Fig. 4.

Inhibitory activity of griffithsin at early stages of MERS-CoV infection. (A) Schematic of experimental time course. Infection of Huh-7 cells was divided into four steps: the initial viral inoculation (attachment) step was performed at 4 °C for 1 h in presence (conditions b and e) or absence (PBS in a, c, and d) of 1 μg/mL griffithsin, using an m.o.i. of 10. During the second step, the viral inoculum was removed and cells were washed three times with cold PBS and kept at 4 °C for 1 h, enabling further binding of virus to cell surfaces, in presence (c and e) or absence (a, b, and d) of 1 μg/mL griffithsin. For the third step, the cells were placed at 37 °C for 1 h allowing for internalization of virions to occur, in presence (d and e) or absence (a, b, and c) of 1 μg/mL griffithsin. The infection was left to proceed in a last 8 h step at 37 °C in absence griffithsin. (B) Immunofluorescence assay of MERS-CoV-infected Huh-7 cells with griffithsin present at different stages of infection. Cells were infected and treated as described in (A) then fixed, immunolabeled and analyzed as described previously in Fig. 2. (C) Quantification of MERS-CoV-positive Huh-7 cells for each condition tested. Cells were analyzed as described in Fig. 2. Results are expressed as percentage infected cells, with error bars representing SD from the average of three independent experiments. Data were statistically analyzed using two-tailed Student's t-test comparing results with non-treated condition (a) with the following convention for p-value significance: not significant (n.s.), p > 0.05; very highly significant (***), p ≤ 0.001.