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 associated with ND10s were also altered in HRSVinfected cells. ND10 constituents TAR and RAD50 were significantly decreased in abundance, but the major constituent, the PML protein, was not detected by LC-MS/MS. Because PML is an ND10 marker protein, it was used to validate the abundance and localization of ND10s in HRSV-infected cells. Western blot analysis indicated that the abundance of PML protein increased in the nucleus of HRSV-infected cells. This was confirmed by indirect immunofluorescence confocal microscopy that showed an increased number of ND10s in the nucleus of HRSV-infected cells. These results are in contrast to a previous report for A549 cells infected with HRSV A2 strain (26) . However, in the current analysis, indirect immunofluorescence confocal microscopy positively identified HRSVinfected cells and showed that tagged PML-I and PML-II proteins remain in the nucleus. However, this does not preclude that certain isoforms may be more abundant in the cytoplasm of infected cells. Other negative sense RNA viruses have also been shown to interact with ND10s. For example, in rabies virus-infected cells, ND10s became larger, and the expression of rabies virus P protein led to the sequestration of PML in the cytoplasm, which resulted in an increased viral titer, presumably through the ablation of an antiviral response (108) . Along with the observations described above and the contrasting results from previous studies, it is important to note that ND10s are dynamic nuclear structures whose composition is highly dependent on the metabolic state of the cell.