In Vitro Anti‐Hepatitis C Virus (HCV) Resistance Selection and Characterization

Hongmei Mo1

1 Gilead Sciences, Inc., Foster City, California
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13B.5
DOI:  10.1002/0471141755.ph13b05s53
Online Posting Date:  June, 2011
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Abstract

Understanding the resistance profile for an antiviral drug is essential in the drug discovery process. The selection of drug‐resistant mutant viruses is critical in characterizing the resistance of hepatitis C virus (HCV) to anti‐HCV agents, and can be examined in HCV replicon systems. Three basic methods are employed in this unit to select HCV resistance replicons: (i) cells containing HCV replicons are cultured at low density in the presence of G418 and a fixed concentration of the investigational drug; (ii) cells containing HCV replicons are passaged in the presence of a fixed concentration of investigational drug, but in the absence of G418; (iii) cells containing HCV replicons are passaged in the presence of a gradually increasing concentration of an investigational drug in the presence of G418 for several weeks. The cells from each passage are then harvested and stored for phenotypic and genotypic characterization. The protocols in this unit describe the techniques necessary to select and characterize drug‐ resistant HCV variants and estimate the frequency of drug resistance in a viral population. Curr. Protoc. Pharmacol. 53:13B.5.1‐13B.5.27. © 2011 by John Wiley & Sons, Inc.

Keywords: HCV; resistance selection; phenotype and genotype

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Maintenance and Passage of Replicon Cells
  • Basic Protocol 2: Resistance Selection Method I: Selection of Anti‐HCV Compound Resistant Colonies and Determination of Resistance Frequency
  • Basic Protocol 3: Resistance Selection Method II: Selection of Compound‐Resistant Replicons and Monitoring of Antiviral Potency
  • Basic Protocol 4: Resistance Selection Method III: Selection of HCV Resistance by Passage of Replicon Cells in the Presence of Compound and G418
  • Sequencing Characterization of Drug‐Resistant HCV Replicons
  • Basic Protocol 5: Extraction of Total RNA from the Selected Replicon Cells
  • Basic Protocol 6: Amplification of the Target Gene by Reverse Transcription (cDNA Synthesis) Followed by PCR (RT‐PCR)
  • Basic Protocol 7: Phenotypic Characterization of Cells Containing Replicons Resistant to Anti‐HCV Agents
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Maintenance and Passage of Replicon Cells

  Materials
  • HCV replicon–containing cells (Apath, http://www.apath.com/, or ReBlikon GmbH)
  • DMEM growth medium (see recipe)
  • Phosphate buffered saline (PBS), pH 7.4
  • Trypsin/EDTA: 0.05% (w/v) trypsin/0.02% (w/v) EDTA in PBS
  • 162‐cm2 flasks, tissue culture treated, sterile, vent cap/straight neck, nonpyrogenic, polystyrene (Corning)
  • Inverted tissue culture microscope
  • 5‐ml disposable serological pipets
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 2: Resistance Selection Method I: Selection of Anti‐HCV Compound Resistant Colonies and Determination of Resistance Frequency

  Materials
  • HCV replicon–containing cells (Apath, http://www.apath.com/, or ReBlikon GmbH)
  • DMEM growth medium (see recipe)
  • Phosphate buffered saline (PBS), pH 7.4
  • Trypsin/EDTA: 0.05% (w/v) trypsin/0.02% (w/v) EDTA in PBS
  • Compounds of interest for anti‐HCV activity (one example of a reference compound for the experimental control is A‐7982759; Mo et al., )
  • Freezing medium: culture medium containing 20% fetal bovine serum (FBS) and 10% DMSO
  • Isopropanol
  • 95% ethanol
  • Crystal violet stain: 2% (w/v) crystal violet in 10% (v/v) ethanol
  • 75‐cm2 and 162‐cm2 flasks, tissue culture treated, sterile, vent cap/straight neck, nonpyrogenic, polystyrene (Corning)
  • Inverted tissue culture microscope
  • Hemacytometer or automated cell counter
  • 10‐cm or 15‐cm disposable cell culture petri dishes
  • 24‐well, 48‐well, and 96‐well cell culture plates
  • Cryotubes
  • Cryogenic freezing container (e.g., Mr. Frosty from Nalgene)
  • –140°C freezer or liquid N 2 tank
  • Manual colony counter (Reichart, Mannostat, Bantex, Sci ED), light box, and marking pen or stylus or automated colony counter (Alpha Innotech, Spiral Biotech, New Brunswick Scientific)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 3: Resistance Selection Method II: Selection of Compound‐Resistant Replicons and Monitoring of Antiviral Potency

  Materials
  • HCV replicon–containing cells (Apath (http://www.apath.com/, or ReBlikon GmbH)
  • DMEM growth medium (see recipe)
  • Phosphate‐buffered saline (PBS), pH 7.4
  • Trypsin/EDTA: 0.05% (w/v) trypsin/0.02% (w/v) EDTA in PBS
  • Potential anti‐HCV compound(s) and appropriate solvent(s)
  • RLT RNA lysis buffer (Qiagen)
  • RNeasy spin column kit (Qiagen)
  • 75‐cm2 and 162‐cm2 flasks, tissue culture treated, sterile, vent cap/straight neck, nonpyrogenic, polystyrene (Corning)
  • Inverted tissue culture microscope
  • Hemacytometer or automated cell counter
  • Additional reagents and equipment for RT‐PCR (Fraga et al., )
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 4: Resistance Selection Method III: Selection of HCV Resistance by Passage of Replicon Cells in the Presence of Compound and G418

  Materials
  • HCV replicon–containing cells (Apath, http://www.apath.com/, or ReBlikon GmbH)
  • DMEM growth medium (see recipe)
  • Phosphate buffered saline (PBS), pH 7.4
  • Trypsin/EDTA: 0.05% (w/v) trypsin/0.02% (w/v) EDTA in PBS
  • Geneticin (G418; Invitrogen, cat. no. 10131‐035)
  • Potential anti‐HCV compound(s) and appropriate solvent(s)
  • RLT RNA lysis buffer (Qiagen)
  • 75‐cm2 and 162‐cm2 flasks, tissue culture treated, sterile, vent cap/straight neck, nonpyrogenic, polystyrene (Corning)
  • Inverted tissue culture microscope
  • Hemacytometer or automated cell counter
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 5: Extraction of Total RNA from the Selected Replicon Cells

  Materials
  • The resistance replicon‐containing cells to be characterized (see Basic Protocols 2,3, and 4)
  • DMEM growth medium (see recipe)
  • Phosphate‐buffered saline (PBS), pH 7.4
  • RNeasy Mini Kit (Qiagen)
  • 2‐mercaptoethanol (2‐ME)
  • 100% ethanol
  • 6‐well tissue culture plate
  • Hemacytometer
  • NanoDrop spectrophotometer

Basic Protocol 6: Amplification of the Target Gene by Reverse Transcription (cDNA Synthesis) Followed by PCR (RT‐PCR)

  Materials
  • RNA template: purified total RNA from the selected HCV replicon cells ( protocol 5)
  • First‐round and nested PCR primers, forward and reverse: the primers are specifically designed according to the flanking sequences of the target gene, using primer‐design programs
  • Reverse transcriptase kit (e.g., Superscript III RT, Invitrogen Life Technologies)
  • PCR kit (e.g., High Fidelity PCR Enzyme Platinum Pfx, Invitrogen Life Technologies) including:
    • 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP)
    • 10× PCR buffer
    • 50 mM MgCl 2
    • Nested PCR primers, forward and reverse
    • Platinum Taq DNA polymerase
  • Sizing standards (1 kb DNA ladder; New England Biolabs, cat. no. N3232S)
  • PCR product cleanup kit (e.g., Qiagen)
  • 200‐µl PCR tubes
  • Thermal cycler
  • Sequencher 4.0 sequence analysis software (Gene Codes Corp.)
  • Additional reagents and equipment for agarose/ethidium bromide gel electrophoresis (Voytas, ) and spectrophotometric quantitation of DNA (Voytas, )
NOTE: Thaw all reagents at room temperature or 4°C and place in a cooling block or on ice.

Basic Protocol 7: Phenotypic Characterization of Cells Containing Replicons Resistant to Anti‐HCV Agents

  Materials
  • Cells containing resistance replicon
  • Cells containing wild‐type replicon
  • DMEM growth medium (see recipe) without G418
  • Trypsin/EDTA: 0.05% (w/v) trypsin/0.02% (w/v) EDTA in PBS
  • Phosphate‐buffered saline (PBS), pH 7.4
  • Dimethylsulfoxide (DMSO)
  • Test compounds
  • Firefly Luciferase Assay Kit (Promega)
  • 75‐cm 2 tissue culture flasks
  • Hemacytometer
  • 12‐channel pipettor
  • 96‐well white/clear flat‐bottom plates with lids, tissue culture treated (Costar, cat. no. 3610)
  • 96‐well V‐bottom plate (Nunc)
  • 96‐well deep‐well (2 ml/well) plates
  • Titer plate shaker (Millipore)
  • Luminescence reader
  • GraphPad Prism 4.0 software (http://www.graphpad.com)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.
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Figures

Videos

Literature Cited

Literature Cited
   Ali, S., Leveque, V., Le Pogam, S., Ma, H., Philipp, F., Inocencio, N., Smith, M., Alker, A., Kang, H., Najera, I., Klumpp, K., Symons, J., Cammack, N., and Jiang, W.R. 2008. Selected replicon variants with low‐level in vitro resistance to the hepatitis C virus NS5B polymerase inhibitor PSI‐6130 lack cross‐resistance with R1479. Antimicrob. Agents Chemother. 52:4356‐4369.
   Blight, K.J., Kolykhalov, A.A., and Rice, C.M. 2000. Efficient initiation of HCV RNA replication in cell culture. Science 290:1972‐1974.
   Fraga, D., Meulia, T., and Fenster, S. 2008. Real‐time PCR. Curr. Protoc. Essential Lab. Tech. 00:10.3.1‐10.3.34.
   Gallagher, S.R. 2011. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Curr. Protoc. Mol. Biol. 93:A.3D.1‐A.3D.14.
   Koev, G., Dekhtyar, T., Han, L., Yan, P., Ng, T.I., Lin, C.T., Mo, H., and Molla, A. 2007. Antiviral interactions of an HCV polymerase inhibitor with an HCV protease inhibitor or interferon in vitro. Antiviral Res. 73:78‐83.
   Le Pogam, S., Jiang, W.R., Leveque, V., Rajyaguru, S., Ma, H., Kang, H., Jiang, S., Singer, M., Ali, S., Klumpp, K., Smith, D., Symons, J., Cammack, N., and Najera, I. 2006a. In vitro selected Con1 subgenomic replicons resistant to 2′‐C‐methyl‐cytidine or to R1479 show lack of cross resistance. Virology 351:349‐359.
   Le Pogam, S., Kang, H., Harris, S.F., Leveque, V., Giannetti, A.M., Ali, S., Jiang, W.R., Rajyaguru, S., Tavares, G., Oshiro, C., Hendricks, T., Klumpp, K., Symons, J., Browner, M.F., Cammack, N., and Najera, I. 2006b. Selection and characterization of replicon variants dually resistant to thumb‐ and palm‐binding nonnucleoside polymerase inhibitors of the hepatitis C virus. J. Virol. 80:6146‐6154.
   Lenz, O., Verbinnen, T., Lin, T.I., Vijgen, L., Cummings, M.D., Lindberg, J., Berke, J.M., Dehertogh, P., Fransen, E., Scholliers, A., Vermeiren, K., Ivens, T., Raboisson, P., Edlund, M., Storm, S., Vrang, L., de Kock, H., Fanning, G.C., and Simmen, K.A. 2010. In vitro resistance profile of the hepatitis C virus NS3/4A protease inhibitor TMC435. Antimicrob. Agents Chemother. 54:1878‐1887.
   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.
   Lu, L., Pilot‐Matias, T.J., Stewart, K.D., Randolph, J.T., Pithawalla, R., He, W., Huang, P.P., Klein, L.L., Mo, H., and Molla, A. 2004. Mutations conferring resistance to a potent hepatitis C virus serine protease inhibitor in vitro. Antimicrob. Agents Chemother. 48:2260‐2266.
   Lu, L., Dekhtyar, T., Masse, S., Pithawalla, R., Krishnan, P., He, W., Ng, T., Koev, G., Stewart, K., Larson, D., Bosse, T., Wagner, R., Pilot‐Matias, T., Mo, H., and Molla, A. 2007. Identification and characterization of mutations conferring resistance to an HCV RNA‐dependent RNA polymerase inhibitor in vitro. Antiviral Res. 76:93‐97.
   McCown, M.F., Rajyaguru, S., Le Pogam, S., Ali, S., Jiang, W.R., Kang, H., Symons, J., Cammack, N., and Najera, I. 2008. The hepatitis C virus replicon presents a higher barrier to resistance to nucleoside analogs than to nonnucleoside polymerase or protease inhibitors. Antimicrob. Agents Chemother. 52:1604‐1612.
   Mo, H., Lu, L., Pilot‐Matias, T., Pithawalla, R., Mondal, R., Masse, S., Dekhtyar, T., Ng, T., Koev, G., Stoll, V., Stewart, K.D., Pratt, J., Donner, P., Rockway, T., Maring, C., and Molla, A. 2005. Mutations conferring resistance to a hepatitis C virus (HCV) RNA‐dependent RNA polymerase inhibitor alone or in combination with an HCV serine protease inhibitor in vitro. Antimicrob. Agents Chemother. 49:4305‐4314.
   Shi, S.T., Herlihy, K.J., Graham, J.P., Fuhrman, S.A., Doan, C., Parge, H., Hickey, M., Gao, J., Yu, X., Chau, F., Gonzalez, J., Li, H., Lewis, C., Patick, A.K., and Duggal, R. 2008. In vitro resistance study of AG‐021541, a novel nonnucleoside inhibitor of the hepatitis C virus RNA‐dependent RNA polymerase. Antimicrob. Agents Chemother. 52:675‐683.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
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