title: 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.
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.
authors.affiliation: School of Animal and Microbial Sciences, University of Reading, Reading, United Kingdom.
authors.forename: Hongying
authors.initials: H
authors.lastname: Chen
authors.forename: Andrew
authors.initials: A
authors.lastname: Gill
authors.forename: Brian K
authors.initials: BK
authors.lastname: Dove
authors.forename: Stevan R
authors.initials: SR
authors.lastname: Emmett
authors.forename: C Fred
authors.initials: CF
authors.lastname: Kemp
authors.forename: Mark A
authors.initials: MA
authors.lastname: Ritchie
authors.forename: Michael
authors.initials: M
authors.lastname: Dee
authors.forename: Julian A
authors.initials: JA
authors.lastname: Hiscox
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002522:Chlorocebus aethiops; D004220:Disulfides / Q000737:chemistry; D001351:Infectious bronchitis virus / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014709:Vero Cells
chemical_list: D004220:Disulfides; D019590:Nucleocapsid Proteins; D012367:RNA, Viral