Biosensor assay detecting MERS-3CLpro activity in cell culture. (A) Diagram depicting the circularly permuted luciferase construct linked by the protease cleavage site VRLQS for assessing CoV 3CLpro activity. (B) Expression of MERS-pp3CLpro activates the biosensor. HEK293T cells were cotransfected with pGlo-VRLQS and pMERS-pp3CLpro expressing either wild-type or catalytic-mutant (CA) 3CLpro. At 20 h after transfection, cells were lysed and assayed for luciferase activity. The experiment was performed in triplicate, with error bars representing the standard deviations of the means. (C) MERS-CoV 3CLpro activity is detected in a live-cell assay. Cells were transfected as described above, and at 14 h, they were incubated with GloSensor reagent. Luciferase activity was assayed in live cells every hour using a luminometer. The experiment was performed in triplicate, with error bars representing the standard deviations of the means.