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. 1983 Jun;46(3):841–859. doi: 10.1128/jvi.46.3.841-859.1983

Structure of glycosylated and unglycosylated gag and gag-pol precursor proteins of Moloney murine leukemia virus.

C J Saris, J van Eenbergen, R M Liskamp, H P Bloemers
PMCID: PMC256560  PMID: 6602220

Abstract

Precursor polyproteins containing translational products of the gag gene of Moloney murine leukemia virus were purified by gel electrophoresis and cleaved into large fragments by hydroxylamine, mild acid hydrolysis, or cyanogen bromide. The hydroxylamine cleavage method (specific for asparagine-glycine bonds) was adapted especially for this study. The electrophoretic mobility and antigenicity of the fragments and, in some cases, the presence or absence of [35S]methionine revealed detailed information on the structure of Pr65gag, gPr80gag, and Pr75gag (the unglycosylated variant of gPr80gag formed in vivo in the presence of tunicamycin or in vitro in a reticulocyte cell-free system). When compared with Pr65gag, gPr80gag contains 7,000 daltons of additional amino acids, presumably as, or as part of, a leader sequence at or very close to its N terminus. We present evidence that this leader may have replaced part of the p15 sequence. Furthermore, gPr80gag contains three separate carbohydrate groups. One is attached to the presumed leader sequence or to the p15 domain, and two are attached to the p30 domain. Each of the Moloney murine leukemia virus gag precursor proteins Pr65gag, gPr80gag, and Pr75gag corresponds with a read-through product into the pol gene. We designated these products Pr180gag-pol, gPr200gag-pol, and Pr190gag-pol (the unglycosylated variant of gPr200gag-pol), respectively. gPr200gag-pol contains all of the extra amino acids and carbohydrate groups present in gPr80gag and at least one carbohydrate group in its pol sequences.

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Selected References

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

  1. Bandyopadhyay A. K., Levy C. C. Effect of RNA tumor virus-specific protein p30 on reverse transcriptase. Intraspecies and interspecies interaction between reverse transcriptase and p30. J Biol Chem. 1978 Nov 25;253(22):8285–8290. [PubMed] [Google Scholar]
  2. 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]
  3. Bassin R. H., Duran-Troise G., Gerwin B. I., Rein A. Abrogation of Fv-1b restriction with murine leukemia viruses inactivated by heat or by gamma irradiation. J Virol. 1978 May;26(2):306–315. doi: 10.1128/jvi.26.2.306-315.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  5. Bornstein P., Balian G. Cleavage at Asn-Gly bonds with hydroxylamine. Methods Enzymol. 1977;47:132–145. doi: 10.1016/0076-6879(77)47016-2. [DOI] [PubMed] [Google Scholar]
  6. Edwards S. A., Fan H. Sequence relationship of glycosylated and unglycosylated gag polyproteins of Moloney murine leukemia virus. J Virol. 1980 Jul;35(1):41–51. doi: 10.1128/jvi.35.1.41-51.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Edwards S. A., Fan H. gag-Related polyproteins of Moloney murine leukemia virus: evidence for independent synthesis of glycosylated and unglycosylated forms. J Virol. 1979 May;30(2):551–563. doi: 10.1128/jvi.30.2.551-563.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Evans L. H., Dresler S., Kabat D. Synthesis and glycosylation of polyprotein precursors to the internal core proteins of Friend murine leukemia virus. J Virol. 1977 Dec;24(3):865–874. doi: 10.1128/jvi.24.3.865-874.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fan H., Paskind M. Measurement of the sequence complexity of cloned Moloney murine leukemia virus 60 to 70S RNA: evidence for a haploid genome. J Virol. 1974 Sep;14(3):421–429. doi: 10.1128/jvi.14.3.421-429.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jolicoeur P., Rassart E. Fate of unintegrated viral DNA in Fv-1 permissive and resistant mouse cells infected with murine leukemia virus. J Virol. 1981 Feb;37(2):609–619. doi: 10.1128/jvi.37.2.609-619.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jolicoeur P. The Fv-1 gene of the mouse and its control of murine leukemia virus replication. Curr Top Microbiol Immunol. 1979;86:67–122. doi: 10.1007/978-3-642-67341-2_3. [DOI] [PubMed] [Google Scholar]
  12. Kopchick J. J., Karshin W. L., Arlinghaus R. B. Tryptic peptide analysis of gag and gag-pol gene products of Rauscher murine leukemia virus. J Virol. 1979 May;30(2):610–623. doi: 10.1128/jvi.30.2.610-623.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Landon Cleavage at aspartyl-prolyl bonds. Methods Enzymol. 1977;47:145–149. doi: 10.1016/0076-6879(77)47017-4. [DOI] [PubMed] [Google Scholar]
  15. Ledbetter J. A., Nowinski R. C., Eisenman R. N. Biosynthesis and metabolism of viral proteins expressed on the surface of murine leukemia virus-infected cells. Virology. 1978 Nov;91(1):116–129. doi: 10.1016/0042-6822(78)90360-4. [DOI] [PubMed] [Google Scholar]
  16. Ledbetter J. A. Two-dimensional analysis of murine leukemia virus gag-gene polyproteins. Virology. 1979 May;95(1):85–98. doi: 10.1016/0042-6822(79)90403-3. [DOI] [PubMed] [Google Scholar]
  17. Ledbetter J., Nowinski R. C. Identification of the Gross cell surface antigen associated with murine leukemia virus-infected cells. J Virol. 1977 Aug;23(2):315–322. doi: 10.1128/jvi.23.2.315-322.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Naso R. B., Karshin W. L., Wu Y. H., Arlinghaus R. B. Characterization of 40,000- and 25,000-dalton intermediate precursors to Rauscher murine leukemia virus gag gene products. J Virol. 1979 Oct;32(1):187–198. doi: 10.1128/jvi.32.1.187-198.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Perucho M., Molgaard H. V., Shevack A., Pataryas T., Ruiz-Carrillo A. An improved method for the preparation of undegraded polysomes and active messenger RNA from immature chicken erythrocytes. Anal Biochem. 1979 Oct 1;98(2):464–471. doi: 10.1016/0003-2697(79)90168-4. [DOI] [PubMed] [Google Scholar]
  21. Reynolds F. H., Jr, Sacks T. L., Deobagkar D. N., Stephenson J. R. Cells nonproductively transformed by Abelson murine leukemia virus express a high molecular weight polyprotein containing structural and nonstructural components. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3974–3978. doi: 10.1073/pnas.75.8.3974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reynolds R. K., Stephenson J. R. Intracistronic mapping of the murine type C viral gag gene by use of conditional lethal replication mutants. Virology. 1977 Sep;81(2):328–340. doi: 10.1016/0042-6822(77)90149-0. [DOI] [PubMed] [Google Scholar]
  23. Rommelaere J., Donis-Keller H., Hopkins N. RNA sequencing provides evidence for allelism of determinants of the N-, B- or NB-tropism of murine leukemia viruses. Cell. 1979 Jan;16(1):43–50. doi: 10.1016/0092-8674(79)90186-7. [DOI] [PubMed] [Google Scholar]
  24. Schultz A. M., Lockhart S. M., Rabin E. M., Oroszlan S. Structure of glycosylated and unglycosylated gag polyproteins of Rauscher murine leukemia virus: carbohydrate attachment sites. J Virol. 1981 May;38(2):581–592. doi: 10.1128/jvi.38.2.581-592.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schultz A. M., Oroszlan S. Murine leukemia virus gag polyproteins: the peptide chain unique to Pr80 is located at the amino terminus. Virology. 1978 Dec;91(2):481–486. doi: 10.1016/0042-6822(78)90395-1. [DOI] [PubMed] [Google Scholar]
  26. Schultz A. M., Rabin E. H., Oroszlan S. Post-translational modification of Rauscher leukemia virus precursor polyproteins encoded by the gag gene. J Virol. 1979 Apr;30(1):255–266. doi: 10.1128/jvi.30.1.255-266.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
  28. Snyder H. W., Jr, Stockert E., Fleissner E. Characterization of molecular species carrying gross cell surface antigen. J Virol. 1977 Aug;23(2):302–314. doi: 10.1128/jvi.23.2.302-314.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Van Zaane D., Dekker-Michielsen J. A., Bloemers H. P. Virus-specific precursor polypeptides in cells infected with Rauscher leukemia virus: synthesis, identification, and processing. Virology. 1976 Nov;75(1):113–129. doi: 10.1016/0042-6822(76)90011-8. [DOI] [PubMed] [Google Scholar]
  30. Witte O. N., Baltimore D. Relationship of retrovirus polyprotein cleavages to virion maturation studied with temperature-sensitive murine leukemia virus mutants. J Virol. 1978 Jun;26(3):750–761. doi: 10.1128/jvi.26.3.750-761.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Zaanie D., Gielkens A. L., Dekker-michielsen M. J., Bloemers H. P. Virus-specific precursor polypeptides in cells infected with Rauscher leukemia virus. Virology. 1975 Oct;67(2):544–552. doi: 10.1016/0042-6822(75)90454-7. [DOI] [PubMed] [Google Scholar]

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