FIG 8.

Langerin-mediated IAV infection involves entry via Rab5⁺ early endosomes. Cells transfected with expression vectors encoding Rab5 or DN Rab5 S34N were incubated at 37°C for 24 h to allow for protein expression. After incubation, cells were infected with 10⁷ PFU BJx109 at 37°C for 1 h, washed, and incubated a further 6 to 8 h before they were fixed and stained for viral NP. (A) Representative images from confocal microscopy show RFP-labeled Rab5 and Rab5 S34N expression in all cell lines (red, left panels) and newly synthesized viral NP (green, middle panels), as well as a merged image (right panels), including staining for double-stranded nuclei acid using DAPI (blue). White arrows indicate examples of cells showing colocalization of Rab5 and viral NP. Note that cells are present in the middle panel for Lec2-ctrl cells but did not stain for viral NP. (B) Following transfection and IAV infection, cells were detached using 0.75 mM EDTA in PBS, fixed, and stained for viral NP prior to analysis via flow cytometry. Representative dot plots showing RFP-Rab5/RFP-Rab5 S34N expression (vertical axis) and NP expression (horizontal axis) are shown. Gates have been included to identify Rab5/Rab5 S34N⁺ cells (Q1 and Q4) as well as NP⁺ cells (Q2 and Q3). Arrows indicate cells with high expression levels of Rab5 or Rab5 S34N. (C) Analysis of transfected and untransfected CHO-ctrl, Lec2-ctrl, and Lec2-Lg cells shows the percentage of IAV-infected cells in the RFP⁻ fraction of cells transfected with constructs encoding Rab5 (white bars) or DN Rab5 S34N⁺ (black bars) (panel i) and the percentage of IAV-infected cells in the RFP⁺ fraction of cells transfected with constructs encoding Rab5 (white bars) or DN Rab5 S34N⁺ (black bars) (panel ii). Data represent the mean (±1 SD) of triplicate samples and are representative of two independent experiments. Statistical significance was assessed using one-way ANOVA with Tukey's post hoc analysis. ***, P < 0.001; n.s., not significant.