Mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using surface plasmon resonance

Chen, Hongying and Gill, Andrew C. and Dove, Brian K. and Emmett, Stevan R. and Kemp, C. Fred and Ritchie, Mark A. and Dee, Michael and Hiscox, Julian A. (2005) Mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using surface plasmon resonance. Journal of Virology, 79 (2). pp. 1164-1179. ISSN 0022-538X

Full content URL: http://doi.org/10.1128/JVI.79.2.1164-1179.2005

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Abstract

Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphosphorylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5′ end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

Keywords:IBV, Coronavirus, nucleocapsid protein, protein phosphorylation, mass spectrometry
Subjects:C Biological Sciences > C760 Biomolecular Science
Divisions:College of Science
ID Code:29564
Deposited On:01 Dec 2017 08:30

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