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-Several virus-encoded proteins were identified in the nuclear and cytoplasmic fractions. For the nuclear fraction (supplemental Table 6 ), one of these could be predicted, the M protein, as this has well characterized nuclear-cytoplasmic trafficking (69, 109, 110) . Overexpression analysis using fluorescently labeled M2-1 and P proteins indicated that these proteins were present in the nucleus but predominately localized to the cytoplasm (Fig. 13) , and hence this could explain why they were identified in both fractions. The F protein was detected in the cytoplasmic fraction only (supplemental Table 7 ), which may be due to the association of this protein with the cellular membrane. Viral proteins could not be quantified in this experimental system as there were no unlabeled virus peptides of known amount in the isolated fractions with which to compare. However, the comparison of viral peptides with mammalian peptides (some of which may have similar sequences) does allow some putative observations to be made regarding the relative amounts of the identified viral proteins. Examination of the viral protein ratios in the cytoplasmic fraction (supplemental Table 7 ) indicated that the relative protein abundance of the identified proteins reflected their position on the genome and hence the abundance of their corresponding mRNA. This correlates with the relationship between gene position on the genome and abundance of mRNA in the Mononegavirales (111) (112) (113) . This may also explain why no L protein was detected in the LC-MS/MS analysis as this is the least abundant virus protein in HRSVinfected cells. Several other HRSV-encoded proteins were not detected, including the NS-2, SH, and G proteins. This may be a function of their post-translational modifications (e.g. glycosylation of G protein) or indicative of protein stability and turnover inside a cell.