Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1979 Mar;29(3):1131–1141. doi: 10.1128/jvi.29.3.1131-1141.1979

Cell-free synthesis of mouse mammary tumor virus Pr77 from virion and intracellular mRNA.

H H Dahl, C Dickson
PMCID: PMC353273  PMID: 221668

Abstract

Mouse mammary tumor virus (MuMTV) was purified from two cell lines (GR and Mm5MT/c1), and the genomic RNA was isolated and translated in vitro in cell-free systems derived from mouse L cells and rabbit reticulocytes. The major translation product in both systems was a protein with the molecular weight 77,000. Several other products were also detected, among them a 110,000-dalton and in minor amounts a 160,000-dalton protein. All three polypeptides were specifically immunoprecipitated by antiserum raised against the major core protein of MuMTV (p27), but they were not precipitated by antiserum against the virion glycoprotein gp52. Analysis of the in vitro products by tryptic peptide mapping established their relationship to the virion non-glycosylated structural proteins. The 77,000-dalton polypeptide was found to be similar, if not identical, to an analogous precursor isolated from MuMTV-producing cells. Peptide mapping of the 110,000-dalton protein shows that it contains all of the methionine-labeled peptides found in the 77,000-dalton protein plus some additional peptides. We conclude that the products synthesized in vitro from the genomic MuMTV RNA are related to the non-glycosylated virion structural proteins. Polyadenylic acid-containing RNA from MuMTV-producing cells also directed the synthesis of the 77,000-dalton polypeptide in the L-cell system. If this RNA preparation was first fractionated by sucrose gradient centrifugation the 77,000-dalton protein appeared to be synthesized from mRNA with a sedimentation coefficient between 25 and 35S.

Full text

PDF
1131

Images in this article

1135Image
on p.1135
1136Image
on p.1136
1136Image
on p.1136
1137Image
on p.1137
1138Image
on p.1138
1139Image
on p.1139

Selected References

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

  1. BERNHARD W. Electron microscopy of tumor cells and tumor viruses; a review. Cancer Res. 1958 Jun;18(5):491–509. [PubMed] [Google Scholar]
  2. Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  3. Barbacid M., Stephenson J. R., Aaronson S. A. gag Gene of mammalian type-C RNA tumour viruses. Nature. 1976 Aug 12;262(5569):554–559. doi: 10.1038/262554a0. [DOI] [PubMed] [Google Scholar]
  4. Cardiff R. D., Puentes M. J., Young L. J., Smith G. H., Teramoto Y. A., Altrock B. W., Pratt T. S. Serological and biochemical characterization of the mouse mammary tumor virus with localization of p10. Virology. 1978 Mar;85(1):157–167. doi: 10.1016/0042-6822(78)90420-8. [DOI] [PubMed] [Google Scholar]
  5. Dickson C., Atterwill M. Polyproteins related to the major core protein of mouse mammary tumor virus. J Virol. 1978 Jun;26(3):660–672. doi: 10.1128/jvi.26.3.660-672.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dickson C., Puma J. P., Nandi S. Identification of a precursor protein to the major glycoproteins of mouse mammary tumor virus. J Virol. 1975 Jan;17(1):275–282. doi: 10.1128/jvi.17.1.275-282.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duesberg P. H., Blair P. B. Isolation of the nucleic acid of mouse mammary tumor virus (MTV). Proc Natl Acad Sci U S A. 1966 Jun;55(6):1490–1497. doi: 10.1073/pnas.55.6.1490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duesberg P. H., Cardiff R. D. Structural relationships between the RNA of mammary tumor virus and those of other RNA tumor viruses. Virology. 1968 Dec;36(4):696–700. doi: 10.1016/0042-6822(68)90206-7. [DOI] [PubMed] [Google Scholar]
  9. Fine D. L., Arthur L. O., PLOWMAN J. K., Hillman E. A., Klein F. In vitro system for production of mouse mammary tumor virus. Appl Microbiol. 1974 Dec;28(6):1040–1046. doi: 10.1128/am.28.6.1040-1046.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Friedrich R., Morris V. L., Goodman H. M., Bishop J. M., Varmus H. E. Differences between genomes of two strains of mouse mammary tumor virus as shown by partial RNA sequence analysis. Virology. 1976 Jul 15;72(2):330–340. doi: 10.1016/0042-6822(76)90162-8. [DOI] [PubMed] [Google Scholar]
  11. Gielkens A. L., Salden M. H., Bloemendal H. Virus-specific messenger RNA on free and membrane-bound polyribosomes from cells infected with Rauscher leukemia virus. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1093–1097. doi: 10.1073/pnas.71.4.1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gielkens A. L., Van Zaane D., Bloemers H. P., Bloemendal H. Synthesis of Rauscher murine leukemia virus-specific polypeptides in vitro. Proc Natl Acad Sci U S A. 1976 Feb;73(2):356–360. doi: 10.1073/pnas.73.2.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hayward W. S. Size and genetic content of viral RNAs in avian oncovirus-infected cells. J Virol. 1977 Oct;24(1):47–63. doi: 10.1128/jvi.24.1.47-63.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hilgers J., Bentvelzen P. Interaction between viral and genetic factors in murine mammary cancer. Adv Cancer Res. 1978;26:143–195. doi: 10.1016/s0065-230x(08)60087-1. [DOI] [PubMed] [Google Scholar]
  15. Jamjoom G. A., Naso R. B., Arlinghaus R. B. Further characterization of intracellular precursor polyproteins of Rauscher leukemia virus. Virology. 1977 May 1;78(1):11–34. doi: 10.1016/0042-6822(77)90075-7. [DOI] [PubMed] [Google Scholar]
  16. Kerr I. M., Olshevsky U., Lodish H. F., Baltimore D. Translation of murine leukemia virus RNA in cell-free systems from animal cells. J Virol. 1976 May;18(2):627–635. doi: 10.1128/jvi.18.2.627-635.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  18. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  19. Murphy E. C., Jr, Kopchick J. J., Watson K. F., Arlinghaus R. B. Cell-free synthesis of a precursor polyprotein containing both gag and pol gene products by Rauscher murine leukemia virus 35S RNA. Cell. 1978 Feb;13(2):359–369. doi: 10.1016/0092-8674(78)90204-0. [DOI] [PubMed] [Google Scholar]
  20. Naso R. B., Arcement L. J., Wood G., Saunders T. E., Arlinghaus R. B. The cell-free translation of Rauscher leukemia virus RNA into high molecular weight polypeptides. Biochim Biophys Acta. 1975 Mar 10;383(2):195–206. doi: 10.1016/0005-2787(75)90261-0. [DOI] [PubMed] [Google Scholar]
  21. Oppermann H., Bishop J. M., Varmus H. E., Levintow L. A joint produce of the genes gag and pol of avian sarcoma virus: a possible precursor of reverse transcriptase. Cell. 1977 Dec;12(4):993–1005. doi: 10.1016/0092-8674(77)90164-7. [DOI] [PubMed] [Google Scholar]
  22. Owens R. B., Hackett A. J. Tissue culture studies of mouse mammary tumor cells and associated viruses. J Natl Cancer Inst. 1972 Nov;49(5):1321–1332. [PubMed] [Google Scholar]
  23. Parks W. P., Ransom J. C., Young H. A., Scolnick E. M. Mammary tumor virus induction by glucocorticoids. Characterization of specific transcriptional regulation. J Biol Chem. 1975 May 10;250(9):3330–3336. [PubMed] [Google Scholar]
  24. Paterson B. M., Marciani D. J., Papas T. S. Cell-free synthesis of the precursor polypeptide for avian myeloblastosis virus DNA polymerase. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4951–4954. doi: 10.1073/pnas.74.11.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Paucha E., Harvey R., Smith A. E. Cell-free synthesis of simian virus 40 T-antigens. J Virol. 1978 Oct;28(1):154–170. doi: 10.1128/jvi.28.1.154-170.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pawson T., Harvey R., Smith A. E. The size of Rous sarcoma virus mRNAs active in cell-free translation. Nature. 1977 Aug 4;268(5619):416–420. doi: 10.1038/268416a0. [DOI] [PubMed] [Google Scholar]
  27. Pawson T., Martin G. S., Smith A. E. Cell-free translation of virion RNA from nondefective and transformation-defective Rous sarcoma viruses. J Virol. 1976 Sep;19(3):950–967. doi: 10.1128/jvi.19.3.950-967.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  29. Philipson L., Andersson P., Olshevsky U., Weinberg R., Baltimore D., Gesteland R. Translation of MuLV and MSV RNAs in nuclease-treated reticulocyte extracts: enhancement of the gag-pol polypeptide with yeast suppressor tRNA. Cell. 1978 Jan;13(1):189–199. doi: 10.1016/0092-8674(78)90149-6. [DOI] [PubMed] [Google Scholar]
  30. Racevskis J., Sarkar N. H. Synthesis and processing of precursor polypeptides to murine mammary tumor virus structural proteins. J Virol. 1978 Jan;25(1):374–383. doi: 10.1128/jvi.25.1.374-383.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ringold G. M., Yamamoto K. R., Bishop J. M., Varmus H. E. Glucocorticoid-stimulated accumulation of mouse mammary tumor virus RNA: increased rate of synthesis of viral RNA. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2879–2883. doi: 10.1073/pnas.74.7.2879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ringold G. M., Yamamoto K. R., Tomkins G. M., Bishop M., Varmus H. E. Dexamethasone-mediated induction of mouse mammary tumor virus RNA: a system for studying glucocorticoid action. Cell. 1975 Nov;6(3):299–305. doi: 10.1016/0092-8674(75)90181-6. [DOI] [PubMed] [Google Scholar]
  33. Ringold G., Lasfargues E. Y., Bishop J. M., Varmus H. E. Production of mouse mammary tumor virus by cultured cells in the absence and presence of hormones: assay by molecular hybridization. Virology. 1975 May;65(1):135–147. doi: 10.1016/0042-6822(75)90014-8. [DOI] [PubMed] [Google Scholar]
  34. Rothman J. E., Lodish H. F. Synchronised transmembrane insertion and glycosylation of a nascent membrane protein. Nature. 1977 Oct 27;269(5631):775–780. doi: 10.1038/269775a0. [DOI] [PubMed] [Google Scholar]
  35. Salden M. H., Bloemendal H. Translation of avian myeloblastosis virus RNA in a reticulocyte cell-free system. Biochem Biophys Res Commun. 1976 Jan 12;68(1):249–255. doi: 10.1016/0006-291x(76)90036-x. [DOI] [PubMed] [Google Scholar]
  36. Salden M. H., Selten-Versteegen A. M., Bloemendal H. Translation of Rauscher murine leukemia viral RNA. A model for the function of virus-specific messenger. Biochem Biophys Res Commun. 1976 Sep 20;72(2):610–618. doi: 10.1016/s0006-291x(76)80084-8. [DOI] [PubMed] [Google Scholar]
  37. Schochetman G., Long C. W., Oroszlan S., Arthur L., Fine D. L. Isolation of separate precursor polypeptides for the mouse mammary tumor virus glycoproteins and nonglycoproteins. Virology. 1978 Mar;85(1):168–174. doi: 10.1016/0042-6822(78)90421-x. [DOI] [PubMed] [Google Scholar]
  38. Scolnick E. M., Young H. A., Parks W. P. Biochemical and physiological mechanisms in glucocorticoid hormone induction of mouse mammary tumor virus. Virology. 1976 Jan;69(1):148–156. doi: 10.1016/0042-6822(76)90202-6. [DOI] [PubMed] [Google Scholar]
  39. Smith A. E., Smith R., Paucha E. Extraction and fingerprint analysis of simian virus 40 large and small T-antigens. J Virol. 1978 Oct;28(1):140–153. doi: 10.1128/jvi.28.1.140-153.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stacey D. W., Allfrey V. G., Hanafusa H. Microinjection analysis of envelope-glycoprotein messenger activities of avian leukosis viral RNAs. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1614–1618. doi: 10.1073/pnas.74.4.1614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Teramoto Y. A., Cardiff R. D., Lund J. K. The structure of the mouse mammary tumor virus: isolation and characterization of the core. Virology. 1977 Mar;77(1):135–148. doi: 10.1016/0042-6822(77)90413-5. [DOI] [PubMed] [Google Scholar]
  42. Vogt V. M., Eisenman R., Diggelmann H. Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide. J Mol Biol. 1975 Aug 15;96(3):471–493. doi: 10.1016/0022-2836(75)90174-6. [DOI] [PubMed] [Google Scholar]
  43. Wang L., Galehouse D., Mellon P., Duesberg P., Mason W. S., Vogt P. K. Mapping oligonucleotides of Rous sarcoma virus RNA that segregate with polymerase and group-specific antigen markers in recombinants. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3952–3956. doi: 10.1073/pnas.73.11.3952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Waters L. C. Lysine tRNA is the predominant tRNA in murine mammary tumor virus. Biochem Biophys Res Commun. 1978 Apr 14;81(3):822–827. doi: 10.1016/0006-291x(78)91425-0. [DOI] [PubMed] [Google Scholar]
  45. Weiss S. R., Varmus H. E., Bishop J. M. The size and genetic composition of virus-specific RNAs in the cytoplasm of cells producing avian sarcoma-leukosis viruses. Cell. 1977 Dec;12(4):983–992. doi: 10.1016/0092-8674(77)90163-5. [DOI] [PubMed] [Google Scholar]
  46. Witte O. N., Rosenberg N., Paskind M., Shields A., Baltimore D. Identification of an Abelson murine leukemia virus-encoded protein present in transformed fibroblast and lymphoid cells. Proc Natl Acad Sci U S A. 1978 May;75(5):2488–2492. doi: 10.1073/pnas.75.5.2488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zaane D. V., Gielkens A. L., Hesselink W. G., Bloemers H. P. Identification of Rauscher murine leukemia virus-specific mRNAs for the synthesis of gag- and env-gene products. Proc Natl Acad Sci U S A. 1977 May;74(5):1855–1859. doi: 10.1073/pnas.74.5.1855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Závada J., Dickson C., Weiss R. Pseudotypes of vesicular stomatitis virus with envelope antigens provided by murine mammary tumor virus. Virology. 1977 Oct 1;82(1):221–231. doi: 10.1016/0042-6822(77)90045-9. [DOI] [PubMed] [Google Scholar]
  49. von der Helm K., Duesberg P. H. Translation of Rous sarcoma virus RNA in a cell-free system from ascites Krebs II cells. Proc Natl Acad Sci U S A. 1975 Feb;72(2):614–618. doi: 10.1073/pnas.72.2.614. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES