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.

Proteins-Labeled A549 cells were infected with HRSV at an m.o.i. of 1 or mock-infected with supernatant that was prepared in the same way as described for the labeled virus stock. Labeling, validation, fractionation, protein identification, and quantification were conducted as outlined in Fig. 1 . Mock-infected cells were grown in media labeled with R6K4-Medium, and cells infected with virus were grown in media containing R10K8-Heavy. Proteins were identified and quantified at 24 h postinfection. Indirect immunofluorescence confocal microscopy indicated that 60 -70% of cells treated with HRSV were infected (Fig. 1B) . Fluorescence was not detected in mock-infected cells (Fig. 1B) . Cells were enriched into cytoplasmic and nuclear fractions, which were validated by the detection of characteristic cellular (tubulin for the cytoplasm and lamin B1 for the nucleus) (Fig. 1C ) and viral (Fig. 1D ) marker proteins that were enriched in the respective fractions. The protein concentration was determined by BCA assay. Note that equal protein concentrations were loaded, and therefore quantitative comparison of the relative abundance of proteins between the cytoplasmic and nuclear fractions should not be made. Western blot analysis showed that there was no virus present in mock-infected cells (Fig. 1D) .