FIG. 9.

Mutation of the C-terminal N-linked glycosylation site does not enable CCMV US6 to inhibit cell surface expression of HLA-B2705. (A) Schematic representation of the amino acid amino acid substitution introduced into CCMV US6 to generate the CCMV US6 S99A glycosylation mutant. The schematic representations are set up as described in the legend to Fig. 3. (B) Analysis of the effect of CCMV US6 and the chimeric US6 on cell surface HLA-B2705 expression. HeLa-M cells were either transfected with a control vector or cotransfected with a control vector and the HLA-B2705 expression construct or cotransfected with HLA-B2705 and each US6 construct. To detect HLA-B2705 cell surface expression, the transfectants were stained with the ME1 MAb and a Cy5-conjugated secondary antibody; antibody fluorescence was quantified by flow cytometry. The level of HLA-B2705 expression is expressed as a percentage of the MFV of the positive control (cells transfected with HLA-B2705 and control vector) after subtracting the MFV of the negative control (cells transfected with control vector only). Each bar represents the mean level of HLA-B2705 expression calculated from four independent experiments. Error bars show the standard deviations. Statistical analysis was performed using the Mann-Whitney U test. Those samples with a significantly increased level (P ≤ 0.05) of cell surface HLA-B2705 expression compared to that of cells cotransfected with the HCMV US6 and HLA-B2705 vectors are indicated by an asterisk.