FIG 8.

Lec1 cells expressing endocytosis-defective MGL1 show reduced sensitivity to IAV infection. (B) Binding of 5 μg/ml biotin-labeled BJx109 to cells in the presence of 10 mM CaCl₂ was determined by flow cytometry. Representative histograms of triplicate samples are shown, and dashed lines represent cells stained with streptavidin-APC alone (un). MFI (± 1 SD) of BJx109 binding to Lec1-ctrl, Lec1-MGL1, and Lec1-ΔMGL1 cells (14.4 ± 2.8, 48.4 ± 7.1, and 65.9 ± 22.5, respectively). **, P < 0.01 by one-way ANOVA compared to Lec1-ctrl. (C) Monolayers of Lec1-ctrl, Lec1-MGL1, and Lec1-ΔMGL1 were infected with 10⁷ PFU of BJx109 as described in Materials and Methods. Cells were fixed at 8 h, stained, and then examined by immunofluorescence. Data show the mean percent infection (± 1 SD) from 6 independent experiments. (**, P ≤ 0.01; ***, P < 0.001; n.s., not significant; one-way ANOVA). (D) Monolayers of CHO-ctrl (i), Lec1-MGL1 (ii), and Lec1-ΔMGL1 (iii) were incubated with 10⁷ PFU of BJx109 at 4°C for 30 min to allow virus binding and then moved to 37°C. At various times, supernatants were removed and replaced with media containing 5 mM NH₄Cl to prevent further infection. NH₄Cl was not added to mock-treated cells. All cells were fixed at 8 h and stained for expression of viral NP. Data represent the mean percent infection (± 1 SD) and are representative of 3 independent experiments. §, percent infection was <1% of total cells.