title: Screening compounds against HCV based on MAVS/IFN-β pathway in a replicon model.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Blood-Borne Virus, Beijing Institute of Transfusion Medicine, 27(9) Tai Ping Road, Beijing 100850, China.
authors.forename: Qiu-Xia
authors.initials: QX
authors.lastname: Fu
authors.forename: Li-Cui
authors.initials: LC
authors.lastname: Wang
authors.forename: Shuai-Zheng
authors.initials: SZ
authors.lastname: Jia
authors.forename: Bo
authors.initials: B
authors.lastname: Gao
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhou
authors.forename: Juan
authors.initials: J
authors.lastname: Du
authors.forename: Ying-Li
authors.initials: YL
authors.lastname: Wang
authors.forename: Xiao-Hui
authors.initials: XH
authors.lastname: Wang
authors.forename: Jian-Chun
authors.initials: JC
authors.lastname: Peng
authors.forename: Lin-Sheng
authors.initials: LS
authors.lastname: Zhan
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000998:Antiviral Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques; D015153:Blotting, Western; D045744:Cell Line, Tumor; D016572:Cyclosporine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D016899:Interferon-beta / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012093:Replicon; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000469:Alkaline Phosphatase; D000998:Antiviral Agents; D001426:Bacterial Proteins; D016572:Cyclosporine; D058851:GPI-Linked Proteins; D016898:Interferon-alpha; D016899:Interferon-beta; D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C054562:yellow fluorescent protein, Bacteria; C019839:alkaline phosphatase, placental