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.
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.
Chen
Hongying
H
School of Animal and Microbial Sciences, University of Reading, Reading, United Kingdom.
Gill
Andrew
A
Dove
Brian K
BK
Emmett
Stevan R
SR
Kemp
C Fred
CF
Ritchie
Mark A
MA
Dee
Michael
M
Hiscox
Julian A
JA
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
D004220:Disulfides; D019590:Nucleocapsid Proteins; D012367:RNA, Viral