Quantitative proteomic analysis of A549 cells infected with human respiratory syncytial virus subgroup B using SILAC coupled to LC‐MS/MS

Abstract

Human respiratory syncytial virus (HRSV) is a leading cause of serious lower respiratory tract infections in infants. The virus has two subgroups A and B, which differ in prevalence and (nucleotide) sequence. The interaction of subgroup A viruses with the host cell is relatively well characterized, whereas for subgroup B viruses it is not. Therefore quantitative proteomics was used to investigate the interaction of subgroup B viruses with A549 cells, a respiratory cell line. Changes in the cellular proteome and potential canonical pathways were determined using SILAC coupled to LC-MS/MS and Ingenuity Pathway Analysis. To reduce sample complexity and investigate potential trafficking both nuclear and cytoplasmic fractions were analyzed. A total of 904 cellular and six viral proteins were identified and quantified, of which 112 cellular proteins showed a twofold or more change in HRSV-infected cells. Data sets were validated using indirect immunofluorescence confocal microscopy on independent samples. Major changes were observed in constituents of mitochondria including components of the electron transport chain complexes and channels, as well as increases in the abundance of the products of interferon-stimulated genes. This is the first quantitative proteomic analysis of cells infected with HRSV-subgroup B.

Zn-Responsive Proteome Profiling and Time-Dependent Expression of Proteins Regulated by MTF-1 in A549 Cells

“…A549 cells have been extensively used in the characterization of metal exposure and in the proteomic analysis of cells [13] [44]. In our 2DE experiments, A549 cells were incubated with 100 µM ZnSO 4 and harvested after 24 h. This concentration and exposure time and concentration was chosen to ensure that the cells remained approximately 75% confluent without undergoing contact inhibition.…”

Proteomic analysis of mitochondria in respiratory epithelial cells infected with human respiratory syncytial virus and functional implications for virus and cell biology

“…Of particular interest were previously uncharacterised changes in the abundance of mitochondrial proteins quantified 24 h post‐infection (p.i.) in comparison to mock [5,10,11]…”

“…Identifying those cellular proteins critical for HRSV biology and disputing their activity could lead to broader spectrum antiviral compounds being developed: high throughput, quantitative proteomic analysis provides a powerful tool for identifying such intricate protein and pathway‐specific alterations in virus‐infected cells on a large scale [9]. Previous global proteomic analyses of HRSV–host cell interactions highlighted specific cellular proteome changes related to the antiviral response and demonstrated that host cell proteome alterations were confined to specific signalling pathways and processes [5,10,11]. Of particular interest were previously uncharacterised changes in the abundance of mitochondrial proteins quantified 24 h post‐infection (p.i.)…”

Mass spectrometry based proteomic studies on viruses and hosts – A review

“…Heat shock protein 70 (Hsp70) was also identified and found to have an increased abundance in lipid-raft membranes [125]. nucleolus and ND10, were validated by Western blot and indirect immunofluorescence confocal microscopy analysis, respectively [131]. Flotation gradient analysis confirmed the increased level of raft-associated Hsp70 and immunoprecipitation assays validated the interaction between Hsp70 and virus polymerase complex in lipid-rafts [126].…”