Extensive Phosphorylation Flanking the C-Terminal Functional Domains of the Measles Virus Nucleoprotein

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• 作者：Emmanuel J. F. Prodhomme ; Fred Fack ; Dominique Revets ; Patrick Pirrotte ; Jacques R. Kremer ; Claude P. Muller
• 刊名：Journal of Proteome Research
• 出版年：2010
• 出版时间：November 5, 2010
• 年：2010
• 卷：9
• 期：11
• 页码：5598-5609
• 全文大小：551K
• 年卷期：v.9,no.11(November 5, 2010)
• ISSN：1535-3907

文摘

The measles virus nucleoprotein (vNP) is the first and most abundant protein in infected cells. It plays numerous important roles including the encapsidation of genomic viral RNA and the transcription of viral proteins. Intricate interactions with host cell proteins rely on the structural integrity of its functional domains. Although some of these functional domains are known, their structural features are still poorly understood. Here we identified multiple isoforms of measles vNP by two-dimensional differential gel electrophoresis (2D-DIGE) and 2D Western blot. These isoforms were further analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)/TOF using MS (PMF) and MSMS (PSD) and electrospray ionization (ESI)-ion trap using LC−ESI−ion trap MS¹, MS² (neutral loss), MS³ (phosphosite). Both recombinant NP (rNP) and vNP were α-acetylated at the N-terminus. After tryptic or chymotryptic digestion, phosphopeptides were enriched and nine phosphorylation sites were identified and localized in the rNP, seven of which were also phosphorylated in vNP, probably by casein kinase 2. The phosphosites were all found within the intrinsically unstructured C-terminal domain. They clustered around functional domains involved in transcription and replication, as well as in sequences interacting with host-cell proteins. This underlines the importance of these post-translational modifications.

Keywords:

Paramyxoviridae; measles virus; nucleoprotein; post-translational modification; phosphorylation, mass spectrometry