HCMV replication and virus production assays.

HFF were grown in white-bottom 96-well tissue culture plates to 90% confluence and inoculated with ADCREGFP virus at an MOI of 0.001 in DMEM containing 5% fetal calf serum for 2 h. The inoculated cells were washed with phosphate-buffered saline (PBS) and incubated with 100 μl of DMEM containing 5% fetal calf serum with test compounds or DMSO at 37°C and 5% CO₂ for 144 h. After 144 h, cell supernatants were collected for a yield reduction assay (described below), and the infected cells were lysed to measure GFP fluorescence as an indication of the extent of virus replication. For lysis, 200 μl of lysis buffer (25 mM Tris [pH 7.8], 2 mM dithiothreitol [DTT], 2 mM trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 1% Triton X-100, 10% glycerol) was added to each well, and the mixture was incubated for 10 min at 37°C, followed by a 30-min incubation at room temperature on a shaker. GFP relative fluorescence units were determined at an excitation/emission wavelength of 495/515 nm in a Molecular Devices M5e plate reader. For the yield reduction assay, HFF cells were inoculated for 2 h with the supernatants from the antiviral assay. The supernatants were removed and replaced by culture medium. After 144 h, cells were washed with PBS and lysed. GFP units were determined at an excitation/emission wavelength of 495/515 nm.