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.

Identification and Quantification of Cellular Proteins and Experimental Conditions-Potential changes in the host cell cytoplasmic and nuclear proteomes were elucidated for a HRSV subgroup A using optimized conditions to ensure that the majority of cells were infected with HRSV to generate the highest potential differences between the mock-and HRSVinfected cell treatments for LC-MS/MS quantification and data comparison. The laboratory-adapted HRSV A2 strain used in animal-based pathogenesis studies (75) was used in the SILAC study because it replicated to high efficiency in the A549 model cell line. These cells have been used extensively in HRSV studies because of their ability to retain features of alveolar cells (76) . Proteins were identified and quantified at 24 h postinfection as this time point occurs prior to the appearance of a major cytopathic effect; thus, the majority of cells remain viable for downstream processing. In addition, this time point is commonly used in HRSV studies and there- fore allows comparison with the previously published data. For example, microarray studies (24) of HRSV-infected cells have demonstrated a high return of data at this time point. In total, 606 cellular proteins were identified in the nuclear fraction, and 534 proteins were identified in the cytoplasmic fraction along with six viral proteins.