title: Development of a cell-based high-throughput specificity screen using a hepatitis C virus-bovine viral diarrhea virus dual replicon assay.
abstract: The hepatitis C virus (HCV) replicon is a unique system for the development of a high-throughput screen (HTS), since the analysis of inhibitors requires the quantification of a decrease in a steady-state level of HCV RNA. HCV replicon replication is dependent on host cell factors, and any toxic effects may have a significant impact on HCV replicon replication. Therefore, determining the antiviral specificity of compounds presents a challenge for the identification of specific HCV inhibitors. Here we report the development of an HCV/bovine viral diarrhea virus (BVDV) dual replicon assay suitable for HTS to address these issues. The HCV reporter enzyme is the endogenous NS3 protease contained within the HCV genome, while the BVDV reporter enzyme is a luciferase enzyme engineered into the BVDV genome. The HTS uses a mixture of HCV and BVDV replicon cell lines placed in the same well of a 96-well plate and isolated in the same cell backgrounds (Huh-7). The format consists of three separate but compatible assays: the first quantitates the amount of cytotoxicity based upon the conversion of Alamar blue dye via cellular enzymes, while the second indirectly quantitates HCV replicon replication through measurement of the amount of NS3 protease activity present. The final assay measures the amount of luciferase activity present from the BVDV replicon cells, as an indicator of the specificity of the test compounds. This HCV/BVDV dual replicon assay provides a reliable format to determine the potency and specificity of HCV replicon inhibitors.
authors.affiliation: Department of Virology, Bristol Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492-7660, USA. Min.
authors.forename: Donald R
authors.initials: DR
authors.lastname: O'Boyle
authors.forename: Peter T
authors.initials: PT
authors.lastname: Nower
authors.forename: Julie A
authors.initials: JA
authors.lastname: Lemm
authors.forename: Lourdes
authors.initials: L
authors.lastname: Valera
authors.forename: Jin-Hua
authors.initials: JH
authors.lastname: Sun
authors.forename: Karen
authors.initials: K
authors.lastname: Rigat
authors.forename: Richard
authors.initials: R
authors.lastname: Colonno
authors.forename: Min
authors.initials: M
authors.lastname: Gao
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D001908:Diarrhea Viruses, Bovine Viral / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D012093:Replicon / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication; D014966:Xanthenes / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D008156:Luciferases; D010078:Oxazines; D017361:Viral Nonstructural Proteins; D014966:Xanthenes; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C005843:resazurin