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. 1988 Jan;62(1):40–46. doi: 10.1128/jvi.62.1.40-46.1988

Detection of phosphorylated forms of Moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis.

K Ikuta 1, R B Luftig 1
PMCID: PMC250499  PMID: 3334749

Abstract

We detected phosphorylation of the major Moloney murine leukemia virus (M-MuLV) capsid polypeptide, p30, by using 32Pi-labeled virions. This was observed both on two-dimensional polyacrylamide gels directly or on one-dimensional gels of viral lysates that had been immunoprecipitated with monospecific goat anti-p30 serum. The phosphorylation event had been difficult to detect because pp12 the major virion phosphoprotein incorporates almost all of the 32P label added to infected cells (Y. Yoshinaka and R. B. Luftig, Virology 116:181-195, 1982). When immunoprecipitates from M-MuLV lysates labeled with 32Pi were compared with those labeled with [35S]methionine, it was calculated that the degree of phosphorylation at the p30 domain of Pr65gag was only 0.22 to 0.54% relative to phosphorylation at the p12 domain. Two-dimensional gel electrophoresis of the 32P-labeled p30 immunoprecipitates showed that there were three phosphorylated p30 forms with isoelectric points (pIs) of 5.7, 5.8, and 6.0. These forms were generally more acidic than the [35S]methionine-labeled p30 forms, which had pIs of 6.0, 6.1, 6.3 (the major constituent with greater than 80% of the label), and 6.6. The predominant phosphoamino acid of the major phosphorylated p30 form (pI 5.8) was phosphoserine. Further, tryptic peptide analysis of this p30 form showed that only one peptide was predominantly phosphorylated. Based on a comparison of specific labeling of p30 tryptic peptides with [14C]serine, [35S]methionine, and 32Pi, we tentatively assigned the phosphorylation site to a 2.4-kilodalton NH2-terminal peptide containing triple tandem serines spanning the region from amino acids 4 to 24.

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Selected References

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

  1. Aboud M., Wolfson M., Hassan Y., Huleihel M. Rapid purification of extracellular and intracellular Moloney murine leukemia virus. Arch Virol. 1982;71(3):185–195. doi: 10.1007/BF01314870. [DOI] [PubMed] [Google Scholar]
  2. Anders D. G., Consigli R. A. Comparison of nonphosphorylated and phosphorylated species of polyomavirus major capsid protein VP1 and identification of the major phosphorylation region. J Virol. 1983 Oct;48(1):206–217. doi: 10.1128/jvi.48.1.206-217.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  4. Elder J. H., Pickett R. A., 2nd, Hampton J., Lerner R. A. Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein. J Biol Chem. 1977 Sep 25;252(18):6510–6515. [PubMed] [Google Scholar]
  5. Garcea R. L., Ballmer-Hofer K., Benjamin T. L. Virion assembly defect of polyomavirus hr-t mutants: underphosphorylation of major capsid protein VP1 before viral DNA encapsidation. J Virol. 1985 May;54(2):311–316. doi: 10.1128/jvi.54.2.311-316.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hartley J. W., Rowe W. P. Clonal cells lines from a feral mouse embryo which lack host-range restrictions for murine leukemia viruses. Virology. 1975 May;65(1):128–134. doi: 10.1016/0042-6822(75)90013-6. [DOI] [PubMed] [Google Scholar]
  7. Henderson L. E., Krutzsch H. C., Oroszlan S. Myristyl amino-terminal acylation of murine retrovirus proteins: an unusual post-translational proteins modification. Proc Natl Acad Sci U S A. 1983 Jan;80(2):339–343. doi: 10.1073/pnas.80.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ikuta K., Ueda S., Kato S., Hirai K. Monoclonal antibodies reactive with the surface and secreted glycoproteins of Marek's disease virus and herpesvirus of turkeys. J Gen Virol. 1983 Dec;64(Pt 12):2597–2610. doi: 10.1099/0022-1317-64-12-2597. [DOI] [PubMed] [Google Scholar]
  9. Karshin W. L., Arcement L. J., Naso R. B., Arlinghaus R. B. Common precursor for Rauscher leukemia virus gp69/71, p15(E), and p12(E). J Virol. 1977 Sep;23(3):787–798. doi: 10.1128/jvi.23.3.787-798.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Katoh I., Yoshinaka Y., Luftig R. B. Murine leukaemia virus p30 heterogeneity as revealed by two-dimensional gel electrophoresis is not an artefact of the technique. J Gen Virol. 1984 Apr;65(Pt 4):733–741. doi: 10.1099/0022-1317-65-4-733. [DOI] [PubMed] [Google Scholar]
  11. Katoh I., Yoshinaka Y., Luftig R. B. The effect of cerulenin on Moloney murine leukemia virus morphogenesis. Virus Res. 1986 Aug;5(2-3):265–276. doi: 10.1016/0168-1702(86)90023-7. [DOI] [PubMed] [Google Scholar]
  12. 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]
  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. Lai M. M. Phosphoproteins of Rous sarcoma viruses. Virology. 1976 Oct 15;74(2):287–301. doi: 10.1016/0042-6822(76)90336-6. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Lu A. H., Soong M. M., Wong P. K. Maturation of Moloney murine leukemia virus. Virology. 1979 Feb;93(1):269–274. doi: 10.1016/0042-6822(79)90297-6. [DOI] [PubMed] [Google Scholar]
  17. Ludlow J. W., Consigli R. A. Differences in biological activity and structural protein VP1 phosphorylation of polyomavirus progeny resulting from infection of primary mouse kidney and primary mouse embryo cell cultures. J Virol. 1987 Feb;61(2):509–515. doi: 10.1128/jvi.61.2.509-515.1987. [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. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  20. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  21. O'Farrell P. Z., Goodman H. M. Resolution of simian virus 40 proteins in whole cell extracts by two-dimensional electrophoresis: heterogeneity of the major capsid protein. Cell. 1976 Oct;9(2):289–298. doi: 10.1016/0092-8674(76)90119-7. [DOI] [PubMed] [Google Scholar]
  22. Pal B. K., McAllister R. M., Gardner M. B., Roy-Burman P. Comparative studies on the structural phosphoproteins of mammalian type C viruses. J Virol. 1975 Jul;16(1):123–131. doi: 10.1128/jvi.16.1.123-131.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pal B. K., Roy-Burman P. Phosphoproteins: structural components of oncornaviruses. J Virol. 1975 Mar;15(3):540–549. doi: 10.1128/jvi.15.3.540-549.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Ro J. H., Ghosh H. P. Impaired cleavage of the joint gag-pol polyprotein precursor and virion assembly in a temperature-sensitive mutant of Rous sarcoma virus. Virology. 1984 Jun;135(2):489–502. doi: 10.1016/0042-6822(84)90203-4. [DOI] [PubMed] [Google Scholar]
  26. Salunke D. M., Caspar D. L., Garcea R. L. Self-assembly of purified polyomavirus capsid protein VP1. Cell. 1986 Sep 12;46(6):895–904. doi: 10.1016/0092-8674(86)90071-1. [DOI] [PubMed] [Google Scholar]
  27. Schultz A. M., Oroszlan S. In vivo modification of retroviral gag gene-encoded polyproteins by myristic acid. J Virol. 1983 May;46(2):355–361. doi: 10.1128/jvi.46.2.355-361.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Sen A., Sherr C. J., Todaro G. J. Phosphorylation of murine type C viral p12 proteins regulates their extent of binding to the homologous viral RNA. Cell. 1977 Mar;10(3):489–496. doi: 10.1016/0092-8674(77)90036-8. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Strand M., August J. T. Protein kinase and phosphate acceptor proteins in Rauscher murine leukaemia virus. Nat New Biol. 1971 Sep 29;233(39):137–140. doi: 10.1038/newbio233137a0. [DOI] [PubMed] [Google Scholar]
  32. Tan K. B., Sokol F. Phosphorylation of simian virus 40 proteins in a cell-free system. J Virol. 1973 Oct;12(4):696–703. doi: 10.1128/jvi.12.4.696-703.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tan K. B., Sokol F. Structural proteins of simian virus 40: phosphoproteins. J Virol. 1972 Nov;10(5):985–994. doi: 10.1128/jvi.10.5.985-994.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Van Zaane D., Brinkhof J. M., Westenbrink F., Gielkens A. L. Molecular-biological characterization of Marek's disease virus. I. Identification of virus-specific polypeptides in infected cells. Virology. 1982 Aug;121(1):116–132. doi: 10.1016/0042-6822(82)90122-2. [DOI] [PubMed] [Google Scholar]
  35. Yoshinaka Y., Luftig R. B. In vitro phosphorylation of murine leukemia virus proteins: specific phosphorylation of Pr65gag, the precursor of the internal core antigens. Virology. 1982 Jan 15;116(1):181–195. doi: 10.1016/0042-6822(82)90412-3. [DOI] [PubMed] [Google Scholar]

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