A Comprehensive Proteomic View of Responses of A549 Type II Alveolar Epithelial Cells to Human Respiratory Syncytial Virus Infection
2014; Elsevier BV; Volume: 13; Issue: 12 Linguagem: Inglês
10.1074/mcp.m114.041129
ISSN1535-9484
AutoresKeyur A. Dave, Emma L. Norris, Alexander Bukreyev, Madeleine J. Headlam, Ursula J. Buchholz, Toshna Singh, Peter L. Collins, Jeffrey J. Gorman,
Tópico(s)RNA modifications and cancer
ResumoHuman 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. 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. Human respiratory syncytial virus (hRSV) 1The abbreviations used are:hRSVhuman respiratory syncytial virus1Done-dimensional2Dtwo-dimensionalCapcapillaryFhuman repiratory syncytial virus fusion proteinFDRfalse discovery rateGhuman repiratory syncytial virus glycoproteinGOGene OntologyHep2human epithelial type 2IFNinterferonIFIT3interferon-induced protein with tetracopeptide repeats 3IPAIngenuity Pathway AnalysisIRFinterferon-regulatory factorISG15Interferon-induced 17 kDa proteinLhuman repiratory syncytial virus polymerase proteinLTQlinear ion trapMhuman repiratory syncytial virus matrix proteinMS/MStandem mass spectrometryMxAInterferon-induced GTP-binding proteinNS1hRSV nonstructural protein1NS2hRSV nonstructural protein2nUnanoUltraNF-κBnuclear factor kappa-light-chain-enhancer of activated B cellsPEPposterior error probabilitySILACstable isotope labeling by amino acids in cell cultureSOD2Mitochondrial Mn Superoxide Dismutase 2StatSignal transducer and activator of transcriptionTGM2Protein-glutamine gamma-glutamyltransferase 2TNF-αTumor necrosis factor-αVSNvariance-stabilising normalisationWARSTryptophan-tRNA synthetase.1The abbreviations used are:hRSVhuman respiratory syncytial virus1Done-dimensional2Dtwo-dimensionalCapcapillaryFhuman repiratory syncytial virus fusion proteinFDRfalse discovery rateGhuman repiratory syncytial virus glycoproteinGOGene OntologyHep2human epithelial type 2IFNinterferonIFIT3interferon-induced protein with tetracopeptide repeats 3IPAIngenuity Pathway AnalysisIRFinterferon-regulatory factorISG15Interferon-induced 17 kDa proteinLhuman repiratory syncytial virus polymerase proteinLTQlinear ion trapMhuman repiratory syncytial virus matrix proteinMS/MStandem mass spectrometryMxAInterferon-induced GTP-binding proteinNS1hRSV nonstructural protein1NS2hRSV nonstructural protein2nUnanoUltraNF-κBnuclear factor kappa-light-chain-enhancer of activated B cellsPEPposterior error probabilitySILACstable isotope labeling by amino acids in cell cultureSOD2Mitochondrial Mn Superoxide Dismutase 2StatSignal transducer and activator of transcriptionTGM2Protein-glutamine gamma-glutamyltransferase 2TNF-αTumor necrosis factor-αVSNvariance-stabilising normalisationWARSTryptophan-tRNA synthetase. belongs to the Pneumovirus genus of the Pnuemovirinae subfamily of the Paramyxoviridae family of viruses. It has a negative-sense single-stranded RNA genome (1Collins P.L. Chanock R.M. Murphy B.R. "Respiratory Syncytial Virus,".in: Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Fourth Ed. Lippincott-Raven Publishers, Philadelphia2001: 1443-1485Google Scholar, 2Collins P.L. Crowe J.E. J. "Respiratory Syncytial Virus and Metapneumovirus,".in: Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. 5 Ed. Lippincott-Raven Publishers, Philadelphia2007: 1601-1646Google Scholar, 3Collins P.L. Graham B.S. Viral and host factors in human respiratory syncytial virus pathogenesis.J. Virol. 2008; 82: 2040-2055Crossref PubMed Scopus (344) Google Scholar) with 10 distinct genes that encode 11 proteins (1Collins P.L. Chanock R.M. Murphy B.R. "Respiratory Syncytial Virus,".in: Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Fourth Ed. 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C., Hermans, P. W., Quantitative proteome profiling of respiratory virus-infected lung epithelial cells. J. Proteomics 73, 1680–1693Google Scholar) levels. This has included studies with forms of hRSV lacking genes that encode proteins known to impair host innate antiviral responses, such as the nonstructural protein1 (NS1) (24Hastie M.L. Headlam M.J. Patel N.B. Bukreyev A.A. Buchholz U.J. Dave K.A. Norris E.L. Wright C.L. Spann K.M. Collins P.L. Gorman J.J. The human respiratory syncytial virus nonstructural protein 1 regulates type I and type II interferon pathways.Mol. Cell. Proteomics. 2012; 11: 108-127Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Various proteomic studies conducted with hRSV infected cells have made important individual contributions, however, it is arguable that various experimental design features have limited the extents of their impacts. For instance, the use of fractionation of cells into cytoplasmic and nuclear fractions (23Brasier A.R. Spratt H. Wu Z. Boldogh I. Zhang Y. Garofalo R.P. Casola A. Pashmi J. Haag A. Luxon B. Kurosky A. Nuclear heat shock response and novel nuclear domain 10 reorganization in respiratory syncytial virus-infected a549 cells identified by high-resolution two-dimensional gel electrophoresis.J. Virol. 2004; 78: 11461-11476Crossref PubMed Scopus (76) Google Scholar, 25Jamaluddin M. Wiktorowicz J.E. Soman K.V. Boldogh I. Forbus J.D. Spratt H. Garofalo R.P. Brasier A.R. Role of peroxiredoxin 1 and peroxiredoxin 4 in protection of respiratory syncytial virus-induced cysteinyl oxidation of nuclear cytoskeletal proteins.J. Virol. 2010; 84: 9533-9545Crossref PubMed Scopus (43) Google Scholar, 26Munday D.C. Emmott E. Surtees R. Lardeau C.H. Wu W. Duprex W.P. Dove B.K. Barr J.N. Hiscox J.A. Quantitative proteomic analysis of A549 cells infected with human respiratory syncytial virus.Mol. Cell. Proteomics. 2010; 9: 2438-2459Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 27Munday D.C. Surtees R. Emmott E. Dove B.K. Digard P. Barr J.N. Whitehouse A. Matthews D. Hiscox J.A. Using SILAC and quantitative proteomics to investigate the interactions between viral and host proteomes.Proteomics. 2012; 12: 666-672Crossref PubMed Scopus (53) Google Scholar) is likely to have perturbed the ability to reliably quantify protein abundance changes at a global cellular level. Some of these studies may have been limited because of the lack of penetrating coverage of the proteomes using two-dimensional (2D)-gel-based protocols (23Brasier A.R. Spratt H. Wu Z. Boldogh I. Zhang Y. Garofalo R.P. Casola A. Pashmi J. Haag A. Luxon B. Kurosky A. Nuclear heat shock response and novel nuclear domain 10 reorganization in respiratory syncytial virus-infected a549 cells identified by high-resolution two-dimensional gel electrophoresis.J. Virol. 2004; 78: 11461-11476Crossref PubMed Scopus (76) Google Scholar, 24Hastie M.L. Headlam M.J. Patel N.B. Bukreyev A.A. Buchholz U.J. Dave K.A. Norris E.L. Wright C.L. Spann K.M. Collins P.L. Gorman J.J. The human respiratory syncytial virus nonstructural protein 1 regulates type I and type II interferon pathways.Mol. Cell. Proteomics. 2012; 11: 108-127Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 25Jamaluddin M. Wiktorowicz J.E. Soman K.V. Boldogh I. Forbus J.D. Spratt H. Garofalo R.P. Brasier A.R. Role of peroxiredoxin 1 and peroxiredoxin 4 in protection of respiratory syncytial virus-induced cysteinyl oxidation of nuclear cytoskeletal proteins.J. Virol. 2010; 84: 9533-9545Crossref PubMed Scopus (43) Google Scholar, 29van Diepen, A., Brand, H. K., Sama, I., Lambooy, L. H., van den Heuvel, L. P., van der Well, L., Huynen, M., Osterhaus, A. D., Andeweg, A. C., Hermans, P. W., Quantitative proteome profiling of respiratory virus-infected lung epithelial cells. J. Proteomics 73, 1680–1693Google Scholar), with (23Brasier A.R. Spratt H. Wu Z. Boldogh I. Zhang Y. Garofalo R.P. Casola A. Pashmi J. Haag A. Luxon B. Kurosky A. Nuclear heat shock response and novel nuclear domain 10 reorganization in respiratory syncytial virus-infected a549 cells identified by high-resolution two-dimensional gel electrophoresis.J. Virol. 2004; 78: 11461-11476Crossref PubMed Scopus (76) Google Scholar, 25Jamaluddin M. Wiktorowicz J.E. Soman K.V. Boldogh I. Forbus J.D. Spratt H. Garofalo R.P. Brasier A.R. Role of peroxiredoxin 1 and peroxiredoxin 4 in protection of respiratory syncytial virus-induced cysteinyl oxidation of nuclear cytoskeletal proteins.J. Virol. 2010; 84: 9533-9545Crossref PubMed Scopus (43) Google Scholar) and without (24Hastie M.L. Headlam M.J. Patel N.B. Bukreyev A.A. Buchholz U.J. Dave K.A. Norris E.L. Wright C.L. Spann K.M. Collins P.L. Gorman J.J. The human respiratory syncytial virus nonstructural protein 1 regulates type I and type II interferon pathways.Mol. Cell. Proteomics. 2012; 11: 108-127Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 29van Diepen, A., Brand, H. K., Sama, I., Lambooy, L. H., van den Heuvel, L. P., van der Well, L., Huynen, M., Osterhaus, A. D., Andeweg, A. C., Hermans, P. W., Quantitative proteome profiling of respiratory virus-infected lung epithelial cells. J. Proteomics 73, 1680–1693Google Scholar) subcellular fractionation steps. In some instances, proteome coverage may have been limited from an analytical technology standpoint (23Brasier A.R. Spratt H. Wu Z. Boldogh I. Zhang Y. Garofalo R.P. Casola A. Pashmi J. Haag A. Luxon B. Kurosky A. Nuclear heat shock response and novel nuclear domain 10 reorganization in respiratory syncytial virus-infected a549 cells identified by high-resolution two-dimensional gel electrophoresis.J. 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