2.5. Time-Of-Addition Assay

One day prior to infection, Huh-7 cells (5 × 10⁴ cells/well) were seeded in a 12-well tissue culture plate. The next day, cells were infected with MERS-CoV at an MOI 0.02 for 1 h at 37 °C. After 1 h, the unbound virus was removed by washing with phosphate-buffered saline (PBS, pH 7.4). Compounds were added to cells at specific time points during virus infection as follows: pre, 1 h prior to virus infection; co-treatment during virus infection; post, 1 h and 4 h after virus infection. The antiviral activity of the compound was analyzed after 24 h p.i. by measuring the number of infectious viral particles and intracellular viral RNA expression levels using plaque assay and RT-qPCR, respectively.

In addition, the time-of-addition experiment combined with temperature-shifting assay was performed. Briefly, Huh-7 cells were inoculated with MERS-CoV at 4 °C for 1 h (attachment/binding). The unadsorbed virus was removed by washing three times with ice-cold PBS and replenished with fresh culture medium. Subsequently, plates were shifted to 37 °C to allow synchronous entry and infection. Saracatinib was treated during the 4 °C incubation only or added at specific time points during the 37 °C incubation. The antiviral activity of the compound was analyzed after 24 h p.i. by measuring the number of infectious viral particles and intracellular viral RNA expression levels using plaque assay and RT-qPCR, respectively.