title: High-throughput quantitative proteomic analysis of dengue virus type 2 infected A549 cells.
abstract: Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC) in combination with high-throughput mass spectrometry (MS). Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection.
authors.affiliation: School of Cellular and Molecular Medicine Faculty of Medical and Veterinary Sciences, University of Bristol, Bristol, United Kingdom.
authors.forename: Han-Chen
authors.initials: HC
authors.lastname: Chiu
authors.affiliation: School of Cellular and Molecular Medicine Faculty of Medical and Veterinary Sciences, University of Bristol, Bristol, United Kingdom.
authors.forename: Holger
authors.initials: H
authors.lastname: Hannemann
authors.affiliation: Proteomics Facility, Faculty of Medical and Veterinary Sciences, University of Bristol, Bristol, United Kingdom.
authors.forename: Kate J
authors.initials: KJ
authors.lastname: Heesom
authors.affiliation: School of Cellular and Molecular Medicine Faculty of Medical and Veterinary Sciences, University of Bristol, Bristol, United Kingdom.
authors.forename: David A
authors.initials: DA
authors.lastname: Matthews
authors.affiliation: School of Cellular and Molecular Medicine Faculty of Medical and Veterinary Sciences, University of Bristol, Bristol, United Kingdom.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Davidson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003715:Dengue / Q000378:metabolism* / Q000821:virology; D003716:Dengue Virus / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C468925:ERC1 protein, human; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C499863:PRAF2 protein, human; D020543:Proteome; D014764:Viral Proteins