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Phosphorylation of measles virus nucleoprotein upregulates the transcriptional activity of minigenomic RNA
Author(s) -
Hagiwara Kyoji,
Sato Hiroki,
Inoue Yoshihisa,
Watanabe Akira,
Yoneda Misako,
Ikeda Fusako,
Fujita Kentaro,
Fukuda Hiroyuki,
Takamura Chizuko,
KozukaHata Hiroko,
Oyama Masaaki,
Sugano Sumio,
Ohmi Shinobu,
Kai Chieko
Publication year - 2008
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200701051
Subject(s) - nucleoprotein , measles virus , phosphorylation , microbiology and biotechnology , biology , transcription (linguistics) , rna , luciferase , viral replication , paramyxoviridae , gene , virology , virus , biochemistry , measles , transfection , linguistics , philosophy , vaccination , viral disease
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 .