Viral protease assay based on GAL4 inactivation is applicable to high-throughput screening in mammalian cells.

We present an assay for viral proteases that relies on the proteolytic cleavage of substrate leading to the dissociation of the yeast transcription factor GAL4. A consensus substrate for the cytomegalovirus protease is fused between the DNA binding and transactivating domains of GAL4. Proteolysis inactivates the transcription factor which drives a luciferase reporter system. The assay is performed in mammalian cells, has a robust signal-to-noise ratio, and assesses proteolysis in a physiologic context. A unique feature of the assay is its ability to detect inhibitors of viral replication that act on viral targets other than the protease.