FIG. 7.

A chimeric US6 inhibits cell surface expression of HLA-B2705. (A) Schematic representation of the amino acid substitutions introduced into CCMV US6 to generate the chimeric US6 molecule. 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 ± standard deviation (error bar) of HLA-B2705 expression calculated from four independent experiments. Statistical analysis was performed using the Mann-Whitney U test. The sample 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 is indicated by an asterisk. (C) Analysis of the interaction between chimeric and CCMV US6 with TAP. US6 proteins were immunoprecipitated from stable cell lines that expressed either HCMV US6, CCMV US6, or chimeric US6 constructs with the anti-FLAG epitope tag M2 antibody, resolved by SDS-PAGE, transferred onto PVDF membranes. and probed with TAP1 and M2 antibodies. The position of a background band present in all samples, which may correspond to the light chain of the M2 antibody, is indicated by the asterisk.