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.

Together, the indirect immunofluorescence confocal microscopy images supported the observations from the quan- titative proteomic analysis that the abundance of mitochondrial proteins (particularly pore proteins) was altered in HRSV-infected cells. From this, we hypothesized that the mitochondrial permeability transition pores had an altered function in HRSV-infected cells. To test this hypothesis, we made use of a live cell mitochondrial assay in which green fluorescent (calcein AM) dye is maintained in healthy mitochondria (which are also stained red with a MitoTracker dye). As a positive control, cells were treated with the Ca 2Ï© ionophore ionomycin, which results in mitochondrial pore activation from the inner and outer mitochondrial membranes and subsequent loss of green fluorescence. In this live cell assay, it was not possible to use indirect immunofluorescence confocal microscopy to visualize HRSV-infected cells (as the cells could not be made permeable to allow antibody penetration). Therefore, cells were infected at an m.o.i. of 2 to ensure that at least 70% of cells were infected at 24 h postinfection, the assay point (after the experiment, cells were fixed, and infection status confirmed using indirect immunofluorescence confocal microscopy (supplemental Fig. 2) ). The data illustrated that in mock-infected cells the majority of the cells were stained green and red, indicating functional mitochondria (Fig. 9 ). In control cells treated with ionomycin, all of the cells were stained red with no green signal, indicating mitochondrial pore activation (Fig. 9) . In HRSV-infected cells, there was a greater population of predominately red cells than predominately green cells, indicating greater mitochondrial pore activation in cell populations infected with HRSV in comparison with the mock-infected cells (Fig. 9) .