title: Phosphorylation of measles virus nucleoprotein upregulates the transcriptional activity of minigenomic RNA.
abstract: We report the first identification of phosphorylation sites of the nucleoprotein (N) of the family Paramyxoviridae. The N protein is known to be the most abundant protein in infected cells; it constructs the N-RNA complex (nucleocapsid) and supports transcription and replication of viral genomic RNA. To determine the role of phosphorylation of the N protein, we expressed the N protein of the HL strain of measles virus (MV) in mammalian cells and purified the nucleocapsid. After separation of the C-terminal region from the core region, phosphorylated amino acids were assayed using MALDI-TOF/TOF and ESI-Q-TOF MS analyses. Two amino acids, S479 and S510, were shown to be phosphorylated by both methods of analysis. Metabolic labeling of the N protein with (32)P demonstrated that these two sites are the major phosphorylated sites within the MV-N protein. In transcriptional analysis using negative-strand minigenomic RNA containing the ORF of the luciferase gene, mutants of each phosphorylation site showed approximately 80% reduction in luciferase activity compared with the wild-type N, suggesting that the phosphorylation of N protein is important in the activation of the transcription of viral mRNA and/or replication of the genome in vivo.
authors.affiliation: Laboratory Animal Research Center, Graduate School of Frontier Sciences, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Kyoji
authors.initials: K
authors.lastname: Hagiwara
authors.forename: Hiroki
authors.initials: H
authors.lastname: Sato
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Inoue
authors.forename: Akira
authors.initials: A
authors.lastname: Watanabe
authors.forename: Misako
authors.initials: M
authors.lastname: Yoneda
authors.forename: Fusako
authors.initials: F
authors.lastname: Ikeda
authors.forename: Kentaro
authors.initials: K
authors.lastname: Fujita
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Fukuda
authors.forename: Chizuko
authors.initials: C
authors.lastname: Takamura
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kozuka-Hata
authors.forename: Masaaki
authors.initials: M
authors.lastname: Oyama
authors.forename: Sumio
authors.initials: S
authors.lastname: Sugano
authors.forename: Shinobu
authors.initials: S
authors.lastname: Ohmi
authors.forename: Chieko
authors.initials: C
authors.lastname: Kai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000737:chemistry*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D012367:RNA, Viral / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D012367:RNA, Viral; D014764:Viral Proteins; C058314:nucleoprotein, Measles virus