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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 NodaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

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.

Key words

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

Notes

Acknowledgements

The authors thank Dr. J. Tanaka (University of the Ryukyus) for his kind gift of the HCV NS3 inhibitors and Dr. S. Nishikawa (AIST) for providing the expression vector of full length HCV-NS3. The Global COE Program “Center for Practical Chemical Wisdom” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan partially supported this work.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Atsushi Furuta
    • 1
    • 2
  • Kazi Abdus Salam
    • 3
    • 4
  • Hidenori Tani
    • 5
  • Satoshi Tsuneda
    • 1
  • Yuji Sekiguchi
    • 2
  • Nobuyoshi Akimitsu
    • 3
  • Naohiro Noda
    • 1
    • 2
    Email author
  1. 1.Department of Life Science and Medical BioscienceWaseda UniversityTokyoJapan
  2. 2.Biomedical Research InstituteNational Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan
  3. 3.Radioisotope CenterThe University of TokyoTokyoJapan
  4. 4.Department of Biochemistry and Molecular BiologyUniversity of RajshahiRajshahiBangladesh
  5. 5.Research Institute for Environmental Management TechnologyNational Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan

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