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. 2012 May;86(9):4868–4882. doi: 10.1128/JVI.06610-11

Fig 6.

Fig 6

CD98 is important for mature vaccinia virus WR entry into mouse embryonic fibroblasts. (A) CD98 expression in CD98+/+ and CD98−/− MEFs. CD98+/+, CD98−/−, and CD98−/− MEFs stably expressing YFP (CD98−/− YFP) or YFP-CD98 (CD98−/− YFP-CD98) were stained with rat anti-mouse CD98 (for CD98+/+ and CD98−/− cells) or mouse anti-human CD98 (for CD98−/− YFP and CD98−/− YFP-CD98 cells) antibodies and analyzed by FACS analysis. (B) Vaccinia MV binding assays with MEF cells. The above-mentioned MEFs were infected with WR or IA27L MV at an MOI of 20 PFU/cell at 4°C for 60 min, washed, and subsequently fixed with 4% paraformaldehyde. The bound particles were stained by using an anti-L1R antibody (2D5). For each group, viral cores from at least 40 cells were counted to obtain an average number of viral cores/cell; the number of bound virions obtained from CD98+/+ MEFs was used as the 100% control. The experiments were performed three times, and the standard deviations are shown. (C) Vaccinia virus-uncoating assays with MEF cells. The above-mentioned MEFs were infected with WR or IA27L MV at an MOI of 40 PFU/cell, and viral core numbers were determined by staining with an anti-A4 antibody. For each group, viral cores from at least 40 cells were counted to obtain an average number of viral cores/cell; the viral core number obtained from CD98+/+ MEF cells was used as the 100% control. The experiments were performed three times, and the standard deviations are shown. (D) Early gene luciferase expression assays with MEF cells. MEF cells were infected with WR or IA27L virus at an MOI of 5 PFU/cell for 60 min and harvested at 2 h p.i. for early gene luciferase expression assays. Luciferase activities in CD98+/+ MEFs were used as the 100% control. The experiments were performed three times, and the standard deviations are shown. (E) Immunofluorescence microscopy of virus-uncoating assays of CD98+/+ and CD98−/− MEF cells infected with WR or IA27L MV. The core was stained by using an anti-A4 antibody (red). (F) Immunofluorescence microscopy of virus-uncoating assays of CD98−/− MEF cells expressing YFP-CD98 or YFP alone (green). MEF cells were infected with WR MV at an MOI of 40 PFU/cell, and viral cores were stained with an anti-A4 antibody (red) as described above for panel E.