Evaluation of Compound Activity Against Hepatitis C Virus in Replicon Systems

Guangwei Yang1, Mingjun Huang1

1 Achillion Pharmaceuticals, New Haven, Connecticut
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13B.1
DOI:  10.1002/0471141755.ph13b01s50
Online Posting Date:  September, 2010
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Abstract

Described in this unit are protocols for evaluation of new chemical entities for activity against hepatitis C virus (HCV) using HCV replicon systems. While agents designed to target NS3 protease, NS3 helicase, and NS5B RNA polymerase can be evaluated in biochemical assays, it is important to confirm their inhibitory effect on HCV RNA replication using HCV replicon systems, especially since replication involves many components besides these enzymes. Screening of compound libraries using replicon systems has led to the discovery of replication inhibitors, which act through different viral targets. Curr. Protoc. Pharmacol. 50:13B.1.1‐13B.1.21. © 2010 by John Wiley & Sons, Inc.

Keywords: HCV; replicon; inhibitor

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Evaluation of Compound Anti‐HCV Activity by Indirect Measurement of a Reporter
  • Alternate Protocol 1: Alternative Compound Dilution Schemes
  • Basic Protocol 2: Evaluation of Compound Anti‐HCV Activity in Transiently Transfected Replicons
  • Alternate Protocol 2: Evaluation of Compound Anti‐HCV Activity by Direct Measurement of Replicon RNA Using Dot‐Blot Method
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Evaluation of Compound Anti‐HCV Activity by Indirect Measurement of a Reporter

  Materials
  • Huh‐Luc/neo cell line licensed from ReBLikon GmbH
  • Maintenance medium (see recipe)
  • Assay medium (see recipe)
  • Phosphate‐buffered saline (PBS; Cellgro, cat. no. 21‐030‐CM)
  • Trypsin/EDTA (Cellgro, cat. no. 25‐053‐CI)
  • Compound stock solutions (typically prepared in DMSO)
  • CellTiter 96 AQ ueous One Solution (Promega, cat. no. G3581)
  • Bright‐Glo Luciferase Assay System (Promega, cat. no. E2620) or other appropriate luciferase assay system
  • 75‐cm2 flasks
  • 37°C, 5% CO 2 incubator
  • Multichannel pipets
  • Single‐channel pipets
  • Pipet tips
  • 50‐ml Falcon tubes, sterile
  • Centrifuge
  • 96‐well flat‐bottom tissue culture plates
  • 1.2‐ml microtiter tubes and boxes (for compound dilution; Corning, cat. no. 4408)
  • Vmax plate reader (Molecular Devices)
  • Paper towels
  • 96‐well opaque white plates
  • 1450 Microbeta Liquid Scintillation & Luminescence Counter (Perkin Elmer)
  • Prism software (GraphPad Software)
  • Additional reagents and equipment for counting cells (Phelan, )
NOTE: All equipment and solutions coming into contact with cells must be sterile, with proper sterile technique used throughout the procedure.

Alternate Protocol 1: Alternative Compound Dilution Schemes

  Materials
  • Compound stock solutions
  • Dimethyl sulfoxide (DMSO)
  • Assay medium (see recipe)
  • 96‐well plates (V bottom for compound dilution)
  • Multichannel pipets
  • Single‐channel pipets
  • Pipet tips
  • 96‐well tissue culture plates (U bottom for compound dilution)
NOTE: All equipment and solutions coming into contact with cells must be sterile and proper sterile technique should be when executing this protocol.

Basic Protocol 2: Evaluation of Compound Anti‐HCV Activity in Transiently Transfected Replicons

  Materials
  • pFK I341 PI‐Luc/NS3‐3′/ET (a plasmid serving as a template for in vitro synthesis of the FK I341 PI‐Luc/NS3‐3′/ET replicon RNA) obtained from ReBLikon GmbH
  • ScaI restriction enzyme and its buffer for plasmid linearization (NEB, cat. no. R0122S)
  • RNase, DNase‐free distilled H 2O
  • 1% agarose gel
  • PCR purification kit (Qiagen, cat. no. 28106)
  • TE buffer, pH 7.6 (Quality Biological, cat. no. 351‐010‐060)
  • MEGAscript T7 high yield transcription kit (Ambion, cat. no. AM1333) containing:
    • ATP solution
    • CTP solution
    • GTP solution
    • UTP solution
    • 10× reaction buffer
    • Eznyme mix
    • Nuclease‐free water
    • 5 M ammonium acetate
  • DNase I
  • Phenol/chloroform saturated solution, pH 6.7 (MP Biomedicals, cat. no. 802520)
  • Chloroform
  • Isopropanol
  • 70% (v/v) ethanol
  • Huh7‐Lunet cells licensed from ReBLikon GmbH (Mainz, Germany)
  • Growth medium (see recipe)
  • Phosphate‐buffered saline (PBS; Cellgro, cat. no. 21‐030‐CM)
  • Trypsin‐EDTA (Cellgro, cat. no. 25‐053‐CI)
  • Electroporation solution: just before use add ATP stock and glutathione stock to Cytomix (see recipe) to make a final concentration of 2 mM ATP and 5 mM glutathione
  • 200 mM ATP stock (Sigma, cat. no. A‐2383): adjust pH to 7.6 with KOH, sterilize by filtration, and store in aliquots up to 1 year at –20°C
  • 500 mM glutathione stock, reduced (Aldrich, cat. no. G‐4705): adjust pH to 7.6 with KOH, sterilize by filtration and store in aliquots up to 1 year at –20°C
  • Microcentrifuge tubes, sterile
  • Multichannel channel pipets
  • Single‐channel pipets
  • Pipet tips
  • Microcentrifuge
  • 37°C incubator
  • 50‐ml Falcon tubes
  • Electroporation cuvette with 0.4‐cm gap (Molecular BioProducts, cat. no. 5540)
  • Gene Pulser apparatus (Bio‐Rad)
  • 96‐well flat‐bottom tissue culture plate
  • Prism software (GraphPad Software)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ), determining DNA concentration (Gallagher and Desjardins, ), and counting cells using a hemacytometer (Phelan, )
NOTE: Unless indicated, all transfection steps are performed at room temperature.NOTE: All equipment and solutions coming into contact with cells must be sterile and proper sterile technique should be employed throughout this assay procedure.NOTE: During synthesis of RNA, common practices to avoid RNA degradation should be implemented (Kingston, ).NOTE: Although no particular requirements are noted, all materials used for electroporation should be separated from those used for the experiments where RNase is added.

Alternate Protocol 2: Evaluation of Compound Anti‐HCV Activity by Direct Measurement of Replicon RNA Using Dot‐Blot Method

  Materials
  • Stably transfected replicon cell line, e.g., Huh‐9‐13, (ReBLikon GmbH)
  • RNasin RNase Inhibitor (Promega, cat. no. N2615)
  • 1 M Dithiothreitol (DTT)
  • RNL solution (see recipe)
  • Phosphate‐buffered saline (PBS; Cellgro, cat. no. 21‐030‐CM)
  • Plasmid HCV1b‐NS5.1
  • XhoI restriction enzyme and 10× restriction enzyme buffer no. 2 for plasmid linearization
  • SP6 RNA polymerase and 10× SP6 RNA polymerase buffer (NEB, cat. no. M0207S)
  • pHCV1b‐NS5.1 (a plasmid serving as a template for in vitro synthesis of a riboprobe)
  • Bovine serum albumin (BSA), 100×
  • RNase, DNase‐free distilled H 2O
  • DNA size markers
  • QIAquick PCR purification kit (Qiagen, cat. no. 28104)
  • α‐32P UTP (Perkin Elmer Life Sciences, cat. no. BLU007X)
  • 5× rNTP: 2.5 mM each ATP, GTP, CTP, and 0.1 mM UTP
  • tRNA carrier (Sigma‐Aldrich, cat. no. R5636)
  • RQ1 DNase (Promega, cat. no. M6101)
  • Stop buffer (see recipe)
  • Ethanol
  • Hybridization buffer (see recipe)
  • Salmon sperm DNA (Invitrogen, cat. no. 15632‐011)
  • Washing solution: 0.1× SSC and 0.1% SDS
  • Multichannel channel pipets
  • Single‐channel pipets
  • Pipet tips
  • 96‐well U‐bottom tissue culture plates (Matrix, cat. no. 4924)
  • Centrifuge with a plate holder
  • Nylon membrane (Whatman, Nytran SPC)
  • Filter paper
  • Hybridization equipment
  • 96‐well manifold system (Whatman, cat. no. 10447900)
  • UV cross‐linker
  • Screw‐cap microcentrifuge tubes, sterile
  • Microcentrifuge
  • 37°C heat block
  • 40°C incubator
  • Vortex
  • Laboratory survey meter
  • Nylon mesh
  • MicroBeta Liquid Scintillation and P‐32 Filtermat Cassette (Perkin Elmer, cat. no. 1450‐118)
  • Prism software (GraphPad Software)
NOTE: All equipment and solutions coming into contact with cells must be sterile and proper sterile technique should be employed throughout the assay.NOTE: During synthesis of RNA, common practices to avoid RNA degradation should be implemented (Gilman, ).NOTE: Precautions for handling the radioactive materials should also be applied (Meisenhelder and Bursik, ).
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Figures

Videos

Literature Cited

Literature Cited
   Blight, K.J., Kolykhalov, A.A., and Rice, C.M. 2000. Efficient initiation of HCV RNA replication in cell culture. Science 290:1972‐1974.
   Blight, K.J., McKeating, J.A., Marcotrigiano, J., and Rice, C.M. 2003. Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. J. Virol. 77:3181‐3190.
   Eldrup, A.B., Allerson, C.R., Bennett, C.F., Bera, S., Bhat, B., Bhat, N., Bosserman, M.R., Brooks, J., Burlein, C., Carroll, S.S., Cook, P.D., Getty, K.L., MacCoss, M., McMasters, D.R., Olsen, D.B., Prakash, T.P., Prhavc, M., Song, Q., Tomassini, J.E., and Xia, J. 2004. Structure‐activity relationship of purine ribonucleosides for inhibition of hepatitis C virus RNA‐dependent RNA polymerase. J. Med. Chem. 47:2283‐2295.
   Friebe, P., Boudet, J., Simorre, J.P., and Bartenschlager, R. 2005. Kissing‐loop interaction in the 3′ end of the hepatitis C virus genome essential for RNA replication. J. Virol. 79:380‐392.
   Gallagher, S.R. and Desjardins, P.R. 2006. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Curr. Protoc. Mol. Biol. 76:A.3D.1‐A.3D.21.
   Gilman, M. 2002. Preparation of cytoplasmic RNA from tissue culture cells. Curr. Protoc. Mol. Biol. 58:4.1.1‐4.1.5.
   Guo, J.T., Bichko, V.V., and Seeger, C. 2001. Effect of alpha interferon on the hepatitis C virus replicon. J. Virol. 75:8516‐8523.
   Hoofnagle, J.H. and Seeff, L.B. 2006. Peginterferon and ribavirin for chronic hepatitis C. N. Engl. J. Med. 355:2444‐2451.
   Ikeda, M., Yi, M., Li, K., and Lemon, S.M. 2002. Selectable subgenomic and genome‐length dicistronic RNAs derived from an infectious molecular clone of the HCV‐N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J. Virol. 76:2997‐3006.
   Kato, N., Sugiyama, K., Namba, K., Dansako, H., Nakamura, T., Takami, M., Naka, K., Nozaki, A., and Shimotohno, K. 2003a. Establishment of a hepatitis C virus subgenomic replicon derived from human hepatocytes infected in vitro. Biochem. Biophys. Res. Commun. 306:756‐766.
   Kato, T., Date, T., Miyamoto, M., Furusaka, A., Tokushige, K., Mizokami, M., and Wakita, T. 2003b. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125:1808‐1817.
   Kingston, R.E. 2002. Preparation and Analysis of RNA. Curr. Protoc. Mol. Biol. Chapter 4.
   Kishine, H., Sugiyama, K., Hijikata, M., Kato, N., Takahashi, H., Noshi, T., Nio, Y., Hosaka, M., Miyanari, Y., and Shimotohno, K. 2002. Subgenomic replicon derived from a cell line infected with the hepatitis C virus. Biochem. Biophys. Res. Commun. 293:993‐999.
   Krieger, N., Lohmann, V., and Bartenschlager, R. 2001. Enhancement of hepatitis C virus RNA replication by cell culture‐adaptive mutations. J. Virol. 75:4614‐4624.
   Lamarre, D., Anderson, P.C., Bailey, M., Beaulieu, P., Bolger, G., Bonneau, P., Bos, M., Cameron, D.R., Cartier, M., Cordingley, M.G., Faucher, A.M., Goudreau, N., Kawai, S.H., Kukolj, G., Lagace, L., LaPlante, S.R., Narjes, H., Poupart, M.A., Rancourt, J., Sentjens, R.E. St. George, R., Simoneau, B., Steinmann, G., Thibeault, D., Tsantrizos, Y.S., Weldon, S.M., Yong, C.L., and Llinas‐Brunet, M. 2003. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature 426:186‐189.
   Lindenbach, B.D., Evans, M.J., Syder, A.J., Wolk, B., Tellinghuisen, T.L., Liu, C.C., Maruyama, T., Hynes, R.O., Burton, D.R., McKeating, J.A., and Rice, C.M. 2005. Complete replication of hepatitis C virus in cell culture. Science 309:623‐626.
   Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L., and Bartenschlager, R. 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110‐113.
   Lohmann, V., Korner, F., Dobierzewska, A., and Bartenschlager, R. 2001. Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J. Virol. 75:1437‐1449.
   Lohmann, V., Hoffmann, S., Herian, U., Penin, F., and Bartenschlager, R. 2003. Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J. Virol. 77:3007‐3019.
   Meisenhelder, J. and Bursik, S. 2007. Safe use of radioisotopes. Curr. Protoc. Mol. Biol. 79:A.1F.1‐A.1F.18.
   Moradpour, D., Penin, F., and Rice, C.M. 2007. Replication of hepatitis C virus. Nat. Rev. Microbiol. 5:453‐463.
   Murray, E.M., Grobler, J.A., Markel, E.J., Pagnoni, M.F., Paonessa, G., Simon, A.J., and Flores, O.A. 2003. Persistent replication of hepatitis C virus replicons expressing the beta‐lactamase reporter in subpopulations of highly permissive Huh7 cells. J. Virol. 77:2928‐2935.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Pietschmann, T., Lohmann, V., Kaul, A., Krieger, N., Rinck, G., Rutter, G., Strand, D., and Bartenschlager, R. 2002. Persistent and transient replication of full‐length hepatitis C virus genomes in cell culture. J. Virol. 76:4008‐4021.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   Vrolijk, J.M., Kaul, A., Hansen, B.E., Lohmann, V., Haagmans, B.L., Schalm, S.W., and Bartenschlager, R. 2003. A replicon‐based bioassay for the measurement of interferons in patients with chronic hepatitis C. J. Virol. Methods 110:201‐209.
   Wakita, T., Pietschmann, T., Kato, T., Date, T., Miyamoto, M., Zhao, Z., Murthy, K., Habermann, A., Krausslich, H.G., Mizokami, M., Bartenschlager, R., and Liang, T.J. 2005. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11:791‐796.
   Wasley, A. and Alter, M.J. 2000. Epidemiology of hepatitis C: geographic differences and temporal trends. Semin. Liver Dis. 20:1‐16.
   Yi, M., Bodola, F., and Lemon, S.M. 2002. Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein. Virology 304:197‐210.
   Zhao, Y., Sanchez, A., Nie, X., Liu, D., Hou, X., Fabrycki, J., Phadke, A., Deshpande, M., Huang, M., and Yang, W. 2008. A highly sensitive and reproducible HCV RNA hybridization method valuable for antiviral drug discovery. J. Virol. Methods 151:154‐157.
   Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D.R., Wieland, S.F., Uprichard, S.L., Wakita, T., and Chisari, F.V. 2005. Robust hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. U.S.A. 102:9294‐9299.
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