Generation and Quantitation of Infectious Hepatitis C Virus Derived from Cell Culture (HCVcc)

Matthew S. Paulson1

1 Gilead Sciences, Foster City, California
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13B.4
DOI:  10.1002/0471141755.ph13b04s51
Online Posting Date:  December, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The development of robust genotype 1b and genotype 1a hepatitis C virus (HCV) replicon systems has enabled the convenient in vitro study of part of the virus life cycle. This unit describes detailed protocols for generating and measuring infectious HCV, or cell‐culture‐derived infectious HCV (HCVcc). The HCVcc infectious system has two essential components: (1) cells that are permissive to de novo infection and allow effective replication of the full virus life cycle; and (2) a virus genome that has robust and efficient replication in tissue culture. The assays in this unit are based on protocols designed for Huh‐7‐derived cell lines that allow robust replication of HCV and are permissive to infection. These protocols are important for the implementation of drug discovery efforts relative to the entire infectious virus life cycle. Curr. Protoc. Pharmacol. 51:13B.4.1‐13B.4.19. © 2010 by John Wiley & Sons, Inc.

Keywords: HCV; infection; cell culture

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Maintenance and Proliferation of Huh‐7‐Derived Cell Lines in Tissue Culture
  • Basic Protocol 2: Production of Electroporation Competent In Vitro Transcribed Hepatitis C Virus RNA
  • Basic Protocol 3: Electroporation of Hepatitis C RNA into Permissive Cells for Virus Production
  • Basic Protocol 4: Harvesting and Storing HCVcc
  • Methods of Detection of HCVcc‐Infected Cells
  • Basic Protocol 5: Immunofluorescence‐Based Detection of HCVcc
  • Alternate Protocol 1: Immunochemistry‐Based Detection of HCVcc
  • Basic Protocol 6: Determination of Virus Titers from Collected Stocks
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Maintenance and Proliferation of Huh‐7‐Derived Cell Lines in Tissue Culture

  Materials
  • Huh‐7‐derived cells (e.g., Huh‐7.5, Apath; Huh‐Lunet‐CD81, ReBLikon)
  • DMEM complete medium (e.g., Invitrogen): Dulbecco's modified Eagle's medium (with 4.5 g/liter glucose, without pyruvate) supplemented with 2 mM L‐glutamine (or GLUTAMAX version), supplemented with nonessential amino acids, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10% fetal bovine serum (e.g., Hyclone); store for no more than 6 weeks at 4°C (heat to 37°C before use)
  • Phosphate buffered saline (PBS; e.g., Invitrogen)
  • Trypsin/EDTA buffer (e.g., Invitrogen): 0.05% (v/v) trypsin, 0.02% (v/v) EDTA in phosphate‐buffered saline
  • Cell culture plates and cell‐culture dishes or flasks (e.g., VWR Scientific, or Fisher Scientific
  • Hemacytometer
  • Microscope

Basic Protocol 2: Production of Electroporation Competent In Vitro Transcribed Hepatitis C Virus RNA

  Materials
  • cDNA of virus construct (e.g., JFH‐1, Toray)
  • Restriction enzyme (XbaI) and buffer (e.g., New England Biolabs)
  • DNA purification spin column kit (e.g., MiniElute kit, Qiagen) containing:
    • PB buffer
    • PE buffer
  • RNase‐free water (e.g., Ambion)
  • 1:1 (v/v) phenol/chloroform
  • 3 M sodium acetate, pH 5.2
  • 70% and 100% ethanol
  • T7 RNA transcription kit (e.g., Ambion Megascript T7 kit or T7 transcription reagents from Epicenter Technologies) containing:
    • Each NTP
    • 10× reaction buffer
    • T7 RNA polymerase enzyme
    • DNase I
  • RNA purification kit (e.g., RNAeasy, Qiagen)
  • RNase‐free RNA precipitation reagents:
    • 20 µg/ml glycogen
    • 3 M sodium acetate pH, 5.2
    • 70% and 100% ethanol
  • Agarose/ethidium bromide gel
  • 37°C water bath
  • Microcentrifuge
  • RNase‐free 1.5‐ml microcentrifuge tubes
  • RNase‐free pipet tips
  • Vortexer
  • Spectrophotometer
  • Gel electrophoresis apparatus

Basic Protocol 3: Electroporation of Hepatitis C RNA into Permissive Cells for Virus Production

  Materials
  • Permissive cells (∼2 × 107 cells for every two electroporations; see protocol 1)
  • Phosphate buffered saline (PBS; e.g., Invitrogen), room temperature
  • 1× trypsin for tissue culture (e.g., Invitrogen)
  • Appropriate complete cell culture medium, 37°C (see protocol 1)
  • RNase‐free PBS, ice cold
  • In vitro transcribed RNA (see protocol 2)
  • Aspirator
  • 37°C water bath
  • 15‐ and 50‐ml tubes
  • Centrifuge, 4°C
  • Hemacytometer
  • 1.5‐ml sterile, RNase‐free tubes
  • 0.2‐ or 0.4‐cm gap cuvettes (BioRad)
  • Electroporator (BioRad)
  • 75‐ or 162‐cm2 tissue culture flasks (optional)
  • 96‐well microtiter plates (optional)

Basic Protocol 4: Harvesting and Storing HCVcc

  Materials
  • Virus‐infected cell culture (see protocol 3)
  • 1 M HEPES, pH 7.2‐7.5 (e.g., Invitrogen)
  • 0.22‐µm filter system
  • Vacuum system

Basic Protocol 5: Immunofluorescence‐Based Detection of HCVcc

  Materials
  • Permissive cells for HCV infection (see protocol 1)
  • Tissue culture medium (see protocol 1)
  • Virus (see protocol 4)
  • 10% bleach
  • Glacial solution of 1:1 (v/v) methanol/acetone
  • PBS
  • Primary antibody (e.g., anti‐core, clone 7B2/I11, Austral Biologicals or anti‐NS5A, clone 9E10, Apath)
  • PBS/3% (w/v) BSA, prepare fresh
  • Secondary antibody (e.g., goat anti‐mouse Alexa 585, Molecular Devices)
  • DAPI or Hoechst stain (Molecular Probes)
  • 96‐well black plates
  • 1.5‐ml tubes
  • Aspirator
  • Fluorescence microscope with capabilities to detect secondary antibodies (if one is not available, see protocol 6)

Alternate Protocol 1: Immunochemistry‐Based Detection of HCVcc

  Materials
  • Permissive cells for HCV infection (see protocol 1)
  • Tissue culture medium (see protocol 1)
  • Virus
  • 10% bleach
  • PBS
  • Glacial solution of methanol (e.g., Sigma), –20°C
  • PBS containing 0.1% Tween‐20 (e.g., Sigma)
  • Blocking buffer: PBS/1% BSA and 0.2% skim milk‐make fresh and mix well into solution
    • 1% (w/v) BSA
    • 0.2% (w/v) skim milk (powder purchased from a supermarket)
  • 3% (v/v) solution of hydrogen peroxide (H 2O 2) in PBS
  • Primary antibody (e.g., anti‐NS5A, clone 9E10, Apath or anti‐core, clone 7B2/I11, Austral Biologicals)
  • Secondary antibody (e.g., goat anti‐mouse horseradish peroxidase (HRP), Jackson Immuno Research or ImmPRESS, Vector Labs)
  • DAB substrate kit (DAKO, cat. no. K3468) or similar
  • 3,3′ diaminobenzidine (DAB) chromogen (e.g., Vector Labs)
  • Hematoxylin (e.g., Richard Allan hematoxylin 2, ThermoFisher Scientific), optional
  • 70%, 95%, and 100% ethanol, optional
  • Xylene, optional
  • Permount (Fisher), optional
  • 96‐well black plates or microscope slides
  • 1.5‐ml tubes
  • 37°C incubator
  • Aspirator
  • Coverslips
  • Light microscope
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Blight, K.J., McKeating, J.A., and Rice, C.M. 2002. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J. Virol. 76:13001‐13014.
   Centers for Diseas Control and Prevention. 2001. Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV, and HIV and Recommendations for Postexposure Prophylaxis, vol. 50. pp. 1‐67. MMWR.
   Cheng, G., Yang, H., Chan, K., Corsa, A., Pokrovskii, M., Paulson, M., Bahador, G., Zhong, W., and Delaney, I.V.W. 2010. Selection of clinically‐relevant protease inhibitor resistant viruses using the HCV infection system. Antimicrob. Agents Chemother. In press.
   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.
   Gottwein, J.M., Scheel, T.K., Hoegh, A.M., Lademann, J.B., Eugen‐Olsen, J., Lisby, G., and Bukh, J. 2007. Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology 133:1614‐1626.
   Gottwein, J.M., Scheel, T.K., Jensen, T.B., Lademann, J.B., Prentoe, J.C., Knudsen, M.L., Hoegh, A.M., and Bukh, J. 2009. Development and characterization of hepatitis C virus genotype 1‐7 cell culture systems: Role of CD81 and scavenger receptor class B type I and effect of antiviral drugs. Hepatology 49:364‐377.
   Heller, T., Saito, S., Auerbach, J., Williams, T., Moreen, T., Jazwinski, A., Cruz, B., Jeurkar, N., Sapp, R.K., Luo, G., and Liang, J. 2005. An in vitro model of hepatitis C virion production. Proc. Natl. Acad. Sci. U.S.A. 102:2579‐2583.
   Kaul, A., Worz, I., and Bartenschlager, R. 2009. Adaptation of the hepatitis C virus to cell culture. Methods Mol. Biol. 510:361‐372.
   Koev, G. and Kati, W. 2008. The emerging field of HCV drug resistance. Expert Opin. Investig. Drugs 17:303‐319.
   Kolykhalov, A.A., Mihalik, K., Feinstone, S.M., and Rice, C.M. 2000. Hepatitis C virus‐encoded enzymatic activities and conserved RNA elements in the 3′ nontranslated region are essential for virus replication in vivo. J. Virol. 74:2046‐2051.
   Konstantinidis, A.K., Richardson, P.L., Kurtz, K.A., Tripathi, R., Chen, C.M., Huang, P., Randolph, J., Towne, D., Donnelly, J., Warrior, U., Middleton, T., and Kati, W.M. 2007. Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease. Anal. Biochem. 368:156‐167.
   Koutsoudakis, G., Kaul, A., Steinmann, E., Kallis, S., Lohmann, V., Pietschmann, T., and Bartenschlager, R. 2006. Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses. J. Virol. 80:5308‐5320.
   Koutsoudakis, G., Herrmann, E., Kallis, S., Bartenschlager, R., and Pietschmann, T. 2007. The level of CD81 cell surface expression is a key determinant for productive entry of hepatitis C virus into host cells. J. Virol. 81:588‐598.
   Lindenbach, B.D. 2009. Measuring HCV infectivity produced in cell culture and in vivo. Methods Mol. Biol. 510:329‐336.
   Lindenbach, B.D. and Rice, C.M. 2003. Molecular biology of flaviviruses. Adv. Virus Res. 9:23‐61.
   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.
   Mao, S.S., DiMuzio, J., McHale, C., Burlein, C., Olsen, D., and Carroll, S.S. 2008. A time‐resolved, internally quenched fluorescence assay to characterize inhibition of hepatitis C virus nonstructural protein 3‐4A protease at low enzyme concentrations. Anal. Biochem. 373:1‐8.
   Pietschmann, T. 2009. Full‐length infectious HCV chimeras. Methods Mol. Biol. 510:347‐359.
   Pietschmann, T., Zayas, M., Meuleman, P., Long, G., Appel, N., Koutsoudakis, G., Kallis, S., Leroux‐Roels, G., Lohmann, V., and Bartenschlager, R. 2009. Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations. PLoS Pathog. 5:e100475.
   Quinkert, D., Bartenschlager, R., and Lohmann, V. 2005. Quantitative analysis of the hepatitis C virus replication complex. J. Virol. 79:13594‐13605.
   Reed, L.J. and Muench, H. 1938. A simple method of estimating 50% end‐points. Am. J. Hyg. 27:493‐497.
   Russell, R.S., Meunier, J.C., Takikawa, S., Faulk, K., Engle, R.E., Bukh, J., Purcell, R.H., and Emerson, S.U. 2008. Advantages of a single‐cycle production assay to study cell culture‐adaptive mutations of hepatitis C virus. Proc. Natl. Acad. Sci. U.S.A. 105:4370‐4375.
   Sabariegos, R., Picazo, F., Domingo, B., Franco, S., Martinez, M.A., and Llopis, J. 2009. Fluorescence resonance energy transfer‐based assay for characterization of hepatitis C virus NS3‐4A protease activity in live cells. Antimicrob. Agents Chemother. 53:728‐734.
   Scheel, T.K., Gottwein, J.M., Jensen, T.B., Prentoe, J.C., Hoegh, A.M., Alter, H.J., Eugen‐Olsen, J., and Bukh, J. 2008. Development of JFH1‐based cell culture systems for hepatitis C virus genotype 4a and evidence for cross‐genotype neutralization. Proc. Natl. Acad. Sci. U.S.A. 105:997‐1002.
   Sudo, K., Yamaji, K., Kawamura, K., Nishijima, T., Kojima, N., Aibe, K., Shimotohno, K., and Shimizu, Y. 2005. High‐throughput screening of low molecular weight NS3‐NS4A protease inhibitors using a fluorescence resonance energy transfer substrate. Antivir. Chem. Chemother. 16:385‐392.
   Tellinghuisen, T.L. and Lindenbach, B.D. 2009. Reverse transcription PCR‐based sequence analysis of hepatitis C virus replicon RNA. Methods Mol. Biol. 510:165‐175.
   Tscherne, D.M., Evans, M.J., von Hahn, T., Jones, C.T., Stamataki, Z., McKeating, J.A., Lindenbach, B.D., and Rice, C.M. 2007. Superinfection exclusion in cells infected with hepatitis C virus. J. Virol. 81:3693‐3703.
   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.
   Yi, M., Villanueva, R.A., Thomas, D.L., Wakita, T., and Lemon, S.M. 2006. Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc. Natl. Acad. Sci. U.S.A. 103:2310‐2315.
   Zhang, J., Chung, T., and Oldenburg, K. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4:67‐73.
   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.
Key References
   Blight et al., 2002. See above.
  Describes how the Huh‐7.5 cell line was derived and expanded.
   Koutsoudakis et al., 2007. See above.
  Describes how the level of CD81 cell surface expression is a key determinant for productive entry of HCVcc into tissue culture cells.
   Lindenbach, 2009. See above.
  Provides a detailed description of virus quantification.
   Lindenbach et al., 2005. See above.
  These three references were the first publications describing infection and production of HCVcc.
   Wakita et al., 2005. See above.
   Zhong et al., 2005. See above.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library