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.
Highlights
Virus-host interactions involve complex interplay between viral and host factors, rendering them an ideal target for proteomic analysis
In mock-infected cells, vimentin was distributed in the cytoplasm with enrichment in the perinuclear region (Fig. 8B), whereas in infectious bronchitis virus (IBV)-infected cells that form syncytia, this enrichment was lost (Fig. 8B), again validating the change in the amount of vimentin determined by the SILAC analysis (Table II). -Tubulin was identified as being 2.2-fold increased in the nucleolar fraction from IBV-infected cells by SILAC
Changes in Nucleolar Proteome in Response to Virus Infection Are Not Conserved—The interaction of virus infection with the nucleolus has been predicted to alter the nucleolar proteome [23, 25, 51]. This hypothesis was investigated in a recent quantitative proteomics analysis using SILAC coupled to LC-MS/MS that revealed only specific nucleolar proteins changed in abundance in response to adenovirus infection rather than total global changes [52]
Summary
Virus and Cells—Vero cells were grown at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and penicillin/streptomycin. The generation of the peak list, SILAC- and extracted ion currentbased quantitation, calculated posterior error probability and false discovery rate based on search engine results, peptide to protein group assembly, and data filtration and presentation were carried out using MaxQuant. A cutoff of 2.0 was set to identify genes whose expression was significantly differentially regulated These genes, called focus genes, were overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. Assay for Detection of NF-B—80% confluent cell monolayers in 12-well dishes were co-transfected with 100 ng of NF-B or positive regulatory domain II (PRDII) reporter plasmid and 10 ng of Renilla reporter plasmid per well using Lipofectamine 2000 (Invitrogen) in serum-free media according to the manufacturer’s instructions. Control plates of transfection mixture only and media only were included to rule out background reactivity (data not shown)
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