Abstract
Aim:
To develop a sensitive assay for screening compounds against hepatitis C virus (HCV).
Methods:
The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP), which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence. Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy. Cellular localization and protein levels were examined by Western blotting.
Results:
HCV NS3/4A protease cleaved eYFP-MAVS from mitochondria to block the activation of interferon (IFN)-β promoter, thus resulting in downregulation of SEAP activity. The decrease in SEAP activity was proportional to the dose of active NS3/4A protease. Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A.
Conclusion:
Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors. This system will constitute a new tool to allow the efficient screening of HCV inhibitors.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing / genetics
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Adaptor Proteins, Signal Transducing / metabolism*
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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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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Biosensing Techniques*
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Blotting, Western
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Cell Line, Tumor
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Cyclosporine / pharmacology
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Dose-Response Relationship, Drug
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Drug Evaluation, Preclinical
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GPI-Linked Proteins / genetics
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GPI-Linked Proteins / metabolism
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Genes, Reporter
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Hepacivirus / drug effects*
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Hepacivirus / enzymology
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Hepacivirus / genetics
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Humans
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Interferon-alpha / pharmacology
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Interferon-beta / genetics*
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Isoenzymes / genetics
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Isoenzymes / metabolism
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Microscopy, Fluorescence
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Promoter Regions, Genetic
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Recombinant Fusion Proteins / metabolism
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Replicon
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Signal Transduction / drug effects*
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Time Factors
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Transcriptional Activation
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Transfection
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Viral Nonstructural Proteins / genetics
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Viral Nonstructural Proteins / metabolism*
Substances
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Adaptor Proteins, Signal Transducing
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Antiviral Agents
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Bacterial Proteins
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GPI-Linked Proteins
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Interferon-alpha
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Isoenzymes
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Luminescent Proteins
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NS3 protein, hepatitis C virus
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Recombinant Fusion Proteins
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Viral Nonstructural Proteins
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yellow fluorescent protein, Bacteria
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Interferon-beta
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Cyclosporine
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Alkaline Phosphatase
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alkaline phosphatase, placental