Phosphorylation of measles virus nucleoprotein upregulates the transcriptional activity of minigenomic RNA.
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.
Hagiwara
Kyoji
K
Laboratory Animal Research Center, Graduate School of Frontier Sciences, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Sato
Hiroki
H
Inoue
Yoshihisa
Y
Watanabe
Akira
A
Yoneda
Misako
M
Ikeda
Fusako
F
Fujita
Kentaro
K
Fukuda
Hiroyuki
H
Takamura
Chizuko
C
Kozuka-Hata
Hiroko
H
Oyama
Masaaki
M
Sugano
Sumio
S
Ohmi
Shinobu
S
Kai
Chieko
C
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*
D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D012367:RNA, Viral; D014764:Viral Proteins; C058314:nucleoprotein, Measles virus