Quantitative Proteomic Analysis of A549 Cells Infected with Human Respiratory Syncytial Virus

Munday, Diane C.; Emmott, Edward; Surtees, Rebecca; Lardeau, Charles-Hugues; Wu, Weining; Duprex, W. Paul; Dove, Brian K.; Barr, John N.; Hiscox, Julian A.

A, a diagrammatic representation of the methodology used in this analysis. Stable isotope (medium and heavy)-labeled amino acids were incorporated into newly synthesized cellular proteins. A549 cells were infected with HRSV (heavy) alongside a mock infection (medium), and 24 h postinfection subcellular fractionation was used to enrich the cytoplasmic and nuclear proteins. The infection efficiency and the fraction purity were then validated prior to sample preparation and LC-MS/MS analysis. B, indirect immunofluorescence confocal microscopy validation of HRSV infection efficiency in A549 cells 24 h postinfection. Staining with the HRSV antibody combination in mock-infected cells is shown as a control. Different magnifications are presented. Scale bars, 20 m. C, Western blot confirmation of representative proteins enriched from the cytoplasmic and the nuclear fractions of mock-infected (M) and HRSVinfected (I) cells. Membrane-immobilized proteins were detected with tubulin (ϳ55 kDa) and lamin B1 (ϳ58 kDa) antibodies. Tubulin and lamin B1 were predominately localized in the cytoplasmic and nuclear fractions, respectively. D, Western blot confirmation of HRSV infection (and lack thereof in mock-infected cells). An anti-HRSV-specific polyclonal primary antibody was detected by an HRP conjugate. The locations of molecular mass markers (kDa) are indicated on the left, and the tentative assignments of proteins are indicated on the right. RSV, respiratory syncytial virus.