FIG 4.

Mutations in the intracellular domain of langerin do not alter binding to IAV but do prevent IAV infection of Lec2-lg cells. (A) Nucleotide and deduced amino acid sequences of the cytoplasmic domain of langerin showing sequences for wild-type langerin (Lg WT), as well as mutated forms of langerin containing either a single mutation at position 23 which changes proline to isoleucine (Lg P23I) or a deletion corresponding to the first 30 amino acids of the intracellular domain (ΔLg). (B) Flow cytometry was used to determine cell surface expression of langerin on Lec2 cells expressing WT (Lec2-Lg, black histogram), P23I mutant (Lec2-Lg P23I, gray histogram), and ΔLg mutant (Lec2-ΔLg, dark gray histogram) forms of langerin. Lec2-ctrl cells were included to confirm the specificity of binding. (C) Binding of 5 μg/ml of purified BJx109 to Lec2 cells expressing mutated forms of langerin was determined by flow cytometry. (i) Representative histograms of BJx109 binding to Lec2-Lg, Lec2-Lg P23I, and Lec2-ΔLg in the presence of 20 mM Ca²⁺ (black histograms) or 5 mM EDTA (gray histograms) are shown. Unstained cells were included as a negative control (white histograms). (ii) Geometric means (±1 SD) from triplicate samples of BJx109 incubated with cells in buffer containing 20 mM Ca²⁺ (black bars) or 5 mM EDTA (hatched bars) are shown. Unstained cells (white bars) were included for comparison. **, P < 0.01; ***, P < 0.001. (D) Cells were infected either with 10⁷ PFU of BJx109 for 8 h and then fixed and stained for expression of IAV NP (panel i) or with 10⁵ FFU of RSV for 18 h and then fixed and stained for expression of RSV F protein (panel ii). Data show the mean percent infection (±1 SD). Data were analyzed by one-way ANOVA with Tukey's post hoc analysis, ***, significantly reduced compared to results for Lec2-Lg (P < 0.001). No significant differences were observed between cells infected with RSV. n.s., not significant.