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. 2000 Jul 15;349(Pt 2):417–425. doi: 10.1042/0264-6021:3490417

Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system.

F Héricourt 1, S Blanc 1, V Redeker 1, I Jupin 1
PMCID: PMC1221164  PMID: 10880340

Abstract

All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST sequence.

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Selected References

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

  1. Asara J. M., Allison J. Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts. J Am Soc Mass Spectrom. 1999 Jan;10(1):35–44. doi: 10.1016/S1044-0305(98)00129-9. [DOI] [PubMed] [Google Scholar]
  2. Bairoch A., Bucher P., Hofmann K. The PROSITE database, its status in 1995. Nucleic Acids Res. 1996 Jan 1;24(1):189–196. doi: 10.1093/nar/24.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Behrens S. E., Tomei L., De Francesco R. Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J. 1996 Jan 2;15(1):12–22. [PMC free article] [PubMed] [Google Scholar]
  4. Blanc S., Schmidt I., Kuhl G., Esperandieu P., Lebeurier G., Hull R., Cerutti M., Louis C. Paracrystalline structure of cauliflower mosaic virus aphid transmission factor produced both in plants and in a heterologous system and relationship with a solubilized active form. Virology. 1993 Nov;197(1):283–292. doi: 10.1006/viro.1993.1589. [DOI] [PubMed] [Google Scholar]
  5. Bransom K. L., Wallace S. E., Dreher T. W. Identification of the cleavage site recognized by the turnip yellow mosaic virus protease. Virology. 1996 Mar 1;217(1):404–406. doi: 10.1006/viro.1996.0131. [DOI] [PubMed] [Google Scholar]
  6. Bransom K. L., Weiland J. J., Dreher T. W. Proteolytic maturation of the 206-kDa nonstructural protein encoded by turnip yellow mosaic virus RNA. Virology. 1991 Sep;184(1):351–358. doi: 10.1016/0042-6822(91)90851-2. [DOI] [PubMed] [Google Scholar]
  7. Buck K. W. Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv Virus Res. 1996;47:159–251. doi: 10.1016/S0065-3527(08)60736-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cahoreau C., Garnier L., Djiane J., Devauchelle G., Cerutti M. Evidence for N-glycosylation and ubiquitination of the prolactin receptor expressed in a baculovirus-insect cell system. FEBS Lett. 1994 Aug 22;350(2-3):230–234. doi: 10.1016/0014-5793(94)00772-1. [DOI] [PubMed] [Google Scholar]
  9. Castellanos-Serra L., Proenza W., Huerta V., Moritz R. L., Simpson R. J. Proteome analysis of polyacrylamide gel-separated proteins visualized by reversible negative staining using imidazole-zinc salts. Electrophoresis. 1999 Apr-May;20(4-5):732–737. doi: 10.1002/(SICI)1522-2683(19990101)20:4/5<732::AID-ELPS732>3.0.CO;2-Q. [DOI] [PubMed] [Google Scholar]
  10. Chaabihi H., Ogliastro M. H., Martin M., Giraud C., Devauchelle G., Cerutti M. Competition between baculovirus polyhedrin and p10 gene expression during infection of insect cells. J Virol. 1993 May;67(5):2664–2671. doi: 10.1128/jvi.67.5.2664-2671.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ciechanover A. The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 1998 Dec 15;17(24):7151–7160. doi: 10.1093/emboj/17.24.7151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Daniels P. R., Sanders C. M., Maitland N. J. Characterization of the interactions of human papillomavirus type 16 E6 with p53 and E6-associated protein in insect and human cells. J Gen Virol. 1998 Mar;79(Pt 3):489–499. doi: 10.1099/0022-1317-79-3-489. [DOI] [PubMed] [Google Scholar]
  13. Deiman B. A., Kortlever R. M., Pleij C. W. The role of the pseudoknot at the 3' end of turnip yellow mosaic virus RNA in minus-strand synthesis by the viral RNA-dependent RNA polymerase. J Virol. 1997 Aug;71(8):5990–5996. doi: 10.1128/jvi.71.8.5990-5996.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Deiman B. A., Séron K., Jaspars E. M., Pleij C. W. Efficient transcription of the tRNA-like structure of turnip yellow mosaic virus by a template-dependent and specific viral RNA polymerase obtained by a new procedure [corrected]. J Virol Methods. 1997 Mar;64(2):181–195. doi: 10.1016/s0166-0934(96)02166-0. [DOI] [PubMed] [Google Scholar]
  15. Duclos B., Marcandier S., Cozzone A. J. Chemical properties and separation of phosphoamino acids by thin-layer chromatography and/or electrophoresis. Methods Enzymol. 1991;201:10–21. doi: 10.1016/0076-6879(91)01004-l. [DOI] [PubMed] [Google Scholar]
  16. Fraser M. J. Expression of eukaryotic genes in insect cultures. In Vitro Cell Dev Biol. 1989 Mar;25(3 Pt 1):225–235. doi: 10.1007/BF02628459. [DOI] [PubMed] [Google Scholar]
  17. Fujimuro M., Sawada H., Yokosawa H. Production and characterization of monoclonal antibodies specific to multi-ubiquitin chains of polyubiquitinated proteins. FEBS Lett. 1994 Aug 1;349(2):173–180. doi: 10.1016/0014-5793(94)00647-4. [DOI] [PubMed] [Google Scholar]
  18. Gargouri-Bouzid R., David C., Haenni A. L. The 3' promoter region involved in RNA synthesis directed by the turnip yellow mosaic virus genome in vitro. FEBS Lett. 1991 Dec 2;294(1-2):56–58. doi: 10.1016/0014-5793(91)81342-6. [DOI] [PubMed] [Google Scholar]
  19. Goldbach R., Wellink J. Evolution of plus-strand RNA viruses. Intervirology. 1988;29(5):260–267. doi: 10.1159/000150054. [DOI] [PubMed] [Google Scholar]
  20. Hong Y., Hunt A. G. RNA polymerase activity catalyzed by a potyvirus-encoded RNA-dependent RNA polymerase. Virology. 1996 Dec 1;226(1):146–151. doi: 10.1006/viro.1996.0639. [DOI] [PubMed] [Google Scholar]
  21. Hwang S. B., Park K. J., Kim Y. S., Sung Y. C., Lai M. M. Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization. Virology. 1997 Jan 20;227(2):439–446. doi: 10.1006/viro.1996.8357. [DOI] [PubMed] [Google Scholar]
  22. Joyce C. M., Steitz T. A. Polymerase structures and function: variations on a theme? J Bacteriol. 1995 Nov;177(22):6321–6329. doi: 10.1128/jb.177.22.6321-6329.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kadaré G., Drugeon G., Savithri H. S., Haenni A. L. Comparison of the strategies of expression of five tymovirus RNAs by in vitro translation studies. J Gen Virol. 1992 Feb;73(Pt 2):493–498. doi: 10.1099/0022-1317-73-2-493. [DOI] [PubMed] [Google Scholar]
  24. Kadaré G., Rozanov M., Haenni A. L. Expression of the turnip yellow mosaic virus proteinase in Escherichia coli and determination of the cleavage site within the 206 kDa protein. J Gen Virol. 1995 Nov;76(Pt 11):2853–2857. doi: 10.1099/0022-1317-76-11-2853. [DOI] [PubMed] [Google Scholar]
  25. Kamps M. P. Determination of phosphoamino acid composition by acid hydrolysis of protein blotted to Immobilon. Methods Enzymol. 1991;201:21–27. doi: 10.1016/0076-6879(91)01005-m. [DOI] [PubMed] [Google Scholar]
  26. Koonin E. V. The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses. J Gen Virol. 1991 Sep;72(Pt 9):2197–2206. doi: 10.1099/0022-1317-72-9-2197. [DOI] [PubMed] [Google Scholar]
  27. Kratzer R., Eckerskorn C., Karas M., Lottspeich F. Suppression effects in enzymatic peptide ladder sequencing using ultraviolet - matrix assisted laser desorption/ionization - mass spectormetry. Electrophoresis. 1998 Aug;19(11):1910–1919. doi: 10.1002/elps.1150191109. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Lanker S., Valdivieso M. H., Wittenberg C. Rapid degradation of the G1 cyclin Cln2 induced by CDK-dependent phosphorylation. Science. 1996 Mar 15;271(5255):1597–1601. doi: 10.1126/science.271.5255.1597. [DOI] [PubMed] [Google Scholar]
  30. Liao P. C., Leykam J., Andrews P. C., Gage D. A., Allison J. An approach to locate phosphorylation sites in a phosphoprotein: mass mapping by combining specific enzymatic degradation with matrix-assisted laser desorption/ionization mass spectrometry. Anal Biochem. 1994 May 15;219(1):9–20. doi: 10.1006/abio.1994.1224. [DOI] [PubMed] [Google Scholar]
  31. Lin R., Beauparlant P., Makris C., Meloche S., Hiscott J. Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability. Mol Cell Biol. 1996 Apr;16(4):1401–1409. doi: 10.1128/mcb.16.4.1401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Marchal C., Haguenauer-Tsapis R., Urban-Grimal D. A PEST-like sequence mediates phosphorylation and efficient ubiquitination of yeast uracil permease. Mol Cell Biol. 1998 Jan;18(1):314–321. doi: 10.1128/mcb.18.1.314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Morch M. D., Boyer J. C., Haenni A. L. Overlapping open reading frames revealed by complete nucleotide sequencing of turnip yellow mosaic virus genomic RNA. Nucleic Acids Res. 1988 Jul 11;16(13):6157–6173. doi: 10.1093/nar/16.13.6157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Morch M. D., Drugeon G., Szafranski P., Haenni A. L. Proteolytic origin of the 150-kilodalton protein encoded by turnip yellow mosaic virus genomic RNA. J Virol. 1989 Dec;63(12):5153–5158. doi: 10.1128/jvi.63.12.5153-5158.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mouches C., Bove C., Bove J. M. Turnip yellow mosaic virus-RNA replicase: partial purification of the enzyme from the solubilized enzyme-template complex. Virology. 1974 Apr;58(2):409–423. doi: 10.1016/0042-6822(74)90076-2. [DOI] [PubMed] [Google Scholar]
  36. Neufeld K. L., Richards O. C., Ehrenfeld E. Expression and characterization of poliovirus proteins 3BVPg, 3Cpro, and 3Dpol in recombinant baculovirus-infected Spodoptera frugiperda cells. Virus Res. 1991 May;19(2-3):173–188. doi: 10.1016/0168-1702(91)90044-v. [DOI] [PubMed] [Google Scholar]
  37. Neufeld K. L., Richards O. C., Ehrenfeld E. Purification, characterization, and comparison of poliovirus RNA polymerase from native and recombinant sources. J Biol Chem. 1991 Dec 15;266(35):24212–24219. [PubMed] [Google Scholar]
  38. Payne J. M., Laybourn P. J., Dahmus M. E. The transition of RNA polymerase II from initiation to elongation is associated with phosphorylation of the carboxyl-terminal domain of subunit IIa. J Biol Chem. 1989 Nov 25;264(33):19621–19629. [PubMed] [Google Scholar]
  39. Poch O., Sauvaget I., Delarue M., Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 1989 Dec 1;8(12):3867–3874. doi: 10.1002/j.1460-2075.1989.tb08565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rechsteiner M., Rogers S. W. PEST sequences and regulation by proteolysis. Trends Biochem Sci. 1996 Jul;21(7):267–271. [PubMed] [Google Scholar]
  41. Redeker V., Rossier J., Frankfurter A. Posttranslational modifications of the C-terminus of alpha-tubulin in adult rat brain: alpha 4 is glutamylated at two residues. Biochemistry. 1998 Oct 20;37(42):14838–14844. doi: 10.1021/bi981335k. [DOI] [PubMed] [Google Scholar]
  42. Rogers S., Wells R., Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science. 1986 Oct 17;234(4774):364–368. doi: 10.1126/science.2876518. [DOI] [PubMed] [Google Scholar]
  43. Singh R. N., Dreher T. W. Turnip yellow mosaic virus RNA-dependent RNA polymerase: initiation of minus strand synthesis in vitro. Virology. 1997 Jul 7;233(2):430–439. doi: 10.1006/viro.1997.8621. [DOI] [PubMed] [Google Scholar]
  44. Strauss J. H., Strauss E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994 Sep;58(3):491–562. doi: 10.1128/mr.58.3.491-562.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Varshavsky A. The N-end rule: functions, mysteries, uses. Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12142–12149. doi: 10.1073/pnas.93.22.12142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vierstra R. D. Proteolysis in plants: mechanisms and functions. Plant Mol Biol. 1996 Oct;32(1-2):275–302. doi: 10.1007/BF00039386. [DOI] [PubMed] [Google Scholar]
  47. Weiland J. J., Dreher T. W. Cis-preferential replication of the turnip yellow mosaic virus RNA genome. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6095–6099. doi: 10.1073/pnas.90.13.6095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weiland J. J., Dreher T. W. Infectious TYMV RNA from cloned cDNA: effects in vitro and in vivo of point substitutions in the initiation codons of two extensively overlapping ORFs. Nucleic Acids Res. 1989 Jun 26;17(12):4675–4687. doi: 10.1093/nar/17.12.4675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wellink J., van Kammen A. Proteases involved in the processing of viral polyproteins. Brief review. Arch Virol. 1988;98(1-2):1–26. doi: 10.1007/BF01321002. [DOI] [PubMed] [Google Scholar]
  50. Wilm M., Shevchenko A., Houthaeve T., Breit S., Schweigerer L., Fotsis T., Mann M. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature. 1996 Feb 1;379(6564):466–469. doi: 10.1038/379466a0. [DOI] [PubMed] [Google Scholar]
  51. de Groot R. J., Rümenapf T., Kuhn R. J., Strauss E. G., Strauss J. H. Sindbis virus RNA polymerase is degraded by the N-end rule pathway. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8967–8971. doi: 10.1073/pnas.88.20.8967. [DOI] [PMC free article] [PubMed] [Google Scholar]

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