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.
Lawler
J F
JF
Departments of Neuroscience, Pharmacology and Molecular Sciences, and Psychiatry, Johns Hopkins University School of Medicine, 725 N., Wolfe Street, Baltimore, Maryland 21205-2185, USA.
Snyder
S H
SH
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000378:metabolism* / Q000502:physiology; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000032:analysis; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism* / Q000502:physiology
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011480:Protease Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D008156:Luciferases; D012697:Serine Endopeptidases; C071910:assemblin