Figure 4.

Compound 5d inhibits syncytium formation mediated by herpes simplex virus (HSV) surface glycoproteins. (a, b) HSV‐1‐infected or HSV‐2‐infected (multiplicity of infection = 3.0) Vero cells were treated with or without compound 5d (5, 10, 20, and 40 μM) after virus adsorption. At 16 hr p.i., the cells were fixed and then stained with haematoxylin–eosin solution. The inhibition of syncytium formation of (a) HSV‐1 and (b) HSV‐2 was observed using light microscopy. Bar represents 50 μm. (c, d) Plots quantifying syncytium formation in (c) HSV‐1‐ and (d) HSV‐2‐infected cells with different treatments. The relative areas of the syncytium in the different fields were measured using ImageJ (NIH) v.1.33u (USA), and the relative syncytium percentages were determined relative to the non‐treated virus control group. The data shown represent the results of five independent experiments. (e) HSV‐2 (multiplicity of infection = 3.0)‐infected Vero cells were added with compound 3a, 5a, or 5d (20 μM) after adsorption and incubated at 37°C for 16 hr. Then the cells were fixed and stained with haematoxylin–eosin solution. Bar represents 50 μm. (f) Drug affinity responsive target stability assay analysis for 5d. HSV‐2 infected Vero cell lysates were incubated with or without compound 5d (60 μM) for 1 hr and then digested with pronase (1, 2.5, and 5 μg·ml⁻¹) for 30 min. Protein samples were separated by SDS‐PAGE and immunoblotted with the indicated antibodies. The results shown are representative of five independent experiments. *P < 0.05, significantly different from virus control group