Rotaviruses: Extraction and Isolation of RNA, Reassortant Strains, and NSP4 Protein

Krystle A. Yakshe1, Zachary D. Franklin1, Judith M. Ball1

1 Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15C.6
DOI:  10.1002/9780471729259.mc15c06s37
Online Posting Date:  May, 2015
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Abstract

Rotavirus (RV) contains 11 double‐stranded RNA segments that encode for twelve structural and nonstructural proteins. The separation and isolation of viral RNA is a necessary precursor for many experimental techniques and can be useful for rapid RV RNA typing and sequencing of different rotavirus strains. The segmented genome enables RV to recombine easily. These recombinant viruses are essential for many purposes, including generation of potential vaccine strains. Rotavirus gene 10 expresses the viral enterotoxin, NSP4, which has been the focus of several studies due to the influence of NSP4 on rotavirus replication, morphogenesis, and pathogenesis. This unit will describe the isolation and separation of viral RNAs, the production characterization of recombinant RV in culture, and the expression and isolation of NSP4 in mammalian and insect cells. © 2015 by John Wiley & Sons, Inc.

Keywords: rotavirus; reassortants; RNA electrophoresis; NSP4; peptide antibodies; chromatography

     
 
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Table of Contents

  • Introduction
  • Biosafety
  • Basic Protocol 1: Generation of Reassortant Rotavirus Strains from Parental Rotavirus Strains
  • Basic Protocol 2: Phenol‐Chloroform Extraction of RNA for Electrophoretic Typing of Rotavirus Double‐Stranded RNA Segments
  • Alternate Protocol 1: Extraction of Rotavirus RNA from Cultured Cells Using TRIzol
  • Alternate Protocol 2: Extraction of Rotavirus RNA from Cell Culture Supernatant Using QIAamp Viral RNA Kit
  • Basic Protocol 3: Polyacrylamide Gel Electrophoresis (PAGE) for Electrotyping Rotavirus Strains
  • Basic Protocol 4: Purification of Rotavirus NSP4 from the Media of Cultured Mammalian Cells
  • Support Protocol 1: Dot Blot Analysis
  • Alternate Protocol 3: Production of NSP4 in Sf9 Cells Using a Recombinant Baculovirus
  • Support Protocol 2: Quaternary Methylamine (QMA) Anion‐Exchange Chromatography for Semi‐Purification of NSP4 from Sf9 Cell Lysates
  • Support Protocol 3: Generate and Enrich NSP4‐Specific Antibody for Use as Ligand in the NSP4 Affinity Column
  • Support Protocol 4: Affinity Chromatography of Semi‐Purified NSP4 Using Purified NSP4 Antibody as Ligand
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of Reassortant Rotavirus Strains from Parental Rotavirus Strains

  Materials
  • MA104 cells (ATCC #CRL‐2378)
  • Minimum essential medium (MEM), 1× and 2×, supplemented with L‐glutamine, non‐essential amino acids (NEAA), sodium pyruvate, pen‐strep, or equivalent (see recipe)
  • Fetal bovine serum (FBS)
  • Ice
  • Plaque‐purified parental RV strains with known concentration based on plaque‐forming units (PFU)
  • Worthington trypsin (1 mg/ml)
  • Serum‐free MEM (above) or phosphate‐buffered saline (PBS; see recipe)
  • Ice bath
  • Low‐melt agarose [it is recommended that you use SeaPlaque (Lonza) or GenePure (ISC BioExpress)]
  • 6‐well tissue culture plates with lids
  • 37°C water bath
  • 15‐ml polypropylene conical tubes
  • Vortex mixer
  • Platform rocker
  • −70°C or lower freezer
  • Sonicator with a cup horn attachment, ice bath sonicator (Missonix sonicator 300 or equivalent)
  • Inverted light microscope
  • High‐speed
  • Centrifuge
  • Sterile Serological pipets
  • Sterile flint glass Pasteur pipets
  • Suction bulb
  • Roller bottles or flasks
  • Additional reagents and equipment for this procedure are included in protocol 5 of unit (Arnold et al., )

Basic Protocol 2: Phenol‐Chloroform Extraction of RNA for Electrophoretic Typing of Rotavirus Double‐Stranded RNA Segments

  Materials
  • Fecal samples
  • 0.1% sodium acetate buffer, pH 5 (see recipe)
  • 1% (w/v) sodium dodecyl sulfate (SDS)
  • Phenol mixture (see recipe)
  • Chloroform
  • 1.5‐ and 0.6‐ml microcentrifuge tubes
  • Vortex mixer
  • Benchtop microcentrifuge, refrigerated

Alternate Protocol 1: Extraction of Rotavirus RNA from Cultured Cells Using TRIzol

  Materials
  • RV‐infected MA104 cells grown in 75‐cm2 or 25‐cm2 flasks
  • TRIzol (Life Technologies)
  • Chloroform
  • Isopropyl alcohol
  • Sterile deionized water
  • −70° or lower and −20°C freezers
  • Pipettors
  • Filtered pipet tips
  • 50‐ml conical tubes, sterile
  • Sonicator with a cup horn attachment, ice bath sonicator, (Misonix sonicator 300) or equivalent
  • High‐speed centrifuge and microcentrifuge
  • Screw‐cap 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Benchtop microcentrifuge

Alternate Protocol 2: Extraction of Rotavirus RNA from Cell Culture Supernatant Using QIAamp Viral RNA Kit

  Additional Materials (also see protocol 3)
  • QIAamp viral RNA kit (Qiagen, cat. no. 52904)
  • NOTE: Alternatively, the centrifuge may be placed in a refrigerated space.

Basic Protocol 3: Polyacrylamide Gel Electrophoresis (PAGE) for Electrotyping Rotavirus Strains

  Materials
  • 30% acrylamide stock (see recipe)
  • Resolving gel buffer (see recipe)
  • 10% ammonium persulfate (APS) in sterile dH 2O
  • TEMED
  • Sterile dH 2O
  • Stacking gel buffer (see recipe)
  • Tris‐glycine running buffer (see recipe)
  • 100‐bp marker and lambda‐HindIII marker
  • PAGE sample loading buffer (see recipe)
  • Silver Stain kit (Thermo Scientific Pierce, cat. no. 24612)
  • 95% ethanol
  • Stoppered vacuum flask
  • 0.75‐mm mini‐gel glass plates using 95% ethanol and a lint‐free cloth
  • 25‐ml Erlenmeyer flasks
  • Serological pipets
  • Absorbent filter paper
  • Mini‐PROTEAN gel setup (Bio‐Rad) or equivalent
  • Small glass dish to fit the gel
  • Cellophane
  • Larger glass dish to fit the cellophane for gel drying
  • Gel drying frames (The Gel Company)
  • 1.5‐in. binder clips
  • Ruler

Basic Protocol 4: Purification of Rotavirus NSP4 from the Media of Cultured Mammalian Cells

  Materials
  • Caco‐2 cells (ATCC #HTB‐37) or MA104 cells (ATCC #CRL‐2378.1)
  • Rotavirus strain of choice
  • Minimum Essential medium, supplemented, with 10% fetal bovine serum (see recipe)
  • 0.1 N NaOH
  • Milli‐Q water
  • Concanavalin A Sepharose 4B (GE Healthcare, cat. no. 71‐7077‐00 AG)
  • Con A binding buffer (see recipe)
  • Con A elution buffer (see recipe)
  • 0.1 M acetic acid buffer, pH 6, with 1 mM sodium, calcium, manganese, and magnesium salts
  • 20% ethanol
  • Sterile dH 2O
  • Mono S start buffer (see recipe)
  • 1 M NaCl
  • 0.2% sodium azide (see recipe)
  • Mono S elution buffer (see recipe)
  • 160‐cm2 Tissue culture flasks
  • Benchtop centrifuge and ultra‐centrifuge (with swinging‐bucket rotor or fixed‐angle rotor)
  • Ultra‐centrifugal filter with 10‐kD cutoff (Amicon, cat. no. UFC901008 or equivalent)
  • Small‐tipped pipets
  • 50‐ml conical tubes
  • 38‐ml Ultracentrifuge 25×89‐mm Ultra‐Clear tubes (Beckman‐Coulter)
  • −70°C or lower freezer
  • Peristaltic pump
  • Glass Econo‐Column (Bio‐Rad) or equivalent
  • Econo‐Column flow adaptor (Bio‐Rad) or equivalent
  • Mono S 5/50 GL column for FPLC (GE Healthcare Life Sciences, cat. no. 17‐5168‐01)
  • Additional reagents and equipment for SDS‐PAGE and Coomassie Blue staining ( ; Manns, ) and immunoblotting (Blaho et al., )

Support Protocol 1: Dot Blot Analysis

  Materials
  • PBS, pH 7.5
  • PBS with 0.05% Tween 20
  • 10% nonfat dry milk in PBS
  • Primary NSP4 or peptide antibody in 2% nonfat dry milk in PBS
  • Secondary antibody (goat anti‐rabbit‐HRPO) in 2% nonfat dry milk in PBS
  • Bio‐dot apparatus (Bio‐Rad cat. No. 170‐6545) if desired; vacuum
  • Absorbent blotting paper
  • Nitrocellulose membrane, 0.45‐μm (Bio‐Rad)
  • Chemiluminescent kit, HRP substrate (Millipore)
  • Film processor or gel documentation imager

Alternate Protocol 3: Production of NSP4 in Sf9 Cells Using a Recombinant Baculovirus

  Materials
  • Baculovirus pAC461‐G10 plasmid‐expressing gene 10 of RV (Tian et al., ) or equivalent
  • Bac to Bac or BaculoDirect system (Life Technologies)
  • Spodoptera frugiperda 9 (Sf9) cells (ATCC #CRL‐1711)
  • Hink's medium, supplemented with 10% fetal bovine serum and antibiotic/antimycotic (see recipe)
  • Cellfectin II reagent (Life Technologies, cat. no. 10362‐100)
  • Hink's medium, unsupplemented (no FBS, no antibiotic/antimycotic) (Life Technologies)
  • Hink's medium, supplemented with only 10% fetal bovine serum (see recipe)
  • Sucrose solutions prepared in sterile Milli‐Q water (see recipes)
  • 2× Hink's medium (see recipe)
  • Sterile Milli‐Q water
  • Trypan Blue in PBS
  • SeaPlaque Agarose (Lonza, cat. no. 50101)
  • Fetal bovine serum (FBS)
  • Neutral Red solution (see recipe)
  • Phosphate‐buffered saline (see recipe)
  • Sf9 lysis buffer (see recipe)
  • 100× penicillin‐streptomycin‐amphotericin B (Lonza, cat. no. 17745)
  • 35‐mm plates
  • Vortex mixer
  • 27°C incubator; no CO 2 required
  • Beckman Ultracentrifuge 25×89–mm Ultra‐Clear open top tubes that can hold up to 38 ml, or equivalent
  • Beckman Ultracentrifuge with SW‐28 rotor or equivalent
  • 28‐G needle equipped with a 5‐ml syringe
  • Spinner flasks
  • Hemacytometer
  • Sterile 50‐ml conical tubes
  • Low‐speed centrifuge
  • 6‐well tissue culture plates
  • 1.5‐ml microcentrifuge tubes
  • 37°C water bath
  • Humidified chamber
  • Inverted light microscope
  • Additional reagents and equipment for constructing a bacmid plasmid and collecting the medium containing the recombinant bacculovirus (Tian et al., 1995)

Support Protocol 2: Quaternary Methylamine (QMA) Anion‐Exchange Chromatography for Semi‐Purification of NSP4 from Sf9 Cell Lysates

  Materials
  • AccellPlus Quaternary Methylamine Anion (QMA) packing (Waters, cat. no. WAT010742)
  • HPLC‐grade methanol (filtered through 0.45‐μm filter)
  • HPLC Milli‐Q water (filtered through 0.45‐μm filter)
  • 0.2% sodium azide (see recipe)
  • QMA Solution A: 0.1 M Tris·Cl, pH ∼5.0, sterile filtered (see recipe)
  • QMA Solution B: 0.1 M Tris‐Base, pH ∼10.0, sterile filtered (see recipe)
  • QMA Solution C: 2.0 M NaCl, sterile filtered (see recipe)
  • QMA Solution D: Milli‐Q H 2O, sterile filtered
  • 50 mM NH 4HCO 3
  • Dry ice
  • Ethanol
  • Advanced Purification 2 (AP‐2) column (20 × 300 mm) (Waters, cat. no. WAT027503)
  • Support stand
  • Utility clamps
  • Waters FPLC or equivalent (capable of accurately generating specific gradients and a 4.5 ml/min flow rate)
  • Bath sonicator with a cup horn attachment (Misonix sonicator 300 or equivalent)
  • 0.22‐μm filters
  • Fraction collector that automatically collects eluting fractions by time or volume
  • Test tubes for collection that fit the fraction collector utilized and hold up to 5 ml volume
  • Dialysis tubing (12,000 MWCO, Spectra/Por CE) or dialysis cassettes (Slide‐A‐lyzer Dialysis Cassette, 10 kD MWCO, Thermo Scientific, cat. no. 66810)
  • 2‐liter beakers
  • Stir bar
  • External UV detector or the detector that is included in the FPLC
  • Lyophilizer
  • Desiccator

Support Protocol 3: Generate and Enrich NSP4‐Specific Antibody for Use as Ligand in the NSP4 Affinity Column

  Materials
  • NSP4‐specific immune‐dominant synthetic peptides
  • Keyhole limpet hemocyanin (KLH)
  • Glutaraldehyde (EM grade)
  • 1 M glycine, pH 7.5
  • Saturated ammonium sulfate
  • New Zealand white rabbits
  • Phosphate‐buffered saline (PBS; see recipe)
  • 50 mM ammonium bicarbonate
  • Protein A‐Sepharose
  • 50 mM glycine, pH 3.0
  • Dry ice
  • Ethanol
  • CnBr‐activated Sepharose 4B (GE Healthcare, cat. no. 17‐0430‐01), 4% agarose, average bead size of 90 μm, coupling capacity of 25 to 60 mg of α‐chymotrypsinogen, stable pH 3 to 11
  • NSP4‐specific, purified antibody or other ligand (5‐10 mg of protein per ml of column gel)
  • 1 mM HCl, pH 3
  • Coupling buffer (see recipe)
  • Blocking buffer (see recipe)
  • Washing buffers of alternate pH (see recipe)
  • Binding buffer (sterile filtered PBS, pH 7.5; see recipe)
  • Equilibration buffer (see recipe)
  • 0.5 M NaOH, optional
  • Elution buffers (pH 4.5 and 3.0; see reciperecipes)
  • 1 M Tris·Cl, pH 8.0, sterile
  • Dialysis bag (Spectra/Por CE)
  • Dialysis tubing or dialysis cassette (3000 MWCO)
  • Lyophilizer
  • Emulsifying needle
  • Centrifuge
  • Dialysis tubing, 12‐kD MWCO, or dialysis cassette (Slide‐A‐Lyzer Dialysis Cassette, 10 kD MWCO, Thermo Scientific, cat. no. 66810)
  • Rotator
  • Peristaltic pump (Cole Parmer or equivalent), optional
  • Desiccator
  • Sintered glass filter, porosity of G3, or Büchner funnel no. 6
  • Spectrophotometer
  • Stoppered flask
  • Glass column with adaptor
  • UV detector (Isco UA6 or equivalent)
  • Chart recorder
  • Fraction collector
  • Collection tubes
NOTE: If availability of pure NSP4 is problematic, synthetic peptides are a good alternative. Previous studies have shown peptides to be highly immunogenic and reactive to the native protein if carefully selected. However, the peptides must be cross‐linked to a carrier protein such as keyhole limpet hemocyanin (KLH) prior to immunization of the laboratory animals.

Support Protocol 4: Affinity Chromatography of Semi‐Purified NSP4 Using Purified NSP4 Antibody as Ligand

  Materials
  • Purified NSP4‐specific IgG (see protocol 10)
  • CnBr‐activated Sepharose 4B (GE Healthcare, cat. no. 17‐0430‐01), 4% agarose, average bead size of 90 μm, coupling capacity of 25 to 60 mg of α‐chymotrypsinogen, stable pH 3 to 11
  • 1 mM HCl
  • Coupling solution (see recipe)
  • Blocking buffer (see recipe)
  • Washing buffers of alternate pH (see recipes)
  • Binding buffer (sterile filtered PBS, pH 7.5; see recipe)
  • Elution buffers (pH 4.5 and 3.0; see reciperecipes)
  • 0.02% sodium azide
  • 50 mM ammonium bicarbonate (NH 4HCO 3)
  • Dry ice
  • Ethanol
  • 0.05 M ammonium bicarbonate in dH 2O
  • NSP4‐specific, purified antibody or other ligand (5 to 10 mg of protein per ml of column gel)
  • 1 M Glycine, pH 7.5
  • MicroBCA kit (Thermo Pierce, cat. no. 23235)
  • Balance
  • Sintered glass filter, porosity of G3, or Büchner funnel no. 6
  • Spectrophotometer
  • Stoppered flask
  • Rotator
  • Centrifuge
  • Glass column with or without adaptor
  • Peristaltic pump (Cole Parmer or equivalent)
  • Fraction collector
  • UV detector (Isco or equivalent)
  • Chart recorder
  • Collection tubes
  • Dialysis tubing, 12,000 MWCO, or dialysis cassette (Slide‐A‐Lyzer Dialysis Cassette, 10,000 MWCO; Thermo Scientific, cat. no. 66810)
  • Dialysis tubing or dialysis cassette (3000 MWCO)
  • 2‐liter beakers
  • Stir bars
  • Lyophilizer
  • Desiccator
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Figures

Videos

Literature Cited

Literature Cited
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