Screening compounds against HCV based on MAVS/IFN-β pathway in a replicon model

World J Gastroenterol. 2010 Nov 28;16(44):5582-7. doi: 10.3748/wjg.v16.i44.5582.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Alkaline Phosphatase / genetics
  • Alkaline Phosphatase / metabolism
  • Antiviral Agents / pharmacology*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Biosensing Techniques*
  • Blotting, Western
  • Cell Line, Tumor
  • Cyclosporine / pharmacology
  • Dose-Response Relationship, Drug
  • Drug Evaluation, Preclinical
  • GPI-Linked Proteins / genetics
  • GPI-Linked Proteins / metabolism
  • Genes, Reporter
  • Hepacivirus / drug effects*
  • Hepacivirus / enzymology
  • Hepacivirus / genetics
  • Humans
  • Interferon-alpha / pharmacology
  • Interferon-beta / genetics*
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Fluorescence
  • Promoter Regions, Genetic
  • Recombinant Fusion Proteins / metabolism
  • Replicon
  • Signal Transduction / drug effects*
  • Time Factors
  • Transcriptional Activation
  • Transfection
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Antiviral Agents
  • Bacterial Proteins
  • GPI-Linked Proteins
  • Interferon-alpha
  • Isoenzymes
  • Luminescent Proteins
  • NS3 protein, hepatitis C virus
  • Recombinant Fusion Proteins
  • Viral Nonstructural Proteins
  • yellow fluorescent protein, Bacteria
  • Interferon-beta
  • Cyclosporine
  • Alkaline Phosphatase
  • alkaline phosphatase, placental