Human cytomegalovirus (HCMV, strain AD169) was prepared as described previously [15 (link)]. For virus production and measurement of viral genomic DNA, HFF were grown to a confluent monolayer and infected with HCMV at a multiplicity of infection (MOI) of 0.01 to 0.05. After 1.5 h, the viral inoculum was replaced by fresh growth medium. The viral supernatant was harvested upon visibility of a total cytopathic effect, usually 8–10 days post infection, and stored at −80 xB0;C. After thawing, cellular material was removed by centrifugation at 3000× g for 20 min. Infectivity of the viral inoculum was then measured using a plaque assay [16 (link)]. HCMV strain TS15-rN (kindly provided by Sallie R. Permar) was used to infect ARPE-19 cells.
For infection of cell cultures with HCMV AD169, cells were seeded into 6-well plates overnight, and inoculated at MOI 1 (if not indicated otherwise) for 1 h at 37 °C. After viral adsorption, the cell layer was rinsed and fresh growth medium including 10% FBS was added to the cells. In LL-37 treatment experiments (see below), growth medium containing 2% FBS was resupplied after inoculation. For ganciclovir-treated control wells, ganciclovir (Merck Millipore, Burlington, MA, USA) was added to the growth media at a concentration of 10 μg/mL after removal of the inoculum.
For infection of cell cultures with HCMV AD169, cells were seeded into 6-well plates overnight, and inoculated at MOI 1 (if not indicated otherwise) for 1 h at 37 °C. After viral adsorption, the cell layer was rinsed and fresh growth medium including 10% FBS was added to the cells. In LL-37 treatment experiments (see below), growth medium containing 2% FBS was resupplied after inoculation. For ganciclovir-treated control wells, ganciclovir (Merck Millipore, Burlington, MA, USA) was added to the growth media at a concentration of 10 μg/mL after removal of the inoculum.