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.

Fractions-The LC-MS/MS analysis identified several viral proteins in the nuclear and cytoplasmic fractions (supplemental Tables 6 and 7, respectively). Although the data tables show a quantitative ratio between HRSV proteins and mock-infected cells, in reality this should be infinite and is a result of comparison with cellular peptides of similar sequence. HRSV replication is cytoplasmic, therefore, the potential presence of viral proteins in the nucleus may be considered an unusual observation. However, HRSV M protein has been shown to localize to the nucleus and contains nuclear-cytoplasmic trafficking signals (9, (67) (68) (69) . The presence of viral proteins in the nuclear fraction either may have been an artifact of the fractionation procedure or may reflect the subcellular localization of a protein. To investigate whether the HRSV P and M2-1 proteins were present in the nucleus, overexpression analysis was utilized where these proteins were expressed as C-terminal fluorescent fusion proteins tagged with cyan (ECFP). The direct fluorescence confocal microscopy analysis indicated that both the P and the M2-1 proteins localized predominately to the cytoplasm, but some fluorescent signal was observed in the nucleus but excluded from the nucleolus (Fig. 13 ). This correlates with the identification of these proteins in the nuclear fraction. ECFP was localized throughout the cell. Note that ECFP and the fusion proteins have been false colored green postimage capture for increased clarity. DISCUSSION HRSV infection results in multiple changes in the host cell, and the aim of this study was to provide an unbiased global overview of these effects. The quantitative proteomics and the subsequent bioinformatics analysis using IPA allowed alterations in the host cell proteome to be mapped in HRSVinfected cells. This study serves to reflect the existing in vitro and in vivo data and highlights new interactions. To our knowledge, no such SILAC-coupled LC-MS/MS study has been applied to HRSV or to any other single-stranded negative sense RNA virus. Despite the potential of this technique and transferable application to the comparison of treated with non-treated experimental conditions (41, 70) , a few studies have used this methodology to investigate the interactions of viruses with the host cell. These include investigations of the interaction of hepatitis C virus (HCV) with cell lipid rafts (71), pseudorabies virus-infected cells (72) , coronavirus-infected cells (37) , and alterations to the nucleolar proteome in adenovirus- (73) and coronavirus (74)-infected cells.