FIG. 4.

Residues 89 to 108 of HCMV US6 are required for TAP inhibition, but not TAP binding. (A) Analysis of the effect of HCMV US6 tetra-alanine mutants on the cell surface expression of HLA-B2705. 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 tetra-alanine HCMV US6 mutant. 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 cell surface 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. (C) Analysis of the interaction between HCMV US6 tetra-alanine mutants and TAP. HeLa-M cells were transfected with either the control vector, full-length US6, or US6 tetra-alanine construct. US6 proteins were immunoprecipitated with the anti-FLAG epitope tag antibody M2, resolved by SDS-PAGE, transferred onto PVDF membranes, and probed with either a TAP1-specific or M2 antibody. The position of a background band present in all samples that may correspond to the light chain of the M2 antibody is indicated by an asterisk.