Fig 8.

Triple depletion of HFs increases wt HCMV replication and gene expression. (A) HCMV AD169 replication in depleted and nondepleted HFs. Cells were infected with HCMV AD169 at an MOI of 0.1, supernatants were harvested at 9 days postinfection, and relative virus titers were determined by standard plaque assay on HFs. (B) Kinetics of viral IE protein expression in depleted and nondepleted cells. Cells were infected with HCMV AD169 at an MOI of 0.5, and cell lysates were sampled at the indicated time points. Samples were resolved on a 7.5% polyacrylamide gel, and membranes were probed using anti-IE1/2 MAb E13. The loading control was antiactin A5060 antibody (Sigma-Aldrich). (C) Immunofluorescence analysis of the proportions of cells expressing IE1. Cells were infected with HCMV AD169 at an MOI of 0.01, fixed at 48 h postinfection, and simultaneously stained with mouse anti-IE1/2 MAb E13 and rabbit anti-PML antibodies. Secondary antibodies were Alexa Fluor 488 donkey anti-mouse and Alexa Fluor 647 goat anti-rabbit antibodies. PML served as a marker for depletion. Bar, 100 μm.