Figure 5

Downregulation of B7-H6 during HCMV infection is mediated through lysosomal degradation. (A) ARPE19 cells overexpressing B7-H6-FLAG were infected with HCMV US19Stop or left uninfected, and were treated with 50 nM Concanamycin A (CCMA) or left untreated (DMSO). The cells were then stained with DAPI and antibodies against the FLAG-tag or the lysosomal marker CD107A, and visualized using confocal microscopy. GFP marks infected cells. Infected cells boundaries are marked by a yellow line. Yellow arrows mark intracellular accumulations of B7-H6-FLAG. (B) ARPE19 cells overexpressing B7-H6-FLAG were infected with HCMV US19Stop or left uninfected, and were treated with 50 nM Concanamycin A (CCMA) or left untreated (DMSO). The cells were then stained with DAPI and antibodies against the FLAG-tag or the ER marker PDI, and visualized using confocal microscopy. GFP marks infected cells. Infected cells boundaries are delineated using a yellow line. Yellow arrows mark intracellular accumulations of B7-H6-FLAG. (C) Quantification of the FLAG-tag fluorescence intensity shown in A. Averages of 45–85 cells from each category are presented, error bars represent standard error of the mean. A student’s t test was performed in order to evaluate significance. ***p-value < 0.0005.