Bioluminescence technologies to detect calicivirus protease activity in cell-free system and in infected cells

Feline calicivirus (FCV) is an important veterinary pathogen and causes respiratory disease in cats. Because it grows well in cell culture, FCV is often used as a model virus of non-culturable caliciviruses. In this study, a cell-free and two cell culture-based biosensor assay systems were established to detect FCV protease activity. The assays utilize luciferase sensor technology or second-generation bioluminescence resonance energy transfer (BRET(2)). A luciferase sensor was designed to contain an FCV protease cleavage motif within the permutated luciferase (GloSensor). The BRET(2)-based probe contained the same cleavage motif flanked by a renilla luciferase and a variant of green fluorescent protein. To confirm the specificity of these assay systems, GloSensor or a BRET(2)-based probe containing a mutation in the cleavage motif was also constructed. In a cell-free assay, GloSensor showed increased luminescence in proportion to the amount of FCV protease, while no signal change was observed when the construct harboring the mutant cleavage motif was used. A feline cell line stably expressing GloSensor or the BRET(2)-based probe was established. Increased levels of GloSensor luminescence, and decreased levels of BRET(2) signals were observed according to input FCV titers. In contrast, no significant signal change was observed in the cells stably expressing the mutant cleavage motif. GloSensor and the BRET(2)-based probe were capable of detecting the inhibitory activity of ribavirin in FCV-infected cells. Our results demonstrate that these biosensors are useful to detect FCV protease activity induced in infected cells, and well worth consideration for screening of anti-FCV protease compounds in cell-free system as well as anti-FCV compounds in cultured cells.