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Journal of Virology, January 2005, p. 1164-1179, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.1164-1179.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Hongying Chen,1 Andrew Gill,2 Brian K. Dove,3 Stevan R. Emmett,3 C. Fred Kemp,1 Mark A. Ritchie,2,4 Michael Dee,2 and Julian A. Hiscox3,5*

School of Animal and Microbial Sciences, University of Reading, Reading,1 Division of Molecular Biology, Institute for Animal Health, Compton,2 Molecular and Cellular Biology Research Group, Faculty of Biological Sciences,3 Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds,5 Waters Corporation, Micromass MS Technologies Centre, Wythenshawe, Manchester, United Kingdom4

Received 3 September 2003/ Accepted 5 July 2004

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.

* Corresponding author. Mailing address: School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, United Kingdom. Phone: 44 0113 343 5582. Fax: 44 0113 343 3167. E-mail: j.a.hiscox{at}leeds.ac.uk.

Journal of Virology, January 2005, p. 1164-1179, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.1164-1179.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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