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

Nigel Bourne, Richard Pyles, Min Kyung Yi, Ronald L. Veselenak, Melissa M. Davis, Stanley M. Lemon

Research output: Contribution to journalArticle

32 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)76-82
Number of pages7
JournalAntiviral Research
Volume67
Issue number2
DOIs
StatePublished - Aug 2005

Fingerprint

Replicon
Hepacivirus
Antiviral Agents
Alkaline Phosphatase
RNA
Virus Replication
HIV Long Terminal Repeat
tat Gene Products
Human Immunodeficiency Virus Proteins
Culture Media
Real-Time Polymerase Chain Reaction
HIV
Costs and Cost Analysis
Cell Line
Polymerase Chain Reaction

Keywords

  • Alkaline phosphatase
  • Antiviral activity
  • Hepatitis C virus

ASJC Scopus subject areas

  • Virology
  • Pharmacology

Cite this

Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system. / Bourne, Nigel; Pyles, Richard; Yi, Min Kyung; Veselenak, Ronald L.; Davis, Melissa M.; Lemon, Stanley M.

In: Antiviral Research, Vol. 67, No. 2, 08.2005, p. 76-82.

Research output: Contribution to journalArticle

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