1887

Journal of General Virology header logo

Volume 59, Issue 2

Processing of the Gene Products of Moloney Murine Leukaemia Virus Free

Abstract

SUMMARY

The initial gene polyproteins present in Moloney murine leukaemia virus (M-MuLV)-infected NIH/3T3 cells were examined to determine their relationship to each other as well as their role in generating gene products gp70, p15(E) and p12(E). Steady-state labelling with [H]glucosamine revealed anti-gp69/71 immunoprecipitable proteins of mol. wt. 93000 (gPr93), 83000 (gPr83) and 70000 (gp70), whereas similar labelling with [H]fucose showed only two bands of anti-gp69/71 immunoprecipitable radioactivity migrating in SDS-polyacrylamide gels with gPr93 and gp70. Pulse-chase experiments employing [H]leucine labelling instead of labelled sugars failed to detect gPr93 using similar techniques. The gPr83 was the only polypeptide detected in 15 min [H]leucine pulselabellings, whereas gPr83, gp70, p15(E) and p12(E) were detected in chase experiments using appropriate antisera in immunoprecipitation experiments. Pretreatment of infected cells with tunicamycin, an inhibitor of glycosylation, allowed the synthesis of a major band at mol. wt. 62000 (Pr62) and a minor band of 73000 mol. wt. at the expense of gPr83. In pulse-chase experiments conducted in the presence of tunicamycin, Pr62 increased during the early chase period but disappeared during the later stages of the chase. No product of Pr62 was detected. Cation-exchange chromatography of tryptic digests of radioactive tyrosine-labelled gPr83, Pr62 and gp70 showed sequence relationships among the three proteins. Comparison of the two-dimensional fingerprints of [H]leucine-labelled gPr83 and the mature proteins gp70 and p12(E) support their precursor-product relationship. Of interest is the observation that gp70 and p12(E) seemed to share a few leucine-containing tryptic peptides. These results provide strong evidence that gPr83 is the primary product of the gene which, upon tunicamycin treatment, is synthesized as a subglycosylated protein, Pr62. It appears that gPr83 undergoes further modification of its core oligosaccharide structure as detected by fucosylation to yield gPr93. Our inability to detect gPr93 by [H]leucine labellings suggests a close chronological relationship between fucosylation and cleavage of the precursor polyprotein, suggesting that cleavage of gPr93 yields gp70 and p15(E). The latter is further cleaved to yield p12(E) plus a polypeptide containing the C-terminal end of p15(E).

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): gp70 , M-MuLV env gene products , MuLV envelope proteins , p12(E) and p15(E)
© Journal of General Virology 1982
Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-59-2-329
1982-04-01
2024-05-10
Loading full text...

Full text loading...

/deliver/fulltext/jgv/59/2/JV0590020329.html?itemId=/content/journal/jgv/10.1099/0022-1317-59-2-329&mimeType=html&fmt=ahah

References

  1. Bolognesi D. P., Montelaro R. C., Frank H., Schafer W. 1978; Assembly of type C oncornaviruses: a model. Science 199:183–186
     [Google Scholar]
  2. Bonner W. M., Laskey R. A. 1974; A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. European Journal of Biochemistry 46:83–88
     [Google Scholar]
  3. Donoghue D. J., Sharp P. A., Weinberg R. A. 1979; An MSV-specific sub-genomic mRNA in MSV-transformed G8-124 cells. Cell 17:53–63
     [Google Scholar]
  4. Edwards A. A., Fan H. 1979; ‘gag’-related polyproteins of Moloney murine leukemia virus: evidence for independent synthesis of glycosylated and unglycosylated forms. Journal of Virology 30:551–563
     [Google Scholar]
  5. Faller D. V., Rommelaere J., Hopkins N. 1978; Large T1 oligonucleotides of Moloney leukemia virus missing in a ‘env’ gene recombinant, H1X, are present on an intracellular 21S Moloney viral RNA species. Proceedings of the National Academy of Sciences of the United States of America 75:2964–2968
     [Google Scholar]
  6. Famulari N. G., Buchhogen D. L., Klenk H-D., Fleissner E. 1976; Presence of murine leukemia virus envelope proteins gp70 and p15(E) in a common polypeptide of infected cells. Journal of Virology 20:501–508
     [Google Scholar]
  7. Fan H., Paskind M. 1974; Measurement of the sequence complexity of cloned Moloney murine leukemia virus 60 to 70S RNA: evidence for a haploid genome. Journal of Virology 14:421–429
     [Google Scholar]
  8. Forchhammer J., Turnock G. 1978; Glycoproteins from murine C-type virus are more acidic in virus derived from transformed cells than from nontransformed cells. Virology 88:177–182
     [Google Scholar]
  9. Green N., Shinnick T. M., Witte O., Ponticelli A., Sutcliffe J. G., Lerner R. A. 1981; Sequence-specific antibodies show that maturation of Moloney leukemia virus envelope polyprotein involves removal of a COOH-terminal peptide. Proceedings of the National Academy of Sciences of the United States of America 78:6023–6027
     [Google Scholar]
  10. Hart G. W., Brew K., Grant G. A., Bradshaw R. A., Lennarz W. J. 1979; Primary structural requirements for the enzymatic formation of the N-glycosidic bond in glycoproteins. Journal of Biological Chemistry 254:9747–9753
     [Google Scholar]
  11. Hu S., Davidson N., Verma I. M. 1977; A heteroduplex study of the sequence relationships between the RNAs of M-MSV and M-MLV. Cell 10:469–477
     [Google Scholar]
  12. Hubbard S. C., Robbins P. W. 1979; Synthesis and processing of protein-linked oligosaccharides in vivo. Journal of Biological Chemistry 254:4568–4576
     [Google Scholar]
  13. Hunt L. A., Etchison J. R., Summers D. F. 1978; Oligosaccharide chains are trimmed during synthesis of the envelope glycoprotein of vesicular stomatitis virus. Proceedings of the National Academy of Sciences of the United States of America 15:754–758
     [Google Scholar]
  14. Karshin W. L., Arcement L. J., Naso R. B., Arlinghaus R. B. 1977; Common precursor for Rauscher leukemia virus gp69/71, p15(E) and p12(E). Journal of Virology 23:787–798
     [Google Scholar]
  15. Kessler S. W. 1975; Rapid isolation of antigens from cells with a Staphylococcal protein A-antibody adsorbent; parameters of the interaction of antigen-antibody complexes with protein A. Journal of Immunology 115:1617–1624
     [Google Scholar]
  16. Kopchick J. J., Karshin W. L., Arlinghaus R. B. 1979; Tryptic peptide analysis of ‘gag’ and ‘gag-pol’ gene products of Rauscher murine leukemia virus. Journal of Virology 30:610–623
     [Google Scholar]
  17. Laskey R. A., Mills A. D. 1975; Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. European Journal of Biochemistry 56:335–341
     [Google Scholar]
  18. Leamnson R. N., Shander M. H. M., Halpern M. S. 1977; A structural protein complex in Moloney leukemia virus. Virology 76:437–439
     [Google Scholar]
  19. Mahoney W. C., Duskin D. 1979; Biological activities of the two major components of tunicamycin. Journal of Biological Chemistry 254:6572–6576
     [Google Scholar]
  20. Murphy E. C. Jr, Campos D., Arlinghaus R. B. 1979; Cell-free synthesis of Rauscher murine leukemia virus ‘gag’ and ‘env’ gene products from separate cellular mRNA species. Virology 93:293–302
     [Google Scholar]
  21. Naso R. B., Arcement L. J., Karshin W. L., Jamjoom G. A., Arlinghaus R. B. 1976; A fuCOSe-deficient glycoprotein precursor to Rauscher leukemia virus gp69/71. Proceedings of the National Academy of Sciences of the United States of America 73:2326–2330
     [Google Scholar]
  22. Ng V. L., Wood T. G., Lyons D. D., Arlinghaus R. B. 1979; Characterization of intracellular precursor polyproteins of Moloney murine leukemia virus. Journal of Virology 32:1051–1056
     [Google Scholar]
  23. Pinter A., Fleissner E. 1975; The presence of disulfide-linked gp70-p15(E) complexes in AKR murine leukemia virus. Virology 83:917–922
     [Google Scholar]
  24. Rothenburg E., Donoghue D. J., Baltimore D. 1978; Analysis of a 5′ leader sequence on murine leukemia virus 21S RNA: heteroduplex mapping with long reverse transcriptase products. Cell 13:435–451
     [Google Scholar]
  25. Rothman J. E., Lodish H. F. 1977; Synchronized transmembrane insertion and glycosylation of a nascent membrane protein. Nature, London 269:775–780
     [Google Scholar]
  26. Schultz A. M., Oroszlan S. 1979; Tunicamycin inhibits glycosylation of precursor polyprotein encoded by ‘env’ gene of Rauscher murine leukemia virus. Biochemical and Biophysical Research Communications 87:1206–1213
     [Google Scholar]
  27. Sutcliffe J. G., Shlnnick T. M., Green N., Liu F. T., Niman H. L., Lerner R. A. 1980; Chemical synthesis of a polypeptide predicted from nucleotide sequence allows detection of a new retroviral gene product. Nature, London 287:801–805
     [Google Scholar]
  28. Syrewicz J. J., Naso R. B., Wang C. S., Arlinghaus R. B. 1972; Purification of large amounts of murine ribonucleic acid tumor viruses produced in roller bottle cultures. Applied Microbiology 24:488–498
     [Google Scholar]
  29. Tabas I., Schlessinger S., Kornfield S. 1978; Processing of high mannose oligosaccharides to form complex type oligosaccharides on the newly synthesized polypeptides of the vesicular stomatitis virus G protein and the IgG heavy chain. Journal of Biological Chemistry 253:716–722
     [Google Scholar]
  30. Takatsuki A., Arima L., Tamura G. 1971; Tunicamycin, a new antibiotic. I. Isolation and characterization of tunicamycin. Journal of Antibiotics 24:215–223
     [Google Scholar]
  31. Turco S. J., Robbins P. W. 1979; The initial stages of processing of protein bound oligosaccharides in vitro. Journal of Biological Chemistry 254:4560–4567
     [Google Scholar]
  32. van De Ven W. J. M., Onnekink C., Vermoken A. J. M., Bloemers H. P. J. 1977; Effect of impaired glycosylation on the synthesis of envelope proteins of Rauscher murine leukemia virus. Virology 82:334–344
     [Google Scholar]
  33. van Zaane D., Gielkins A. L. J., Hesselink W. G., Bloemers H. P. J. 1977; Identification of Rauscher murine leukemia virus-specific mRNAs for the synthesis of ‘gag’- and ‘env’-gene products. Proceedings of the National Academy of Sciences of the United States of America 74:1855–1859
     [Google Scholar]
  34. Vogt V. M., Eisenman R., Diggelmann H. 1975; Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide. Journal of Molecular Biology 96:471–493
     [Google Scholar]
  35. Waechter C. J., Lennarz W. J. 1976; The role of polyprenol-linked sugars in glycoprotein synthesis. Annual Review of Biochemistry 45:95–112
     [Google Scholar]
  36. Witte O. N., Wirth D. F. 1979; Structure of the murine leukemia virus envelope glycoprotein precursor. Journal of Virology 29:735–743
     [Google Scholar]
  37. Witte O. N., Tsukamoto-Adey A., Weissman I. L. 1977; Cellular maturation of oncornavirus glycoproteins: topological arrangement of precursor and product forms in cellular membranes. Virology 76:539–553
     [Google Scholar]
  38. Wood T. G., Lyons D. D., Ng V. L., Murphy E. C. Jr, Arlinghaus R. B. 1980; Characterization of viral polyproteins in cells transformed and producing Moloney murine sarcoma virus. Biochimica et Biophysica Acta 608:215–231
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-59-2-329
Loading
Processing of the env Gene Products of Moloney Murine Leukaemia Virus
J. Gen. Virol. 59, 329 (1982); https://doi.org/10.1099/0022-1317-59-2-329
/content/journal/jgv/10.1099/0022-1317-59-2-329
/content/journal/jgv/10.1099/0022-1317-59-2-329
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error