FIG. 6.

Down-regulation of TNFRI by HCMV is due to receptor relocalization. (A) Uninfected control (CON) U373 cells (a and c) or U373 cells infected overnight with AD169 (5 PFU/cell) (b and d) were fixed and stained with a goat anti-TNFRI antibody (white area) or control goat immunoglobulins (shaded area), and antibodies were detected with PE-conjugated donkey anti-goat immunoglobulins and analyzed by FACS (a and b). A small aliquot of fixed cells were also stained for IE expression (c and d). (B) Cells were also analyzed by indirect immunofluorescence. U373 cells on eight-well compartment slides were infected overnight with AD169 (1 PFU/cell). Slides were fixed and then stained with a mouse monoclonal antibody to TNFRI or an IgG1 isotype-matched control antibody and detected using PE-conjugated sheep anti-mouse immunoglobulins. Cells were then stained with a FITC-conjugated mouse anti-IE72/IE86 monoclonal antibody. A representative field of view from the infected cell well that contains both infected and uninfected cell types is shown. IE staining is shown in panel a, TNFRI staining is shown in panel b, and the merged images are shown in panel c. Control immunoglobulins showed no specific cross-staining (data not shown). (C) U373 cells on eight-well compartment slides were infected overnight with GFP-tagged HCMV (1 PFU/cell). Slides were fixed and then stained with a mouse monoclonal antibody to TNFRI or an IgG1 isotype-matched control antibody,and antibodies were detected using AMCA-conjugated rabbit anti-mouse immunoglobulins. Cells were then stained with an anti-TGN46 antibody which was detected using Alexafluor 56-conjugated donkey anti-sheep immunoglobulins. A representative field of view from the infected cell well that contains both an infected and uninfected cell type is shown. GFP-IE staining (a), TNFRI staining (b), and TGN46 staining (c) are shown. Control immunoglobulins showed no specific cross-staining (data not shown).