Figure 5

NS1 protein of the H3N2 subtype influenza A virus colocalizes with nucleolin in the nucleolus and nucleoplasm and with B23 and fibrillarin in the nucleolus in influenza A/Udorn/72 virus-infected A549 cells. (A) A549 cells were infected with influenza A/Udorn/72 wt viruses (MOI 5) for eight hours, fixed with 3 % paraformaldehyde and stained with rabbit or guinea pig anti-NS1 immunoglobulins, followed by fluorescein isothiocyanate-labeled goat anti-rabbit or anti-guinea pig immunoglobulins. To detect protein co-localization, cells were double-stained with mouse anti-nucleolin and anti-B23 or rabbit anti-fibrillarin immunoglobulins, followed by Rhodamine Red-X-labeled goat anti-mouse or anti-rabbit immunoglobulins, respectively. The colocalization of proteins was visualized with confocal laser microscopy. Only the nuclei of the cells are presented. Bar, 5 μm. (B) The relative distribution of both viral and nucleolar proteins in the nuclei, as illustrated in A, is presented. The relative intensity of fluorescence in the nucleolus and nucleoplasm was quantified by ImageJ software (mean of 30 cells in each case). The quantification was also performed on 30 control (CTR) cells not expressing NS1 for each nucleolar protein.