Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system

• Published in: Antiviral research Vol. 67; no. 2; pp. 76 - 82
• Main Authors: Bourne, Nigel, Pyles, Richard B., Yi, MinKyung, Veselenak, Ronald L., Davis, Melissa M., Lemon, Stanley M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01-08-2005; Elsevier
• Subjects: Alkaline phosphatase; Alkaline Phosphatase - genetics; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antiviral activity; Antiviral agents; Antiviral Agents - pharmacology; Biological and medical sciences; Cell Line; Drug Evaluation, Preclinical; Genes, Reporter; Hepacivirus - drug effects; Hepacivirus - physiology; Hepatitis C virus; Human immunodeficiency virus; Human viral diseases; Infectious diseases; Medical sciences; Microbial Sensitivity Tests; Pharmacology. Drug treatments; Replicon - drug effects; RNA, Viral - biosynthesis; Viral diseases; Viral hepatitis; Virus Replication - drug effects; Hepatitis C virus; Alkaline phosphatase; Antiviral activity; Enzyme; Secretion; Phosphoric monoester hydrolases; Hepatic disease; Esterases; Medical screening; In vitro; Infection; Virus; Screening; Viral disease; Digestive diseases; Hydrolases; Antiviral; Flaviviridae; Hepacivirus; Viral hepatitis C
• ISSN: 0166-3542; 1872-9096
• DOI: 10.1016/j.antiviral.2005.03.006

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.