FIG 4.

Expression of MGL1 by Lec1 cells results in enhanced IAV binding and increased susceptibility to IAV infection. (A) Binding of 5 μg/ml biotin-labeled BJx109 to CHO-ctrl (i), Lec1-ctrl (ii), and Lec1-MGL1 (iii) cells was determined by flow cytometry in the presence of 10 mM CaCl₂ (black histograms) or 0.5 mM EDTA (gray histograms). Representative histograms of triplicate samples are shown, and data were used to calculate mean MFI (± 1 SD) for each cell type in Ca²⁺ or in EDTA: CHO-ctrl, 389.7 ± 19.0 and 400 ± 28.6, respectively; Lec1-ctrl, 16.5 ± 3.2 and 13.5 ± 1.7, respectively; Lec1-MGL1, 77.2 ± 1.4 and 18.8 ± 6.4, respectively (P ≤ 0.001 [***] compared to Lec1-MGL1 in the presence of Ca²⁺; Student's t test). (B) Proteins in cell lysates of Lec1-MGL1 cells were resolved by SDS-PAGE under nonreducing conditions, transferred to PVDF membrane, and probed with purified BJx109 in VOPBA as described in Materials and Methods. Arrow indicates Ca²⁺-dependent binding to a species of ∼35 kDa, corresponding to the molecular mass of MGL1. (C) Lec1-ctrl or Lec1-MGL1 cells were infected with 10⁷ PFU of BJx109, and expression of newly synthesized viral NP (i) or HA (ii) was determined 8 h later. Data represent mean percent infection (± 1 SD) and are representative of 2 independent experiments (***, P ≤ 0.001; Student's t test). (D) Flow cytometry to determine expression of IAV NP (i) or HA (ii) at 8 h after infection with BJx109. Representative histograms from IAV-infected (white histograms) or mock-infected (gray histograms) cells are shown along with the mean percentage of infected cells (± 1 SD) from triplicate samples. A gate was set to include 5% of cells in uninfected controls, and the percentage of infected cells was determined relative to this. Data are representative of 2 independent experiments.