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.

Cells and Virus-Hep2 and A549 cells were obtained from the Health Protection Agency Culture Collections and grown at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen). All cells were tested to ensure there was no contamination with mycoplasma. The media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. For the SILAC experiment, A549 cells were cultured in DMEM containing either 13 C-labeled arginine and 2 D-labeled lysine (R6K4-Medium) or 13 C-and 15 N-labeled arginine and lysine (R10K8-Heavy) for a minimum of seven population doublings. Labeled media were obtained from Dundee Cell Products Ltd. and were supplemented with 10% dialyzed FCS and 1% penicillin-streptomycin. Mock-infected supernatant was prepared using R6K4-Medium-labeled Hep2 cells. The HRSV A2 strain was propagated in R10K8-Heavy-labeled Hep2 cells. Virus was harvested 4 days postinfection and passed through a 0.45-m filter. Virus titer was calculated for A549 cells using an antibody-based methylcellulose plaque assay technique based on previous studies (34, 35) . Plaques were visualized using a goat anti-HRSV primary antibody (ab20745) and a horseradish peroxidase (HRP)-conjugated secondary antibody (ab6741) that were both obtained from Abcam. The latter antibody was detected using the peroxidase substrate 4-chloro-1-naphthol (Pierce). A549 cells were then grown in 500-cm 2 dishes until 60% confluent and mock-infected/infected with virus at a multiplicity of infection (m.o.i.) of 1.