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. 2015 Jun 23;290(32):19833–19843. doi: 10.1074/jbc.M114.635508

FIGURE 1.

FIGURE 1.

There is a requirement for sialic acids to interact with MAG. A, MeWo cells were infected with (+) or without VZV (−) (top panel). 293T cells were transfected with gB or mock-transfected (bottom panel). Cell lysates were analyzed by non-reducing SDS-PAGE after treatment with (+) or without sialidase (−). Proteins were blotted with anti-gB mAb. B, mock- or gB-transfected 293T cells were treated with sialidase (bold line) or vehicle (thin line) and stained with MAG-Ig or anti-gB mAb, followed by flow cytometry analysis. Cells were stained with secondary antibody only (gray area). C, OL cells stably transfected with WT-MAG were exposed to GFP-VZV at a multiplicity of infection of 0.2 for 24 h. Fold-changes in the infection rate are shown. Fold-changes in the infection rate were calculated by dividing the percentage of GFP+ cells in each indicated treatment by the percentage of GFP+ cells infected with mock-treated VZV (%GFP+ cells among the total cells exposed to mock- or sialidase-treated VZV/%GFP+ cells among the total cells exposed to mock-treated VZV). Representative data from three independent experiments are shown. The error bars represent the mean ± S.D. on the basis of triplicate samples, and the p values were calculated using Student's t test. D, 293T cells transfected with MAG and mock-transfected were incubated with VZV virions after mock treatment (thin lines) or treatment with sialidase for 30 min (bold lines), respectively, followed by staining with anti-gB mAb and secondary antibody. Cells were also stained with antibodies only (gray area). E, 293T cells transfected with gB were stained with MAG-Ig after treatment with sialidase for 0 (gray area), 10 (thin line), and 30 min (bold line). Cells were also stained with secondary antibodies only (dotted line). F, 293T effector cells transfected with gH, gL, T7 polymerase, and Renilla luciferase as an internal control as well as gB (gBgHgL) or mock-transfected (gHgL) were cocultured with other 293T target cells transfected with WT-MAG and firefly luciferase driven by the T7 polymerase promoter. gB-transfected cells were treated with sialidase or mock-treated. The relative fusion efficiencies are shown on the basis of representative data from three independent experiments. The error bars represent the mean ± S.D. on the basis of triplicate samples, and p values were calculated using Student's t test.