Colocalization of Different Influenza Viral RNA Segments in the Cytoplasm before Viral Budding as Shown by Single-molecule Sensitivity FISH Analysis
Figure 5
Rab11 is involved in the colocalization of PB2 and NA vRNAs.
(A–C) A549 cells are infected with PR8 virus at MOI = 5 and were fixed at 6, 8 and 10 hpi. Immunofluorescence staining was performed using anti-Rab11 antibody followed by two-color smFISH using Cy5-labeled PB2 probes and Cy3-labeled NA probes. (A) Merged fluorescent images of cells at 6, 8 and 10 hpi are shown on the upper left corners (Maximum Intensity Projection). For each panel, the boxed region is enlarged and shown on the upper right corner. The enlarged images of the PB2 vRNAs (green), NA vRNAs (red), and Rab11 (blue) are shown at the bottom, and the merged image of the PB2 and NA vRNA signals is shown on the middle right. Scale bar = 5 µm. (B) Colocalization efficiency of the PB2 and NA vRNA in the cytoplasm at 6, 8 and 10 hpi. The colocalized vRNAs that colocalize with Rab11 are represented in dark gray while the colocalized vRNAs that are not associated with Rab11 are shown in light gray. The percentages of colocalized vRNAs associated with Rab11 are 31.3% at 6hpi, 44.9% at 8hpi, 38.8% at 10hpi and 24% for random control. Error bars represent standard deviation. (C) Colocalization efficiency of PB2 and NA vRNAs associated with and not associated with Rab11 in cells at 6, 8 and 10 hpi. The random control represents the colocalization efficiency of PB2 and NA vRNA when the vRNAs were randomly classified into Rab11-associated and Rab11-non-associated groups. Error bars denote standard deviations. ***: t-test p value<0.001, **: t-test p-value<0.01 and *: t-test p value<0.05. (D&E) A549 cells were transfected with 1 µg of GFP-rab11-WT or GFP-rab11-DN and infected with PR8 virus at MOI = 5 at 24 hour post transfection. Two-color smFISH using Cy5 probes targeting the PB2 vRNAs and Cy3 probes targeting the NA vRNAs were performed at 8 hpi. (D) Images are shown for cells transfected with WT-Rab11 or DN-Rab11 (Maximum Intensity Projections). Magnified images of the boxed areas are shown: the separate images showing PB2 vRNAs (green), NA vRNAs (red) and GFP-tagged rab11 proteins (blue) are at the bottom; a merged image of the PB2 and NA vRNAs is shown at the middle right while the merged image of the three channels is located on the upper right corner of the panel. Scale bar = 5 µm. (E) Colocalization of PB2 and NA vRNAs in WT-Rab11 or DN-Rab11 expressing cells. The colocalization efficiency of PB2 and NA vRNAs were analyzed for individual cells that were detected with GFP signal (expressing the GFP-tagged rab11 proteins). The black dots symbolize the colocalization efficiency of vRNAs in WT-Rab11 transfected cells while the red squares symbolize the colocalization efficiency in DN-Rab11 transfected cells. The error bars represent standard deviations. ***: t-test p value<0.001.