A fluorescence-based screening assay for identification of hepatitis C virus NS3 helicase inhibitors and characterization of their inhibitory mechanism

Atsushi Furuta, Kazi Abdus Salam, Hidenori Tani, Satoshi Tsuneda, Yuji Sekiguchi, Nobuyoshi Akimitsu, Naohiro Noda

Research output: Contribution to journalArticle

Abstract

Hepatitis C virus (HCV) can establish a chronic infection in the majority of individuals infected, resulting in liver cirrhosis and hepatocellular carcinoma. Because the current standard treatment for HCV infection has limitations in terms of severe side effects, the emergence of drug resistance, and drug–drug interactions, it is desirable to develop novel antivirals that target viral proteins involved in viral replication. HCV nonstructural protein 3 (NS3) helicase, which unwinds double-stranded nucleic acids to yield single-stranded nucleic acids, is one possible target for new drug development, because it plays an essential role in viral replication. In this chapter, we describe a helicase assay based on fluorescence resonance energy transfer (FRET) that can be used for high-throughput screening of HCV NS3 helicase inhibitors. The assay uses a double-stranded RNA (dsRNA) substrate with a fluorophore-labeled strand hybridized to a quencher-labeled strand and monitors the increase in fluorescence intensity resulting from helicase-catalyzed unwinding of the dsRNA substrate. We further describe radioactive assays to directly visualize RNA strands unwound by helicase and to evaluate the ATPase and RNA-binding activities of NS3, which are linked to helicase activity, for characterization of the inhibitory mechanism.

Original languageEnglish
Pages (from-to)211-228
Number of pages18
JournalMethods in Molecular Biology
Volume1259
DOIs
Publication statusPublished - 2015
Externally publishedYes

Keywords

  • Fluorescence assay
  • Fluorescence resonance energy transfer (FRET)
  • Hepatitis C virus
  • High-throughput screening assay
  • Inhibitor
  • Inhibitory mechanism
  • NS3 helicase
  • Radioactive assay

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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