Quantitative proteomic analysis of A549 cells infected with human respiratory syncytial virus.
Human respiratory syncytial virus (HRSV) is a major cause of pediatric lower respiratory tract disease to which there is no vaccine or efficacious chemotherapeutic strategy. Although RNA synthesis and virus assembly occur in the cytoplasm, HRSV is known to induce nuclear responses in the host cell as replication alters global gene expression. Quantitative proteomics was used to take an unbiased overview of the protein changes in transformed human alveolar basal epithelial cells infected with HRSV. Underpinning this was the use of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS, which allowed the direct and simultaneous identification and quantification of both cellular and viral proteins. To reduce sample complexity and increase data return on potential protein localization, cells were fractionated into nuclear and cytoplasmic extracts. This resulted in the identification of 1,140 cellular proteins and six viral proteins. The proteomics data were analyzed using Ingenuity Pathways Analysis to identify defined canonical pathways and functional groupings. Selected data were validated using Western blot, direct and indirect immunofluorescence confocal microscopy, and functional assays. The study served to validate and expand upon known HRSV-host cell interactions, including those associated with the antiviral response and alterations in subnuclear structures such as the nucleolus and ND10 (promyelocytic leukemia bodies). In addition, novel changes were observed in mitochondrial proteins and functions, cell cycle regulatory molecules, nuclear pore complex proteins and nucleocytoplasmic trafficking proteins. These data shed light into how the cell is potentially altered to create conditions more favorable for infection. Additionally, the study highlights the application and advantage of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS for the analysis of virus-host interactions.
Munday
Diane C
DC
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.
Emmott
Edward
E
Surtees
Rebecca
R
Lardeau
Charles-Hugues
CH
Wu
Weining
W
Duprex
W Paul
WP
Dove
Brian K
BK
Barr
John N
JN
Hiscox
Julian A
JA
D002460:Cell Line / Q000821:virology; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007553:Isotope Labeling; D018186:Pneumovirus Infections / Q000378:metabolism*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D018113:Respiratory Syncytial Virus, Human / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014764:Viral Proteins / Q000032:analysis*
D020543:Proteome; D014764:Viral Proteins