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Abstract

Many viruses use their host’s cellular machinery to regulate the functions of viral proteins. The phosphorylation of viral proteins is known to play a role in genome transcription and replication in paramyxoviruses. The paramyxovirus nucleoprotein (N), the most abundant protein in infected cells, is a component of the N–RNA complex and supports the transcription and replication of virus mRNA and genomic RNA. Recently, we reported that the phosphorylation of measles virus N is involved in the regulation of viral RNA synthesis. In this study, we report a rapid turnover of phosphorylation in the Nipah virus N (NiV-N). The phosphorylated NiV-N was hardly detectable in steady-state cells, but was detected after inhibition of cellular protein phosphatases. We identified a phosphorylated serine residue at Ser451 of NiV-N by peptide mass fingerprinting by electrospray ionization–quadrupole time-of-flight mass spectrometry. In the NiV minigenome assay, using luciferase as a reporter gene, the substitution of Ser451 for alanine in NiV-N resulted in a reduction in luciferase activity of approximately 45 % compared with the wild-type protein. Furthermore, the substitution of Ser451 for glutamic acid, which mimics a phosphoserine, led to a more significant decrease in luciferase activity – approximately 81 %. Northern blot analysis showed that both virus transcription and replication were reduced by these mutations. These results suggest that a rapid turnover of the phosphorylation of NiV-N plays an important role in virus transcription and replication.

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2011-09-01
2024-03-29
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References

  1. Asenjo A., Rodríguez L., Villanueva N. 2005; Determination of phosphorylated residues from human respiratory syncytial virus P protein that are dynamically dephosphorylated by cellular phosphatases: a possible role for serine 54. J Gen Virol 86:1109–1120 [View Article][PubMed]
    [Google Scholar]
  2. Asenjo A., González-Armas J. C., Villanueva N. 2008; Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating. Virology 380:26–33 [View Article][PubMed]
    [Google Scholar]
  3. Bankamp B., Horikami S. M., Thompson P. D., Huber M., Billeter M., Moyer S. A. 1996; Domains of the measles virus N protein required for binding to P protein and self-assembly. Virology 216:272–277 [View Article][PubMed]
    [Google Scholar]
  4. Baron M. D., Barrett T. 1997; Rescue of rinderpest virus from cloned cDNA. J Virol 71:1265–1271[PubMed]
    [Google Scholar]
  5. Becker S., Huppertz S., Klenk H.-D., Feldmann H. 1994; The nucleoprotein of Marburg virus is phosphorylated. J Gen Virol 75:809–818 [View Article][PubMed]
    [Google Scholar]
  6. Chan Y. P., Koh C. L., Lam S. K., Wang L. F. 2004; Mapping of domains responsible for nucleocapsid protein–phosphoprotein interaction of henipaviruses. J Gen Virol 85:1675–1684 [View Article][PubMed]
    [Google Scholar]
  7. Chua K. B., Bellini W. J., Rota P. A., Harcourt B. H., Tamin A., Lam S. K., Ksiazek T. G., Rollin P. E., Zaki S. R. et al. 2000; Nipah virus: a recently emergent deadly paramyxovirus. Science 288:1432–1435 [View Article][PubMed]
    [Google Scholar]
  8. Elliott L. H., Kiley M. P., McCormick J. B. 1985; Descriptive analysis of Ebola virus proteins. Virology 147:169–176 [View Article][PubMed]
    [Google Scholar]
  9. Gombart A. F., Hirano A., Wong T. C. 1995; Nucleoprotein phosphorylated on both serine and threonine is preferentially assembled into the nucleocapsids of measles virus. Virus Res 37:63–73 [View Article][PubMed]
    [Google Scholar]
  10. Hagiwara K., Sato H., Inoue Y., Watanabe A., Yoneda M., Ikeda F., Fujita K., Fukuda H., Takamura C. et al. 2008; Phosphorylation of measles virus nucleoprotein upregulates the transcriptional activity of minigenomic RNA. Proteomics 8:1871–1879 [View Article][PubMed]
    [Google Scholar]
  11. Halpin K., Bankamp B., Harcourt B. H., Bellini W. J., Rota P. A. 2004; Nipah virus conforms to the rule of six in a minigenome replication assay. J Gen Virol 85:701–707 [View Article][PubMed]
    [Google Scholar]
  12. Heggeness M. H., Scheid A., Choppin P. W. 1980; Conformation of the helical nucleocapsids of paramyxoviruses and vesicular stomatitis virus: reversible coiling and uncoiling induced by changes in salt concentration. Proc Natl Acad Sci U S A 77:2631–2635 [View Article][PubMed]
    [Google Scholar]
  13. Heggeness M. H., Scheid A., Choppin P. W. 1981; The relationship of conformational changes in the Sendai virus nucleocapsid to proteolytic cleavage of the NP polypeptide. Virology 114:555–562 [View Article][PubMed]
    [Google Scholar]
  14. Hsu C. H., Kingsbury D. W. 1982; Topography of phosphate residues in Sendai virus proteins. Virology 120:225–234 [View Article][PubMed]
    [Google Scholar]
  15. Hsu V. P., Hossain M. J., Parashar U. D., Ali M. M., Ksiazek T. G., Kuzmin I., Niezgoda M., Rupprecht C., Bresee J., Breiman R. F. 2004; Nipah virus encephalitis reemergence, Bangladesh. Emerg Infect Dis 10:2082–2087[PubMed] [CrossRef]
    [Google Scholar]
  16. Johansson K., Bourhis J. M., Campanacci V., Cambillau C., Canard B., Longhi S. 2003; Crystal structure of the measles virus phosphoprotein domain responsible for the induced folding of the C-terminal domain of the nucleoprotein. J Biol Chem 278:44567–44573 [View Article][PubMed]
    [Google Scholar]
  17. Lamb R. A., Parks G. D. 2006; Paramyxoviridae: the viruses and their replication. In Fields Virology, 5th edn. pp. 1449–1496 Edited by Knipe D. M., Howley P. M. Philadelpha, PA: Lippincott Williams & Wilkins;
    [Google Scholar]
  18. Liston P., Batal R., DiFlumeri C., Briedis D. J. 1997; Protein interaction domains of the measles virus nucleocapsid protein (NP). Arch Virol 142:305–321 [View Article][PubMed]
    [Google Scholar]
  19. Longhi S., Receveur-Bréchot V., Karlin D., Johansson K., Darbon H., Bhella D., Yeo R., Finet S., Canard B. 2003; The C-terminal domain of the measles virus nucleoprotein is intrinsically disordered and folds upon binding to the C-terminal moiety of the phosphoprotein. J Biol Chem 278:18638–18648 [View Article][PubMed]
    [Google Scholar]
  20. Mayo M. A. 2002a; A summary of taxonomic changes recently approved by ICTV. Arch Virol 147:1655–1656 [View Article][PubMed]
    [Google Scholar]
  21. Mayo M. A. 2002b ). Virus taxonomy – Houston 2002. Arch Virol 147:1071–1076 [View Article][PubMed]
    [Google Scholar]
  22. Naruse H., Nagai Y., Yoshida T., Hamaguchi M., Matsumoto T., Isomura S., Suzuki S. 1981; The polypeptides of mumps virus and their synthesis in infected chick embryo cells. Virology 112:119–130 [View Article][PubMed]
    [Google Scholar]
  23. Oglesbee M., Tatalick L., Ringler S., Rice J., Krakowka S. 1989; Rapid isolation of morbillivirus nucleocapsid for genomic RNA cDNA cloning and the production of specific core protein antisera. J Virol Methods 24:285–300 [View Article][PubMed]
    [Google Scholar]
  24. Ong S. T., Yusoff K., Kho C. L., Abdullah J. O., Tan W. S. 2009; Mutagenesis of the nucleocapsid protein of Nipah virus involved in capsid assembly. J Gen Virol 90:392–397 [View Article][PubMed]
    [Google Scholar]
  25. Oyama M., Itagaki C., Hata H., Suzuki Y., Izumi T., Natsume T., Isobe T., Sugano S. 2004; Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res 14:10B2048–2052 [View Article][PubMed]
    [Google Scholar]
  26. Oyama M., Kozuka-Hata H., Suzuki Y., Semba K., Yamamoto T., Sugano S. 2007; Diversity of translation start sites may define increased complexity of the human short ORFeome. Mol Cell Proteomics 6:1000–1006 [View Article][PubMed]
    [Google Scholar]
  27. Radecke F., Spielhofer P., Schneider H., Kaelin K., Huber M., Dötsch C., Christiansen G., Billeter M. A. 1995; Rescue of measles viruses from cloned DNA. EMBO J 14:5773–5784[PubMed]
    [Google Scholar]
  28. Segev Y., Ofir R., Salzberg S., Heller A., Weinstein Y., Isakov N., Udem S., Wolfson M., Rager-Zisman B. 1995; Tyrosine phosphorylation of measles virus nucleocapsid protein in persistently infected neuroblastoma cells. J Virol 69:2480–2485[PubMed]
    [Google Scholar]
  29. Sleeman K., Bankamp B., Hummel K. B., Lo M. K., Bellini W. J., Rota P. A. 2008; The C, V and W proteins of Nipah virus inhibit minigenome replication. J Gen Virol 89:1300–1308 [View Article][PubMed]
    [Google Scholar]
  30. Tokui M., Takei I., Tashiro F., Shimada A., Kasuga A., Ishii M., Ishii T., Takatsu K., Saruta T., Miyazaki J. 1997; Intramuscular injection of expression plasmid DNA is an effective means of long-term systemic delivery of interleukin-5. Biochem Biophys Res Commun 233:527–531 [View Article][PubMed]
    [Google Scholar]
  31. Wang L., Harcourt B. H., Yu M., Tamin A., Rota P. A., Bellini W. J., Eaton B. T. 2001; Molecular biology of Hendra and Nipah viruses. Microbes Infect 3:279–287 [View Article][PubMed]
    [Google Scholar]
  32. WHO 2004; Nipah virus outbreak(s) in Bangladesh, January–April 2004. Wkly Epidemiol Rec 79:168–171[PubMed]
    [Google Scholar]
  33. Wu X., Gong X., Foley H. D., Schnell M. J., Fu Z. F. 2002; Both viral transcription and replication are reduced when the rabies virus nucleoprotein is not phosphorylated. J Virol 76:4153–4161 [View Article][PubMed]
    [Google Scholar]
  34. Yoneda M., Guillaume V., Ikeda F., Sakuma Y., Sato H., Wild T. F., Kai C. 2006; Establishment of a Nipah virus rescue system. Proc Natl Acad Sci U S A 103:16508–16513 [View Article][PubMed]
    [Google Scholar]
  35. Zhang X., Bourhis J. M., Longhi S., Carsillo T., Buccellato M., Morin B., Canard B., Oglesbee M. 2005; Hsp72 recognizes a P binding motif in the measles virus N protein C-terminus. Virology 337:162–174 [View Article][PubMed]
    [Google Scholar]
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