title: Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system.
abstract: We describe a phased screening system for discovery of compounds with antiviral activity against hepatitis C virus (HCV). The primary assay utilizes dicistronic subgenomic HCV replicons in which the upstream cistron was modified to express the human immunodeficiency virus (HIV) tat protein. When these replicons are stably transfected into Huh-7-derived cells that express secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV long terminal repeat promoter, there is a strong correlation between intracellular HCV RNA abundance and the activity of SEAP secreted into the culture medium. Thus, active compounds are easily identified by direct enzymatic quantification of SEAP in the medium without post-assay processing. Compounds that reduce SEAP activity without causing cellular toxicity are next evaluated in a second Huh-7-derived cell line constitutively expressing SEAP under control of the tat-HIV promoter axis, independent of HCV RNA replication. This specificity control identifies compounds that cause reductions in SEAP that are unrelated to suppression of HCV RNA replication. Compounds showing HCV-specific activity in primary assays are next evaluated by real-time RT-PCR to directly quantify reductions in HCV RNA. We have found excellent agreement between the SEAP and RT-PCR assays. This phased system provides an efficient and cost-effective screen for compounds with antiviral activity against HCV.
authors.affiliation: Department of Pediatrics, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0436, USA. nibourne@utmb.edu
authors.forename: Nigel
authors.initials: N
authors.lastname: Bourne
authors.forename: Richard B
authors.initials: RB
authors.lastname: Pyles
authors.forename: MinKyung
authors.initials: M
authors.lastname: Yi
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Veselenak
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Davis
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Lemon
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D017930:Genes, Reporter; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology; D008826:Microbial Sensitivity Tests; D012367:RNA, Viral / Q000096:biosynthesis; D012093:Replicon / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D000469:Alkaline Phosphatase