title: A comprehensive proteomic view of responses of A549 type II alveolar epithelial cells to human respiratory syncytial virus infection.
abstract: Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis by nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II, and III interferon, TNF-α and noncanonical NF-κB2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine-/key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious agents on host cells.
authors.affiliation: From the ‡Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Herston, Queensland, 4029 Australia and.
authors.forename: Keyur A
authors.initials: KA
authors.lastname: Dave
authors.affiliation: From the ‡Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Herston, Queensland, 4029 Australia and.
authors.forename: Emma L
authors.initials: EL
authors.lastname: Norris
authors.affiliation: §Respiratory Virus Section, Laboratory of Infectious Diseases, National Institute for Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Bukreyev
authors.affiliation: From the ‡Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Herston, Queensland, 4029 Australia and.
authors.forename: Madeleine J
authors.initials: MJ
authors.lastname: Headlam
authors.affiliation: §Respiratory Virus Section, Laboratory of Infectious Diseases, National Institute for Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892.
authors.forename: Ursula J
authors.initials: UJ
authors.lastname: Buchholz
authors.affiliation: From the ‡Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Herston, Queensland, 4029 Australia and.
authors.forename: Toshna
authors.initials: T
authors.lastname: Singh
authors.affiliation: §Respiratory Virus Section, Laboratory of Infectious Diseases, National Institute for Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Collins
authors.affiliation: From the ‡Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Herston, Queensland, 4029 Australia and jeff.gorman@qimr.edu.au.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Gorman
mesh_terms: D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004847:Epithelial Cells / Q000276:immunology* / Q000378:metabolism / Q000821:virology; D005786:Gene Expression Regulation; D054884:Host-Pathogen Interactions / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007372:Interferons / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D052003:NF-kappa B p52 Subunit / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D059748:Proteolysis; D020543:Proteome / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D020545:Respiratory Mucosa / Q000276:immunology* / Q000378:metabolism / Q000821:virology; D018113:Respiratory Syncytial Virus, Human / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D002457:Cell Extracts; D052003:NF-kappa B p52 Subunit; D010446:Peptide Fragments; D020543:Proteome; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins; D007372:Interferons
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