Separation and Isolation of BTV dsRNA Segments and Viral Proteins

Joseph K.‐K. Li1, I‐Jen Huang2, Emiko Hayama3

1 Department of Biology, Utah State University, Logan, Utah, 2 Taiwan Sugar Research Institute, Taiwan, China, 3 Department of Pediatric Cardiology, Tokyo Women's Medical University, Tokyo, Japan
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15C.5
DOI:  10.1002/9780471729259.mc15c05s25
Online Posting Date:  May, 2012
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Abstract

Bluetongue virus (BTV) genome contains ten double‐stranded RNA segments. The sequence of the plus strand of each of the BTV genomic double‐stranded RNAs is the same as that of its mRNA, which encodes for a single viral protein, except the smallest S4 segment which can encode for two nonstructural proteins, primarily for the release assistance of the viral progeny. The separation and isolation of each BTV dsRNA segment and viral protein have provided extensive data related to its viral infection, pathology, suppression of host cellular functions, and eventual apoptosis of the infected host cells. This cytoplasmic virus is also an animal killer that costs the U.S. livestock industry at least $125 million yearly. However, this virus has no known effect on humans. Thus, it is very safe to carry out investigation with the virus, preferably in a BSL‐2 laboratory. Curr. Protoc. Microbiol. 25:15C.5.1‐15C.5.22. © 2012 by John Wiley & Sons, Inc.

Keywords: Bluetongue virus; BTV protein separation; ds‐RNA genome isolation; viral guanylyltransferase

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Rapid Selective Separation of BTV Viral Proteins and Double‐Stranded RNAs
  • Basic Protocol 2: Gel Electrophoresis of BTV Proteins
  • Support Protocol 1: Concentration and Desalting of Viral Proteins by Precipitation with Trichloroacetic Acid
  • Support Protocol 2: Desalting Viral Proteins by Spin Column Centrifugation
  • Basic Protocol 3: Baculovirus Expression and Purification of the Bluetongue Virus Guanylyltransferase
  • Alternate Protocol 1: E. coli Expression and Purification of the Bluetongue Virus Guanylyltransferase
  • Support Protocol 3: Purification of 6×His‐VP4 from E. coli
  • Support Protocol 4: Purification of Expressed Insoluble and Soluble VP4 Protein
  • Basic Protocol 4: Isolation of BTV dsRNA Segments from Infected Host Cells
  • Basic Protocol 5: Separation of BTV DS‐Stranded RNA Segments Using 2% NuSieve Agarose Gel Electrophoresis
  • Alternate Protocol 2: Separation of BTV Double‐Stranded RNA Segments using SDS‐PAGE
  • Basic Protocol 6: Purification of Individual BTV dsRNA Segments from Preparative Polyacrylamide Slab Gels
  • Basic Protocol 7: Rapid Production of Multiple Alkaline Northern Blots of BTV Viral dsRNA from a Single Polyacrylamide Gel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Rapid Selective Separation of BTV Viral Proteins and Double‐Stranded RNAs

  Materials
  • BTV viral pellet ( protocol 13 in unit 15.4)
  • 0.2 M Tris⋅Cl, pH 8.0 (Sigma; also see appendix 2A)
  • Protein Assay Kit I (BioRad, cat. no. 500‐0001)
  • 5% to 10 % sodium dodecyl sulfate (SDS; see recipe)
  • 0.25 to 0.5 M KCl (Sigma)
  • 25:24:1 phenol/chloroform/isoamyl alcohol ( appendix 2A)
  • 3 M sodium acetate, pH 5.2
  • Ethanol (ThermoFisher)
  • RNA stabilizer (Biomatrica, cat no. 93221‐001; http://www.biomatrica.com/)
  • DEPC‐treated H 2O ( appendix 2A), sterile
  • Phosphate‐buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific; also see appendix 2A)
  • 100°C water bath
  • Biofuge‐12 microcentrifuge (Heraeus Instruments or Baxter Scientific)
  • Whatman 3MM filter paper
  • NanoDrop spectrophotometer (Model ND‐1000; Thermo Scientific)
  • Vacuum desiccator or SpeedVac evaporator

Basic Protocol 2: Gel Electrophoresis of BTV Proteins

  Materials
  • BTV viral pellets ( protocol 1), purified virions, or infected cell lysates
  • 2× SDS sample buffer for protocol 2 (see recipe)
  • Silver staining kit (BioRad)
  • Additional reagents and equipment for SDS‐PAGE including gel staining ( appendix 3M) and desalting protein samples (Support Protocols protocol 31 and protocol 42)

Support Protocol 1: Concentration and Desalting of Viral Proteins by Precipitation with Trichloroacetic Acid

  Materials
  • Trichloroacetic acid (TCA; Sigma or Mallinckrodt)
  • BTV viral pellets ( protocol 1), purified virions, or infected cell lysates)
  • Acetone, cold
  • 2× SDS sample buffer (see recipe)
  • Biofuge‐12 microcentrifuge (Heraeus Instruments or Baxter Scientific)
  • Whatman 3MM filter paper

Support Protocol 2: Desalting Viral Proteins by Spin Column Centrifugation

  Materials
  • BTV viral pellets, purified virions, or infected cell lysates (see protocol 1 or unit 15.4)
  • 2× SDS sample buffer (BioRad)
  • Nanosep 3K mini spin columns (Pall; cat no. 0D003C33)
  • Biofuge‐12 microcentrifuge (Heraeus Instruments or Baxter Scientific)

Basic Protocol 3: Baculovirus Expression and Purification of the Bluetongue Virus Guanylyltransferase

  Materials
  • Total BTV dsRNA ( protocol 1)
  • M1 Primers with Pst‐1 sites:
    • 5′‐ AGTCGACCTGCAGGTTAAAACATGCCTGAG‐ 3′
    • 5′‐ AGTCGACCTGCAGGTAAGTTGTACATGCCC‐ 3′
  • 10× RT‐PCR buffer (see recipe)
  • 10 mM dNTP mix ( appendix 2A)
  • Avian myeloblastosis virus reverse transcriptase (AMV‐RT)
  • Taq DNA polymerase
  • Mineral oil
  • Restriction enzymes (New England BioLab)
    • PstI
    • BamHI
  • Baculovirus expression vector pVL1392 (Invitrogen)
  • E. coli M15 (pREP4, Qiagen)
  • QIA prep Miniprep kit (Qiagen)
  • SF9 insect cells (PharMingen)
  • Linear baculoviral DNA (Baculogold from PharMingen)
  • STE buffer (see recipe)
  • Phosphate‐buffered saline (PBS, Thermo Fisher Scientific; also see appendix 2A)
  • Thermal cycler
  • Beckman GPR Centrifuge (Beckman Coulter)
  • Sonicator (VirSonic)
  • Additional reagents and equipment for purification of BTV dsRNA segments from slab gels (Schuerch et al., , and protocol 12), RT‐PCR amplification (Beverly, ), agarose gel electrophoresis (Voytas, ), DNA sequencing (Shendure et al., ), phenol‐chloroform extraction and ethanol precipitation of DNA ( protocol 1, step 8), generation of the recombinant plasmid, pVL1392BTV13M1, with the baculovirus expression vector pVL1392 and the PstI‐digested PCR product (Seidman et al., ), infection of insect cells (Murphy et al., ) to obtain VP4 production, SDS‐PAGE ( appendix 3M), and immunodetection by immunoblotting (Gallagher et al., )

Alternate Protocol 1: E. coli Expression and Purification of the Bluetongue Virus Guanylyltransferase

  • Total BTV dsRNA ( protocol 1)
  • M1 primers with SmaI sites:
    • 5′‐GACGTCGACCCGGGGATGCCTGAGCCA‐3′
    • 5′‐GACGTCGACCCGGGTAAGTTGTACAT‐3′
  • Restriction enzyme SmaI (New England BioLabs)
  • pQE30 (Qiagen)
  • E. coli M15 (pREP4, Qiagen)
  • 2 mM isopropyl β‐D‐thiogalactopyranoside (IPTG, BioRad)
  • LB medium ( appendix 4A)
  • QIAprep Miniprep Kit (Qiagen)
  • Anti‐VP4 serum (available as a courtesy from Dr. Li's lab; )
  • Additional reagents and equipment for purification of BTV dsRNA segments for preparative polyacrylamide gel segments (Schuerch et al., , and protocol 12), RT‐PCR amplification (Beverly, ), agarose gel electrophoresis (Voytas, ), DNA sequencing (Shendure et al., ), purification of 6× His‐VP4 from E. coli ( protocol 7), SDS‐PAGE ( appendix 3M), and immunodetection by immunoblotting (Gallagher et al., )

Support Protocol 3: Purification of 6×His‐VP4 from E. coli

  Materials
  • Insoluble fraction containing 6×H‐VP4 ( protocol 5)
  • Dissolving buffer (see recipe)
  • Talon metal affinity resin (Clontech, cat. no. 635502)
  • His 60 Ni Superflow resin cartridges (Clontech, cat. no. 635674 for 1 ml and 635678 for 5 ml)
  • 6×His elution buffer (see recipe)
  • 5 or 10‐ml Quik‐Snap plastic column with a pre‐packed bottom disc (Isolab, cat. no. QS‐NP; http://www.isolabgmbh.com/)
  • 5‐ or 10‐ml sterile glass or plastic syringe (Becton Dickinson)
  • Additional reagents and equipment for SDS‐PAGE ( appendix 3M) and immunodetection by immunoblotting (Gallagher et al., )

Support Protocol 4: Purification of Expressed Insoluble and Soluble VP4 Protein

  Materials
  • Lysate of insect ( protocol 5) or E. coli cells ( protocol 6) expressing BTV V4 proteins
  • Solubilization solution for baculovirus expression system (see recipe)
  • 6 M guanidine‐HCl (Sigma)
  • STE Buffer (see recipe)
  • N‐lauroyl sarcosine (Sigma)
  • Reduced glutathione (Sigma)
  • Triton X‐100 (BioRad)
  • Sephadex G‐200 column (Pharmacia)
  • 5 to 50% discontinuous sucrose gradient in STE with a cushion of 66% sucrose (w/w)
  • 12,000‐MWCO dialysis tubing (Fisher, cat. no. 8‐667A)
  • Beckman L8‐70 ultracentrifuge (Beckman)
  • SW41 rotor and 12‐ml ultracentrifuge tubes (Beckman, cat no. 331220)

Basic Protocol 4: Isolation of BTV dsRNA Segments from Infected Host Cells

  Materials
  • BTV‐infected cells infected at different time points (unit 15.4)
  • NTE buffer (see recipe)
  • 10% (v/v) Nonidet P‐40 (NP‐40) stock solution (Sigma or USB)
  • 10% (w/v) sodium deoxycholate (Sigma)
  • 5% (w/v) SDS (BioRad)
  • Phenol:chloroform (1:1, ThermoFisher or EM Sciences)
  • Chloroform:isoamyl alcohol (24:1, EM Sciences or ThermoFisher)
  • Ethanol (ThermoFisher)
  • Sterile DEPC‐treated distilled H 2O ( appendix 2A)
  • 4 M LiCl (Sigma)
  • Poly(T) resin or poly(T) mini‐column (Sigma)
  • Glycogen (Sigma)
  • NTE buffer (see recipe) containing 15% (v/v) ethanol
  • CF‐11 resin (Whatman)
  • TE buffer, pH 8.0 ( appendix 2A)
  • Beckman Allegra‐6R or GPR Centrifuge (Beckman Coulter)
  • Sonicator (VirSonic 50, Virtis)
  • Sterile Kimwipes or Whatman 3MM paper (Whatman)
  • 5‐ or 10‐ml glass or plastic syringe (Becton Dickinson)

Basic Protocol 5: Separation of BTV DS‐Stranded RNA Segments Using 2% NuSieve Agarose Gel Electrophoresis

  Materials
  • 2% NuSieve agarose (FMC Bioproducts)
  • TBE buffer ( appendix 2A)
  • Ethidium bromide (Sigma)
  • 2× loading buffer (BioRad)
  • Horizontal Canalco HSG Chamber (Miles Laboratories)
  • Power supply (Model 2000/200, BioRad)
  • Circulating pump (Geneline Cooler, Beckman)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Alternate Protocol 2: Separation of BTV Double‐Stranded RNA Segments using SDS‐PAGE

  Materials
  • Acrylamide (BioRad)
  • Bisacrylamide (BioRad)
  • Sodium dodecyl sulfate (SDS, BioRad)
  • N,N,N′,N′‐tetramethylediamine (TEMED, Eastman, http://www.eastman.com/)
  • Urea (Mallinckrodt)
  • 0.04% (w/v) ammonium persulfate (Sigma)
  • Loening E Buffer (BioRad)
  • BTV dsRNA sample ( protocol 1)
  • 2× loading buffer (BioRad)
  • 0.5% ethidium bromide (Sigma)
  • BioRad vertical electrophoresis unit (BioRad)
  • Flat‐bottom gel comb
  • Power supply (Model 2000/200, BioRad)
  • Circulating pump (Geneline Cooler, Beckman)
  • Gyrotwister (Labnet)
  • Mineralight Lamp (Model UVS‐11; Ultra‐violet Products Inc.) or UV lamp (BioRad)
  • Additional reagents and equipment for polyacrylamide gel electrophoresis ( appendix 3M)

Basic Protocol 6: Purification of Individual BTV dsRNA Segments from Preparative Polyacrylamide Slab Gels

  Materials
  • Acrylamide (BioRad)
  • Bisacrylamide (BioRad)
  • Sodium dodecyl sulfate (SDS, BioRad)
  • N,N,N′,N′‐tetramethylediamine (TEMED, Eastman, http://www.eastman.com/)
  • Urea (Mallinckrodt)
  • Loening E buffer (BioRad)
  • 0.04% ammonium persulfate
  • BTV dsRNA sample ( protocol 1)
  • Sample buffer for protocol 12 (see recipe)
  • 0.01% (w/v) ethidium bromide
  • dsRNA elution buffer (see recipe)
  • Phenol (Mallinckrodt)
  • Chloroform–isoamyl alcohol (EM Science or Mallinckrodt)
  • Ethanol
  • Preparative slab gel apparatus (30 cm × 16 cm × 3‐5 mm, BioRad)
  • 70°C water bath
  • Eastman Chromagram sheets impregnated with fluorescent indicator (Eastman)
  • UV light (BioRad)
  • Gel Elutor (BioRad)
  • Mortar and pestle, sterile
  • Shaker
  • GF/C filters (0.2 to 0.4 µm, Gilman Sciences)
  • Additional reagents and equipment for polyacrylamide gel electrophoresis ( appendix 3M)

Basic Protocol 7: Rapid Production of Multiple Alkaline Northern Blots of BTV Viral dsRNA from a Single Polyacrylamide Gel

  Materials
  • Viral dsRNA ((Alternate Protocol 3 or protocol 12)
  • 0.25 N HCl
  • 0.2 N NaOH
  • 20× SSC ( appendix 2A)
  • Bio‐Trace nylon membrane for northern blots (Gelman Sciences, cat. no. 66481)
  • Electroblotting apparatus (BioRad Trans‐Blot Cell, cat. no. 170‐3930)
  • Pyrex dish (Fisher)
  • Gel agitator or gyrotwister (Labnet)
  • Whatman 3MM paper (Whatman)
  • Additional reagents and equipment for preparing a 7.5% SDS‐PAGE gel (Alternate Protocol 3) and SDS‐PAGE ( appendix 3M)
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Figures

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Literature Cited

Literature Cited
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