FIG. 5.

Validated HCMV protease inhibitors BI31 (upper panel) and BI36 (lower panel) stimulate cell proliferation in a concentration-dependent manner. (A) RLY07 cells expressing Trp1¹⁹⁴-Me and active HCMV protease (triangles) or Trp1¹⁹⁴-Me and inactive protease (squares) were cultivated either in the absence (0) or presence of increasing concentrations of BI31 and BI36. Cells were grown in 150-μl cultures in a 96-well microplate for 44 h (BI31) and 28 h (BI36) before OD₅₉₅ was measured. Data are expressed as means from three independent experiments ± standard deviations. (B) Western blot analysis with HA antibody showing that application of BI36 prevents cleavage of the Trp1¹⁹⁴-Me substrate by HCMV protease. RLY07 cells were transformed with Trp1¹⁹⁴-Me and inactive protease (positive control), Trp1¹⁹⁴-Me and active protease, or Trp1¹⁹⁴-Me with active protease in the presence of distinct concentrations of BI36, as indicated. RLY07 cells containing two empty plasmids were used as a negative control. The calmodulin antibody served as an internal control for protein amount. DMSO, dimethyl sulfoxide.