Fig 1.

Vaccinia MV particles colocalize with cell surface CD98 in lipid rafts upon vaccinia MV infection. (A) Flow chart of differential IMID-H4/D4 labeling and LC/MS/MS analyses of lipid raft-associated proteins isolated from HeLa cells that were either mock infected or infected with WR strain MV. Experimental details are described in Materials and Methods, and a list of identified and quantified raft-associated cellular proteins is included in Table S1A in the supplemental material. (B) CD98 localizes at plasma membrane lipid rafts. HeLa cells were copatched with mouse anti-human CD98 antibody (green) and the lipid raft marker CTB (CTB-Alexa Fluor 594) (red) without permeabilization, as described in Materials and Methods. (C and D) WR MV copatched with CD98 on cell surface lipid rafts. WR MV was allowed to bind to HeLa cells for 1 h at 4°C and washed, and the cells were transferred to 37°C for 5 min before copatching with mouse anti-human CD98 (green) and rabbit anti-vaccinia virus (cyan) antibodies and the lipid raft marker CTB (red) (C) or with mouse anti-human CD98 (red) or mouse anti-human CD71 (red) and rabbit anti-vaccinia virus (green) antibodies without permeabilization (D). (E) Quantification of the WR MV particle colocalization with CD98 shown in panel D. The data were quantified by calculating the fraction of the total number of WR MV-positive pixels colocalizing with CD98- or CD71-positive pixels per cell. A total of ∼50 cells were quantified, and the statistical analyses were performed by using Student's t test. The P value is shown, where *** indicates a P value of <0.0001.