Fig 5.
The M protein is involved in ribonucleoprotein localization. (A) A549 cells were infected with WT HMPV for 3 h, washed, and subsequently transfected with 1 µg GFP-M (green) and 1 µg mCherryP (red) at 3 hpi. At 24 hpi, live cells were imaged. Shown is a representative single z-slice time-lapse acquired every 1 s, and the selected frames are shown. The white arrows show a vRNP complex moving throughout the cell. (B) mCherryP (red) was observed trafficking along eGFP-tagged α-tubulin (Addgene Plasmid #21039) (green) (70). Scale bar is 1 µm. (C) A549 cells were infected with WT HMPV for 3 h, washed, and subsequently transfected with 1 µg GFP-M (green) and 1 µg mCherryP (red) at 3 hpi. At 24 hpi, live cells were imaged. Scale bar is 1 µm. (D) A549 cells were plated and infected with WT HMPV for 3 h, washed, and subsequently transfected with 2 µg of pCAGGs-mCherryP (red) at 3 hpi. At 6 hpi, 10 µM of PPMOs were added. At 24 hpi, the cells were fixed and stained with Alexa Fluor 647-conjugated WGA (pseudo-colored green) and DAPI (blue) and imaged by confocal microscopy. A single z-slice is shown, and the scale bar is 10 µm. (E) At least 10 cells were measured in each replicate, which were averaged and represented as a single data point, and analyzed to measure the colocalization of mCherryP and WGA. Data are represented as the average of three independent experiments and shown as the mean ± SD (n = 3). *P < 0.05, by a paired t test.