Abstract
Hepatitis C virus (HCV) encodes a viral protease, nonstructural (NS)3/4A, that is critical for virus maturation. Although NS3/4A has emerged as a promising target for anti-HCV drug discovery, no anti-HCV therapy has succeeded yet based on inhibition of NS3/4A. We have previously shown that EG(delta4AB)SEAP, a reporter consisting of enhanced green fluorescent protein (EG), the NS3-NS4A protease decapeptide recognition sequence (delta4AB), and secreted alkaline phosphatase (SEAP), is an efficient reporter for reflecting NS3/4A proteolytic activity inside cells. In this study, we describe the generation and characterization of a stable cell line, 293EEG(delta4AB)SEAP-NS3/4A, which constitutively expresses EG(delta4AB)SEAP reporter protein and NS3/4A protease. The reporter assay is validated with the compound BILN 2061, a specific and potent peptidomimetic inhibitor of the HCV NS3 protease. Additionally, we show here that this cell line allows screening for NS3/4A protease activity of living cells in 96-well plate format, with a Z factor >0.6. Thus, this cell-based assay may be used for high-throughput screening of chemical libraries.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Alkaline Phosphatase / genetics
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Alkaline Phosphatase / metabolism
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Antiviral Agents / pharmacology
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Carbamates / pharmacology
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Carrier Proteins / antagonists & inhibitors*
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Cell Line
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Dose-Response Relationship, Drug
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Drug Evaluation, Preclinical / methods
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Genes, Reporter
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Green Fluorescent Proteins / genetics
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Hepacivirus / drug effects*
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Hepacivirus / growth & development
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Humans
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Intracellular Signaling Peptides and Proteins
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Macrocyclic Compounds / pharmacology
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Plasmids
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Quinolines / pharmacology
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Recombinant Fusion Proteins / metabolism
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Scintillation Counting / methods
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Serine Proteinase Inhibitors / analysis
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Serine Proteinase Inhibitors / pharmacology*
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Thiazoles / pharmacology
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Transfection
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Viral Nonstructural Proteins / antagonists & inhibitors*
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Viral Nonstructural Proteins / genetics
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Viral Nonstructural Proteins / metabolism
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Viral Proteins / antagonists & inhibitors*
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Viral Proteins / genetics
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Viral Proteins / metabolism
Substances
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Antiviral Agents
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BILN 2061
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Carbamates
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Carrier Proteins
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Intracellular Signaling Peptides and Proteins
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Macrocyclic Compounds
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NS3 protein, hepatitis C virus
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NS4A cofactor peptide, Hepatitis C virus
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Quinolines
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Recombinant Fusion Proteins
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Serine Proteinase Inhibitors
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Thiazoles
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Viral Nonstructural Proteins
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Viral Proteins
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enhanced green fluorescent protein
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Green Fluorescent Proteins
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Alkaline Phosphatase