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.
Hepatitis C virus NS3 helicase Fluorescence assay High-throughput screening assay Fluorescence resonance energy transfer (FRET) Inhibitor Radioactive assay Inhibitory mechanism
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
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.
Ghany MG, Nelson DR, Strader DB et al (2011) An update on treatment of genotype 1 chronic hepatitis C virus infection: 2011 practice guideline by the American Association for the Study of Liver Diseases. Hepatology 54:1433–1444PubMedCentralPubMedCrossRefGoogle Scholar
Kwong AD, Rao BG, Jeang K-T (2005) Viral and cellular RNA helicases as antiviral targets. Nat Rev Drug Discov 4:845–853PubMedCrossRefGoogle Scholar
Kim DW, Gwack Y, Han JH et al (1995) C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem Biophys Res Commun 215:160–166PubMedCrossRefGoogle Scholar
Tai CL, Chi WK, Chen DS et al (1996) The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). J Virol 70:8477–8484PubMedCentralPubMedGoogle Scholar
Gwack Y, Kim DW, Han JH et al (1996) Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein. Biochem Biophys Res Commun 225:654–659PubMedCrossRefGoogle Scholar
Gwack Y, Kim DW, Han JH et al (1997) DNA helicase activity of the hepatitis C virus nonstructural protein 3. Eur J Biochem 250:47–54PubMedCrossRefGoogle Scholar
Zhang C, Cai Z, Kim Y-C et al (2005) Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase. J Virol 79:8687–8697PubMedCentralPubMedCrossRefGoogle Scholar
Bjornson KP, Amaratunga M, Moore KJM et al (1994) Single-turnover kinetics of helicase-catalyzed DNA unwinding monitored continuously by fluorescence energy transfer. Biochemistry 33:14306–14316PubMedCrossRefGoogle Scholar
Boguszewska-Chachulska AM, Krawczyk M, Stankiewicz A et al (2004) Direct fluorometric measurement of hepatitis C virus helicase activity. FEBS Lett 567:253–258PubMedCrossRefGoogle Scholar
Tani H, Fujita O, Furuta A et al (2010) Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro. Biochem Biophys Res Commun 393:131–136PubMedCrossRefGoogle Scholar
Salam KA, Furuta A, Noda N et al (2012) Inhibition of hepatitis C virus NS3 helicase by manoalide. J Nat Prod 75:650–654PubMedCrossRefGoogle Scholar
Furuta A, Salam KA, Akimitsu N et al (2014) Cholesterol sulfate as a potential inhibitor of hepatitis C virus NS3 helicase. J Enzyme Inhib Med Chem 29:223–229PubMedCrossRefGoogle Scholar
Fujimoto Y, Salam KA, Furuta A et al (2012) Inhibition of both protease and helicase activities of hepatitis C virus NS3 by an ethyl acetate extract of marine sponge Amphimedon sp. PLoS One 7:e48685PubMedCentralPubMedCrossRefGoogle Scholar
Yamashita A, Salam KA, Furuta A et al (2012) Inhibition of hepatitis C virus replication and viral helicase by ethyl acetate extract of the marine feather star Alloeocomatella polycladia. Mar Drugs 10:744–761PubMedCentralPubMedCrossRefGoogle Scholar
Salam KA, Furuta A, Noda N et al (2013) Psammaplin A inhibits hepatitis C virus NS3 helicase. J Nat Med 67:765–772PubMedCrossRefGoogle Scholar
Furuta A, Salam KA, Hermawan I et al (2014) Identification and biochemical characterization of halisulfate 3 and suvanine as novel inhibitors of hepatitis C virus NS3 helicase from a marine sponge. Mar Drugs 12:462–476PubMedCentralPubMedCrossRefGoogle Scholar
Tani H, Akimitsu N, Fujita O et al (2009) High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon. Biochem Biophys Res Commun 379:1054–1059PubMedCrossRefGoogle Scholar
Nishikawa F, Funaji K, Fukuda K et al (2004) In vitro selection of RNA aptamers against the HCV NS3 helicase domain. Oligonucleotides 14:114–129PubMedCrossRefGoogle Scholar
Copeland RA (2005) Evaluation of enzyme inhibitors in drug discovery. Wiley, New YorkGoogle Scholar