Figure 3. Infection with the M protein mutated virus strain, VSV^ΔM51, blocks NKG2D-ligand surface expression.

A, JE6-1 cells were either mock infected+non-treated with FR901228 (-), mock infected+treated with 20 ng/ml FR901228 for 18 hr (FR) or infected with the indicated MOI of VSV^ΔM51 for 19 hr. The MICA mRNA level was examined by real-time RT-PCR and surface expression of MICA/B by flow cytometry. The MICA mRNA level is displayed as fold expression relative to the mock infected sample. The bar graphs show mean±SD from four and three experiments, respectively. B and C, JE6-1 cells were mock infected (-) or infected with the indicated MOI of VSV^ΔM51 one hr prior to treatment with 20 ng/mL FR901228. 19 hr post infection, the cells were analyzed for MICA/B or ULBP-2 surface expression or intracellular MICA/B expression by flow cytometry. The bar graphs show mean±SD from three experiments. D, The melanoma cell lines, FM-86 and FM-78, were mock infected (-) or infected with 0.1 MOI VSV^ΔM51 for 19 hr and analysed for MICA/B surface expression by flow cytometry. The bar graphs show mean±SD from two experiments. E, The VSV^ΔM51 M protein was sequenced as described in section 2.7. The VSV^ΔM51 clone insert was aligned to the gene bank sequence of M protein (VSV WT; M11754.1; Indiana serotype of VSV). For all the bar graphs, * p<0.05 and *** p<0.0001.