title: Quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals changes in the cytoplasmic, nuclear, and nucleolar proteomes in Vero cells infected with the coronavirus infectious bronchitis virus.
abstract: Virus-host interactions involve complex interplay between viral and host factors, rendering them an ideal target for proteomic analysis. Here we detail a high throughput quantitative proteomics analysis of Vero cells infected with the coronavirus infectious bronchitis virus (IBV), a positive strand RNA virus that replicates in the cytoplasm. Stable isotope labeling with amino acids in cell culture (SILAC) was used in conjunction with LC-MS/MS to identify and quantify 1830 cellular and two viral proteins from IBV-infected cells. Fractionation of cells into cytoplasmic, nuclear, and nucleolar extracts was used to reduce sample complexity and provide information on the trafficking of proteins between the different compartments. Each fraction showed a proportion of proteins exhibiting >or=2-fold changes in abundance. Ingenuity Pathway Analysis revealed that proteins that changed in response to infection could be grouped into different functional categories. These included proteins regulated by NF-kappaB- and AP-1-dependent pathways and proteins involved in the cytoskeleton and molecular motors. A luciferase-based reporter gene assay was used to validate the up-regulation of AP-1- and NF-kappaB-dependent transcription in IBV-infected cells and confirmed using immunofluorescence. Immunofluorescence was used to validate changes in the subcellular localization of vimentin and myosin VI in IBV-infected cells. The proteomics analysis also confirmed the presence of the viral nucleocapsid protein as localizing in the cytoplasm, nucleus, and nucleolus and the viral membrane protein in the cytoplasmic fraction. This research is the first application of SILAC to study total host cell proteome changes in response to positive sense RNA virus infection and illustrates the versatility of this technique as applied to infectious disease research.
authors.affiliation: Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Edward
authors.initials: E
authors.lastname: Emmott
authors.forename: Mark A
authors.initials: MA
authors.lastname: Rodgers
authors.forename: Andrew
authors.initials: A
authors.lastname: Macdonald
authors.forename: Sarah
authors.initials: S
authors.lastname: McCrory
authors.forename: Paul
authors.initials: P
authors.lastname: Ajuh
authors.forename: Julian A
authors.initials: JA
authors.lastname: Hiscox
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002466:Cell Nucleolus / Q000737:chemistry*; D002467:Cell Nucleus / Q000737:chemistry*; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D001351:Infectious bronchitis virus / Q000472:pathogenicity*; D007553:Isotope Labeling / Q000379:methods*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D040901:Proteomics; D053719:Tandem Mass Spectrometry / Q000379:methods; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D014764:Viral Proteins
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