent Moloney and Friend murine leukemia virus pseudotypes of the src recombinant viruses into 6-week-old NIH Swiss mice resulted in the appearance of splenic foci within 2 weeks, splenomegaly and, later after infection (8 to 10 weeks), anemia. Infectious transforming virus could be recovered from the spleens of diseased animals. Such viruses encoded pp60src but not p21ras or mink cell focus-forming virus-related glycoproteins.

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  1. AHLSTROM C. G., BERGMAN S., EHRENBERG B. Neoplasms in guinea pigs induced by an agent in rous chicken sarcoma. Acta Pathol Microbiol Scand. 1963;58:177–190. doi: 10.1111/j.1699-0463.1963.tb01560.x. [DOI] [PubMed] [Google Scholar]
  2. AHLSTROM C. G., FORSBY N. Sarcomas in hamsters after injection with Rous chicken tumor material. J Exp Med. 1962 Apr 1;115:839–852. doi: 10.1084/jem.115.4.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. AHLSTROM C. G., JONSSON N. Temporary growth of Rous sarcoma (strain Mill Hill) in new-born rats and mice. Acta Pathol Microbiol Scand. 1962;54:136–144. doi: 10.1111/j.1699-0463.1962.tb01232.x. [DOI] [PubMed] [Google Scholar]
  4. Altaner C., Svec F. Virus production in rat tumors induced by chicken sarcoma virus. J Natl Cancer Inst. 1966 Dec;37(6):745–752. [PubMed] [Google Scholar]
  5. Anderson S. M., Hanafusa H. Characterization of avian erythroblastosis virus p75. Virology. 1982 Aug;121(1):32–50. doi: 10.1016/0042-6822(82)90116-7. [DOI] [PubMed] [Google Scholar]
  6. Boettiger D., Love D. N., Weiss R. A. Virus envelope markers in mammalian tropism of avian RNA tumor viruses. J Virol. 1975 Jan;15(1):108–114. doi: 10.1128/jvi.15.1.108-114.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bosselman R. A., van Straaten F., Van Beveren C., Verma I. M., Vogt M. Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA. J Virol. 1982 Oct;44(1):19–31. doi: 10.1128/jvi.44.1.19-31.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brugge J. S., Erikson R. L. Identification of a transformation-specific antigen induced by an avian sarcoma virus. Nature. 1977 Sep 22;269(5626):346–348. doi: 10.1038/269346a0. [DOI] [PubMed] [Google Scholar]
  9. Brugge J., Erikson E., Collett M. S., Erikson R. I. Peptide analysis of the transformation-specific antigen from avian sarcoma virus-transformed cells. J Virol. 1978 Jun;26(3):773–782. doi: 10.1128/jvi.26.3.773-782.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chattopadhyay S. K., Lander M. R., Gupta S., Rands E., Lowy D. R. Origin of mink cytopathic focus-forming (MCF) viruses:comparison with ecotropic and xenotropic murine leukemia virus genomes. Virology. 1981 Sep;113(2):465–483. doi: 10.1016/0042-6822(81)90175-6. [DOI] [PubMed] [Google Scholar]
  11. Chirigos M. A., Scott D., Turner W., Perk K. Biological, pathological and physical characterization of a possible variant of a murine sarcoma virus (Moloney). Int J Cancer. 1968 Mar 15;3(2):223–227. doi: 10.1002/ijc.2910030207. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Collett M. S., Erikson R. L. Protein kinase activity associated with the avian sarcoma virus src gene product. Proc Natl Acad Sci U S A. 1978 Apr;75(4):2021–2024. doi: 10.1073/pnas.75.4.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Czernilofsky A. P., Levinson A. D., Varmus H. E., Bishop J. M., Tischer E., Goodman H. M. Nucleotide sequence of an avian sarcoma virus oncogene (src) and proposed amino acid sequence for gene product. Nature. 1980 Sep 18;287(5779):198–203. doi: 10.1038/287198a0. [DOI] [PubMed] [Google Scholar]
  15. Dougherty R. M., Di Stefano H. S. Virus particles associated with "nonproducer" Rous sarcoma cells. Virology. 1965 Nov;27(3):351–359. doi: 10.1016/0042-6822(65)90115-7. [DOI] [PubMed] [Google Scholar]
  16. Dresler S., Ruta M., Murray M. J., Kabat D. Glycoprotein encoded by the Friend spleen focus-forming virus. J Virol. 1979 May;30(2):564–575. doi: 10.1128/jvi.30.2.564-575.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Furth M. E., Davis L. J., Fleurdelys B., Scolnick E. M. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982 Jul;43(1):294–304. doi: 10.1128/jvi.43.1.294-304.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. HARVEY J. J. AN UNIDENTIFIED VIRUS WHICH CAUSES THE RAPID PRODUCTION OF TUMOURS IN MICE. Nature. 1964 Dec 12;204:1104–1105. doi: 10.1038/2041104b0. [DOI] [PubMed] [Google Scholar]
  21. Hanafusa H., Halpern C. C., Buchhagen D. L., Kawai S. Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants. J Exp Med. 1977 Dec 1;146(6):1735–1747. doi: 10.1084/jem.146.6.1735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hanafusa H., Hanafusa T. Further studies on RSV production form transformed cells. Virology. 1968 Apr;34(4):630–636. doi: 10.1016/0042-6822(68)90084-6. [DOI] [PubMed] [Google Scholar]
  23. Hankins W. D., Scolnick E. M. Harvey and Kirsten sarcoma viruses promote the growth and differentiation of erythroid precursor cells in vitro. Cell. 1981 Oct;26(1 Pt 1):91–97. doi: 10.1016/0092-8674(81)90036-2. [DOI] [PubMed] [Google Scholar]
  24. JONSSON N. SARCOMAS IN ALBINO MICE INOCULATED WITH ROUS CHICKEN TUMOUR MATERIAL. Acta Pathol Microbiol Scand. 1964;62:539–556. doi: 10.1111/apm.1964.62.4.539. [DOI] [PubMed] [Google Scholar]
  25. KLEMENT V., VESELY P. TUMOUR INDUCTION WITH THE ROUS SARCOMA VIRUS IN HAMSTERS AND PRODUCTION OF INFECTIOUS ROUS SARCOMA VIRUS IN AN HETEROLOGOUS HOST. Neoplasma. 1965;12:147–153. [PubMed] [Google Scholar]
  26. Klein B., Le Bousse C., Fagg B., Smajda-Joffe F., Vehmeyer K., Mori K. J., Jasmin C., Ostertag W. Effects of myeloproliferative sarcoma virus on the pluripotential stem cell and granulocyte precursor cell populations of DBA/2 mice. J Natl Cancer Inst. 1981 May;66(5):935–940. [PubMed] [Google Scholar]
  27. 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]
  28. Le Bousse-Kerdiles M. C., Smadja-Joffe F., Klein B., Caillou B., Jasmin C. Study of a virus-induced myeloproliferative syndrome associated with tumor formation in mice. Eur J Cancer. 1980 Jan;16(1):43–51. doi: 10.1016/0014-2964(80)90106-1. [DOI] [PubMed] [Google Scholar]
  29. Levinson A. D., Oppermann H., Levintow L., Varmus H. E., Bishop J. M. Evidence that the transforming gene of avian sarcoma virus encodes a protein kinase associated with a phosphoprotein. Cell. 1978 Oct;15(2):561–572. doi: 10.1016/0092-8674(78)90024-7. [DOI] [PubMed] [Google Scholar]
  30. MacDonald M. E., Reynolds F. H., Jr, Van de Ven W. J., Stephenson J. R., Mak T. W., Bernstein A. Anemia- and polycythemia-inducing isolates of Friend spleen focus-forming virus. Biological and molecular evidence for two distinct viral genomes. J Exp Med. 1980 Jun 1;151(6):1477–1492. doi: 10.1084/jem.151.6.1477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mackey J. K., Brackmann K. H., Green M. R., Green M. Preparation and characterization of highly radioactive in vitro labeled adenovirus DNA and DNA restriction fragments. Biochemistry. 1977 Oct 4;16(20):4478–4483. doi: 10.1021/bi00639a023. [DOI] [PubMed] [Google Scholar]
  32. Oliff A. I., Hager G. L., Chang E. H., Scolnick E. M., Chan H. W., Lowy D. R. Transfection of molecularly cloned Friend murine leukemia virus DNA yields a highly leukemogenic helper-independent type C virus. J Virol. 1980 Jan;33(1):475–486. doi: 10.1128/jvi.33.1.475-486.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Oliff A., Linemeyer D., Ruscetti S., Lowe R., Lowy D. R., Scolnick E. Subgenomic fragment of molecular cloned Friend murine leukemia virus DNA contains the gene(s) responsible for Friend murine leukemia virus-induced disease. J Virol. 1980 Sep;35(3):924–936. doi: 10.1128/jvi.35.3.924-936.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Oliff A., Ruscetti S., Douglass E. C., Scolnick E. Isolation of transplantable erythroleukemia cells from mice infected with helper-independent Friend murine leukemia virus. Blood. 1981 Aug;58(2):244–254. [PubMed] [Google Scholar]
  35. Ostertag W., Vehmeyer K., Fagg B., Pragnell I. B., Paetz W., Le Bousse M. C., Smadja-Joffe F., Klein B., Jasmin C., Eisen H. Myeloproliferative virus, a cloned murine sarcoma virus with spleen focus-forming properties in adult mice. J Virol. 1980 Feb;33(2):573–582. doi: 10.1128/jvi.33.2.573-582.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Parada L. F., Tabin C. J., Shih C., Weinberg R. A. Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus ras gene. Nature. 1982 Jun 10;297(5866):474–478. doi: 10.1038/297474a0. [DOI] [PubMed] [Google Scholar]
  37. Peters R. L., Rabstein L. S., VanVleck R., Kelloff G. J., Huebner R. J. Naturally occurring sarcoma virus of the BALB/cCr mouse. J Natl Cancer Inst. 1974 Dec;53(6):1725–1729. [PubMed] [Google Scholar]
  38. Pierce J. H., Aaronson S. A. BALB- and Harvey-murine sarcoma virus transformation of a novel lymphoid progenitor cell. J Exp Med. 1982 Sep 1;156(3):873–887. doi: 10.1084/jem.156.3.873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pragnell I. B., Fusco A., Arbuthnott C., Smadja-Joffe F., Klein B., Jasmin C., Ostertag W. Analysis of the myeloproliferative sarcoma virus genome: limited changes in the prototype lead to altered target cell specificity. J Virol. 1981 Jun;38(3):952–957. doi: 10.1128/jvi.38.3.952-957.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Purchio A. F. Evidence the pp60src, the product of the Rous sarcoma virus src gene, undergoes autophosphorylation. J Virol. 1982 Jan;41(1):1–7. doi: 10.1128/jvi.41.1.1-7.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Robinson H. L. Isolation of noninfectious particles containing Rous sarcoma virus RNA from the medium of Rous sarcoma virus-transformed nonproducer cells. Proc Natl Acad Sci U S A. 1967 Jun;57(6):1655–1662. doi: 10.1073/pnas.57.6.1655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rosenberg N., Baltimore D., Scher C. D. In vitro transformation of lymphoid cells by Abelson murine leukemia virus. Proc Natl Acad Sci U S A. 1975 May;72(5):1932–1936. doi: 10.1073/pnas.72.5.1932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ruscetti S., Troxler D., Linemeyer D., Scolnick E. Three laboratory strains of spleen focus-forming virus: comparison of their genomes and translational products. J Virol. 1980 Jan;33(1):140–151. doi: 10.1128/jvi.33.1.140-151.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. SIMKOVIC D., VALENTOVA N., THURZO V. An in vitro system for the detection of the Rous sarcoma virus in the cells of the rat tumour XC. Neoplasma. 1962;9:104–106. [PubMed] [Google Scholar]
  45. Scher C. D., Scolnick E. M., Siegler R. Induction of erythroid leukaemia by Harvey and Kirsten sarcoma viruses. Nature. 1975 Jul 17;256(5514):225–226. doi: 10.1038/256225a0. [DOI] [PubMed] [Google Scholar]
  46. Scher C. D., Siegler R. Direct transformation of 3T3 cells by Abelson murine leukaemia virus. Nature. 1975 Feb 27;253(5494):729–731. doi: 10.1038/253729a0. [DOI] [PubMed] [Google Scholar]
  47. Scolnick E., Rands E., Aaronson S. A., Todaro G. J. RNA-dependent DNA polymerase activity in five RNA viruses: divalent cation requirements. Proc Natl Acad Sci U S A. 1970 Dec;67(4):1789–1796. doi: 10.1073/pnas.67.4.1789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shih T. Y., Weeks M. O., Young H. A., Scholnick E. M. Identification of a sarcoma virus-coded phosphoprotein in nonproducer cells transformed by Kirsten or Harvey murine sarcoma virus. Virology. 1979 Jul 15;96(1):64–79. doi: 10.1016/0042-6822(79)90173-9. [DOI] [PubMed] [Google Scholar]
  49. Shimotohno K., Temin H. M. Formation of infectious progeny virus after insertion of herpes simplex thymidine kinase gene into DNA of an avian retrovirus. Cell. 1981 Oct;26(1 Pt 1):67–77. doi: 10.1016/0092-8674(81)90034-9. [DOI] [PubMed] [Google Scholar]
  50. Simkovic D. Characteristics of tumors induced in mammals, especially rodents, by viruses of the avian leukosis sarcoma group. Adv Virus Res. 1972;17:95–127. [PubMed] [Google Scholar]
  51. Simkovic D., Popovic M., Svec J., Grófová M., Valentová N. Continuous production of avian sarcoma virus B77 by rat tumour cells in tissue culture. Int J Cancer. 1969 Jan 15;4(1):80–85. doi: 10.1002/ijc.2910040111. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Svoboda J., Hlozánek I. Role of cell association in virus infection and virus rescue. Adv Cancer Res. 1970;13:217–269. doi: 10.1016/s0065-230x(08)60167-0. [DOI] [PubMed] [Google Scholar]
  54. Tabin C. J., Hoffmann J. W., Goff S. P., Weinberg R. A. Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene. Mol Cell Biol. 1982 Apr;2(4):426–436. doi: 10.1128/mcb.2.4.426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Troxler D. H., Ruscetti S. K., Scolnick E. M. The molecular biology of Friend virus. Biochim Biophys Acta. 1980 Sep 22;605(3):305–324. doi: 10.1016/0304-419x(80)90014-1. [DOI] [PubMed] [Google Scholar]
  56. Varmus H. E., Vogt P. K., Bishop J. M. Integration of deoxyribonucleic acid specific for Rous sarcoma virus after infection of permissive and nonpermissive hosts. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3067–3071. doi: 10.1073/pnas.70.11.3067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Vogt P. K. Avian tumor viruses. Adv Virus Res. 1965;11:293–385. doi: 10.1016/s0065-3527(08)60549-7. [DOI] [PubMed] [Google Scholar]
  58. Vogt V. M., Bruckenstein D. A., Bell A. P. Avian sarcoma virus gag precursor polypeptide is not processed in mammalian cells. J Virol. 1982 Nov;44(2):725–730. doi: 10.1128/jvi.44.2.725-730.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Waneck G. L., Rosenberg N. Abelson leukemia virus induces lymphoid and erythroid colonies in infected fetal cell cultures. Cell. 1981 Oct;26(1 Pt 1):79–89. doi: 10.1016/0092-8674(81)90035-0. [DOI] [PubMed] [Google Scholar]
  60. Wei C. M., Gibson M., Spear P. G., Scolnick E. M. Construction and isolation of a transmissible retrovirus containing the src gene of Harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type 1. J Virol. 1981 Sep;39(3):935–944. doi: 10.1128/jvi.39.3.935-944.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Weiss R. A., Wong A. L. Phenotypic mixing between avian and mammalian RNA tumor viruses: I. Envelope pseudotypes of Rous sarcoma virus. Virology. 1977 Feb;76(2):826–834. doi: 10.1016/0042-6822(77)90262-8. [DOI] [PubMed] [Google Scholar]
  62. Zilber L. A. Pathogenicity and oncogenicity of Rous sarcoma virus for mammals. Prog Exp Tumor Res. 1965;7:1–48. doi: 10.1159/000391376. [DOI] [PubMed] [Google Scholar]

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