Poliovirus: Generation, Quantification, Propagation, Purification, and Storage

Cecily P. Burrill1, Vanessa R. Strings1, Raul Andino1

1 Department of Microbiology and Immunology, University of California, San Francisco
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15H.1
DOI:  10.1002/9780471729259.mc15h01s29
Online Posting Date:  May, 2013
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Abstract

Poliovirus (PV) is the prototypical picornavirus. It is a non‐enveloped RNA virus with a small (∼7.5‐kb) genome of positive polarity. It has long served as a model to study RNA virus biology, pathogenesis, and evolution. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro transcribed viral genomes into an appropriate host cell. PV infects many human and non‐human primate cell lines including HeLa and HeLa S3 cells, and can grow to high titer in culture. Protocols for the production, propagation, quantification, and purification of PV are presented. Curr. Protoc. Microbiol. 29:15H.1.1‐15H.1.27. © 2013 by John Wiley & Sons, Inc.

Keywords: poliovirus; picornavirus; plaque assay; titration; virus purification

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of PV from cDNA Plasmid
  • Support Protocol 1: Propagation and Maintenance of cDNA Plasmid
  • Support Protocol 2: Storage, Propagation, and Maintenance of HeLa and HeLa S3 Monolayer Cultures
  • Basic Protocol 2: Quantification of PV Titer by Plaque Assay
  • Alternate Protocol 1: Quantification of PV Titer by TCID50 Assay
  • Basic Protocol 3: Propagation of PV in HeLa or HeLa S3 Monolayer Cultures
  • Alternate Protocol 2: Propagation of PV in HeLa S3 Suspension Cultures
  • Support Protocol 3: Preparation and Growth of HeLa S3 Suspension Cultures
  • Basic Protocol 4: Generation of Clonal PV Stock by Plaque Purification and Amplification
  • Alternate Protocol 3: Generation of Clonal PV Stock by Limiting Dilution and Amplification
  • Basic Protocol 5: Purification of PV by Ultracentrifugation Through Sucrose Cushion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of PV from cDNA Plasmid

  Materials
  • cDNA plasmid prib(+)XpA (see protocol 2)
  • 20 U/µl EcoRI and 10× buffer (New England BioLabs)
  • Nuclease‐free water
  • 6× DNA loading dye
  • 1% agarose TAE gels and running buffer (Voytas, )
  • DNA ladder (e.g., GeneRuler 1‐kb Plus, Fermentas)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • Isopropanol
  • 75% (v/v) ethanol
  • 5× transcription buffer (see recipe)
  • NTP mix (25 mM each NTP)
  • 40 U/µl RNaseOUT (Invitrogen)
  • T7 RNA polymerase
  • 2 U/µl DNase I (RNase‐free; New England BioLabs)
  • 3× LiCl‐EDTA (see recipe)
  • 0.5× TE buffer (see recipe)
  • 2× RNA denaturing loading dye
  • HeLa or HeLa S3 cells (see protocol 3)
  • D‐PBS (PBS without Ca2+ or Mg2+) ( appendix 2A)
  • 0.05% Trypsin‐EDTA
  • 2.5% NCS DMEM/F‐12 (see ), 37°C
  • 1.5‐ml tubes
  • 37°C heat block or water bath
  • NanoDrop spectrophotometer
  • 95°C heat block
  • Hemacytometer
  • Tissue culture microscope (inverted phase‐contrast)
  • 75‐cm2 tissue culture flasks
  • Electroporator (e.g., BTX Electro Cell Manipulator 600) and 4‐mm cuvettes
  • 37°C, 5% CO 2 humidified incubator

Support Protocol 1: Propagation and Maintenance of cDNA Plasmid

  Materials
  • SURE electroporation‐competent cells (Agilent)
  • prib(+)XpA (or other PV cDNA plasmid; check resistance)
  • Sterile water
  • LB‐amp medium (50 to 100 µg/ml) ( appendix 4A)
  • LB‐amp agar plates (50 to 100 µg/ml) ( appendix 4A)
  • Midi or Maxi prep kit (e.g., NucleoBond Xtra, Machery‐Nagel)
  • 1× TE buffer, pH 8.0 (see recipe)
  • Electroporator and 1‐mm cuvettes
  • 1.5‐ml tubes
  • 37°C incubator with shaking
  • NanoDrop spectrophotometer

Support Protocol 2: Storage, Propagation, and Maintenance of HeLa and HeLa S3 Monolayer Cultures

  Materials
  • 10% NCS DMEM/F‐12 (see recipe)
  • HeLa or HeLa S3 cells (ATCC #CCL‐2 or #CCL‐2.2)
  • D‐PBS (PBS without Ca2+ or Mg2+)
  • 0.05% trypsin‐EDTA
  • Freezing DMEM/F‐12 (see recipe)
  • Isopropanol
  • 37°C, 5% CO 2 humidified incubator
  • 15‐ml conical tubes
  • 150‐cm2 flasks
  • Tissue culture microscope (inverted phase‐contrast)
  • 1.5‐ml cryovials
  • Cell freezing container with foam insert
  • Liquid nitrogen dewar

Basic Protocol 2: Quantification of PV Titer by Plaque Assay

  Materials
  • 10% NCS DMEM/F12 (see recipe)
  • Virus stocks/samples to be titered
  • Serum‐free DMEM/F12 (see recipe)
  • 2% (w/v) agarose
  • 2× DMEM/F12 (see recipe)
  • HeLa or HeLa S3 cells (see protocol 3)
  • 2% (w/v) formaldehyde
  • 1% (v/v) bleach solution
  • 0.1% crystal violet stain (see recipe)
  • 6‐well plates
  • 37°C, 5% CO 2 humidified incubator
  • Tissue culture microscope (inverted phase‐contrast)
  • 37°C and 57°C water baths

Alternate Protocol 1: Quantification of PV Titer by TCID50 Assay

  • 96‐well plates
  • Multi‐channel pipettor and tips
  • Sterile pipet reservoirs

Basic Protocol 3: Propagation of PV in HeLa or HeLa S3 Monolayer Cultures

  Materials
  • HeLa or HeLa S3 cells (see protocol 3)
  • 10% NCS DMEM/F‐12 (see recipe)
  • P0 or clonal infecting stock
  • Serum‐free DMEM/F‐12 (see recipe)
  • PBS ( appendix 2A)
  • 2.5% NCS DMEM/F‐12 (see recipe)
  • 150‐cm2 tissue culture flasks
  • 37°C, 5% CO 2 humidified incubator
  • Tissue culture microscope (inverted phase‐contrast)
  • Centrifuge
  • 50‐ml tubes

Alternate Protocol 2: Propagation of PV in HeLa S3 Suspension Cultures

  Materials
  • HeLa S3 cells in suspension (see protocol 8)
  • Serum‐free SMEM (see recipe)
  • P1 or subsequent infecting stock
  • 37°C, 5% CO 2 humidified incubator
  • Centrifuge
  • 500‐ml spinner flask and magnetic stir plate
  • Hemacytometer
  • Tissue culture microscope (inverted phase‐contrast)
  • 50‐ml conical tubes

Support Protocol 3: Preparation and Growth of HeLa S3 Suspension Cultures

  Materials
  • HeLa S3 cells in monolayers (see protocol 3)
  • D‐PBS (PBS without Ca2+ or Mg2+)
  • 0.05% trypsin‐EDTA
  • 10% FBS SMEM (see recipe)
  • 5% FBS/5% NCS SMEM (see recipe)
  • 10% NCS SMEM (see recipe)
  • 37°C, 5% CO 2 humidified incubator
  • Hemacytometer
  • Tissue culture microscope (inverted phase‐contrast)
  • 250‐ml, 1‐liter, and 3‐liter spinner flasks and magnetic stir plate

Basic Protocol 4: Generation of Clonal PV Stock by Plaque Purification and Amplification

  Materials
  • HeLa or HeLa S3 cells (see protocol 3)
  • 10% NCS DMEM/F12 (see recipe)
  • 2% (w/v) agarose
  • 2× DMEM/F12 (see recipe)
  • P0 or other infecting stocks of known titer
  • Serum‐free DMEM/F‐12 (see recipe)
  • PBS ( appendix 2A)
  • TRIzol LS reagent (Invitrogen)
  • Chloroform
  • 20 µg/µl nuclease‐free glycogen
  • Isopropanol
  • 75% (v/v) ethanol
  • Nuclease‐free water
  • 15 U/µl ThermoScript RT (Invitrogen) kit containing:
    • 10 mM dNTP mix
    • 50 µM oligo (dT) 20
    • 50 ng/µl random hexamers
    • 5× cDNA synthesis buffer
    • 0.1 M DTT
    • 40 U/µl RNaseOUT
  • 10 µM PCR primers (51For and 2651Rev; 2439For and 5050Rev; 4790For and 7400Rev; Table 15.1.1)
  • 5 U/µl Taq DNA polymerase and 10× PCR buffer with Mg2+
  • 6× DNA loading dye
  • 1% agarose TAE gels and running buffer (Voytas, )
  • DNA ladder (e.g., GeneRuler 1‐kb Plus, Fermentas)
  • PCR clean‐up kit (e.g., Nucleospin Gel and PCR Clean‐up, Machery‐Nagel)
  • 2.5% NCS DMEM/F12 (see recipe)
  • 10‐cm dishes
  • 37°C, 5% CO 2 humidified incubator
  • Tissue culture microscope (inverted phase‐contrast)
  • 37°C and 57°C water bath
  • 24‐well plates
  • Ethanol‐resistant marker
  • 1.5‐ml tubes
  • NanoDrop spectrophotometer
  • Thermal cycler

Alternate Protocol 3: Generation of Clonal PV Stock by Limiting Dilution and Amplification

  • Viral RNA isolation kit (e.g., ZR Viral RNA Kit, ZymoResearch)
  • Multi‐channel 20‐ to 200‐µl pipettor and tips
  • 96‐well plates
  • Sterile pipet reservoirs
  • 0.5‐ml tubes

Basic Protocol 5: Purification of PV by Ultracentrifugation Through Sucrose Cushion

  Materials
  • ∼200 ml of high‐titer viral stock (see protocol 7)
  • HBSS
  • 10 mg/ml RNase A
  • 30% (w/v) sucrose in HBSS
  • 1× DMEM/F12/5% SDS/20 mM EDTA (see recipe)
  • Virion storage buffer (see recipe)
  • 1 × 3 1/2–in. ultraclear centrifuge tubes (Beckman, cat. no. 344058)
  • Refrigerated ultracentrifuge and SW28 rotor (Beckman)
  • 50‐ml tubes
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Figures

Videos

Literature Cited

Literature Cited
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   Herold, J. and Andino, R. 2000. Poliovirus requires a precise 5′ end for efficient positive‐strand RNA synthesis. J. Virol. 74:6394‐6400.
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   King, A.M.Q., Lefkowitz, E., Adams, M.J., and Carstens, E.B. (eds.) 2011. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses. Academic Press, Elsevier, San Diego, Calif.
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   Knowles, N.J., Hovi, T., King, A.M.Q., and Stanway, G. 2010. Overview of taxonomy. In The Picornaviruses (E. Ehrenfeld, E. Domingo, and R.P. Roos, eds.) pp. 19‐32. ASM Press, Washington, D.C.
   Lindenbach, B.D. 2009. Measuring HCV infectivity produced in cell culture and in vivo. Methods Mol. Biol. 510:329‐336.
   Mendelsohn, C.L., Wimmer, E., and Racaniello, V.R. 1989. Cellular receptor for poliovirus: Molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily. Cell 56:855‐865.
   Racaniello, V.R. and Baltimore, D. 1981. Cloned poliovirus complementary DNA is infectious in mammalian cells. Science 214:916‐919.
   Reed, L.J. and Muench, H. 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hygiene 27:493‐497.
   Ren, R.B., Costantini, F., Gorgacz, E.J., Lee, J.J., and Racaniello, V.R. 1990. Transgenic mice expressing a human poliovirus receptor: A new model for poliomyelitis. Cell 63:353‐362.
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