A High‐Throughput Approach for Studying Virus Replication in Yeast

Judit Pogany1, Tadas Panavas1, Elena Serviene1, Muhammad Shah Nawaz‐ul‐Rehman1, Peter D. Nagy1

1 University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16J.1
DOI:  10.1002/9780471729259.mc16j01s19
Online Posting Date:  November, 2010
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Abstract

Viruses are intracellular pathogens that are dependent on viral and host factors for multiplication. Model hosts, such as yeast, can be very valuable in identifying host factors involved in viral replication. Yeast is also useful for studies on functional interactions of host factors with viral proteins and/or virus nucleic acids. The advantages of using yeast include the availability of a single gene–deletion library and the essential gene library (yTHC); the controllable small‐ or large‐scale expression of viral proteins and nucleic acids; and the rapid growth of yeast strains. Procedures that facilitate high‐throughput analysis of host factors and plant and animal RNA virus replication in yeast, with a plant virus (tombusvirus; TBSV) and an animal virus (nodavirus; FHV) as examples, are described. Curr. Protoc. Microbiol. 19:16J.1.1‐16J.1.15. © 2010 by John Wiley & Sons, Inc.

Keywords: yeast; high‐throughput; transformation; viral RNA extraction; northern blotting; tombusviruses; nodaviruses; host factors

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Yeast High‐Throughput Transformation
  • Basic Protocol 2: High‐Throughput Induction and Extraction of Viral RNA from Yeast
  • Basic Protocol 3: Viral RNA Analysis with Agarose Gel Electrophoresis
  • Basic Protocol 4: Viral RNA Analysis with Northern Blotting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Yeast High‐Throughput Transformation

  Materials
  • YPD (see recipe) containing 200 mg/liter G418 (add G418 to YPD medium only after autoclaving)
  • Yeast libraries in 96 strains/plate format (Open Biosystems): YKO library (single deletions of nonessential genes) or yTHC library (single gene mutations that can be turned off by the addition of doxycycline)
  • 1 M lithium acetate (filter sterilize using a 0.22‐µm filter and store up to 1 year at room temperature in the dark)
  • 50% (w/v) PEG 3350 (filter sterilize using a 0.22‐µm filter and store up to 1 year at room temperature in the dark)
  • 10 mg/ml single‐stranded carrier DNA (sonicate and store up to 1 year at −20°C; Gietz and Woods, )
  • TBSV expression plasmids:
    • pGAD‐His92 plasmid (Clontech)
    • pHisGBK‐His33 plasmid (Clontech)
    • pYC/DI‐72 plasmid (Invitrogen)
  • SC‐ULH/glucose agar plates (see recipe)
  • 96‐well, 2‐ml round‐bottom growth plates
  • Multichannel pipettors (20‐, 300‐, and 1000–µl) or a liquid handling robotic instrument
  • 96‐pin replicator (Phenix Research)
  • Gas permeable sealing membranes for covering plates (USA Scientific)
  • 30°C incubator shaker equipped with plate holders
  • Spectrophotometer (plate format)
  • Table‐top or floor centrifuge equipped with rotor for deep‐well plates
  • Corning storage mats (Fisher Scientific)
  • 42°C water bath

Basic Protocol 2: High‐Throughput Induction and Extraction of Viral RNA from Yeast

  Materials
  • SC‐ULH/galactose medium (see recipe) for TBSV and SC‐UH/glucose/copper medium (see recipe) for FHV
  • 10 mg/ml doxycycline in 50% ethanol
  • Yeast transformants (see protocol 1)
  • 1× yeast RNA extraction buffer (see recipe)
  • Phenol, water saturated
  • Chloroform
  • Absolute and 70% ethanol
  • 3 M sodium acetate, pH 5.3
  • 96‐deep‐well plates
  • Multichannel pipettor and tips
  • Breathable membrane
  • Centrifuge with rotor for 96‐well plates
  • 96‐well storage mats
  • 65°C water bath
  • SpeedVac rotary evaporator
NOTE: Each reagent should be molecular biology grade and RNase‐free.

Basic Protocol 3: Viral RNA Analysis with Agarose Gel Electrophoresis

  Materials
  • 2× RNA loading dye (see recipe)
  • RNA pellet (see protocol 2)
  • TBSV or FHV replicon
  • 1% to 1.5% agarose gel containing 0.5× TBE and 100 mg/liter ethidium bromide
  • 0.5× TBE
  • 96‐well storage mats
  • Multichannel pipettor
  • Large millipede electrophoresis unit with 96‐wells and multichannel pipettor‐compatible (IBI)
  • UV light
  • Gel documentation system

Basic Protocol 4: Viral RNA Analysis with Northern Blotting

  Materials
  • Agarose gel (see protocol 3)
  • 0.5× TBE (see recipe), sterile
  • 20× SSC (see recipe), sterile
  • UltraHyb hybridization buffer (Ambion)
  • Probe (e.g., riboprobes of 160 to 200 nt)
  • 50% formamide
  • Northern wash solution I (see recipe)
  • Northern wash solution II (see recipe)
  • Filter paper, extra thick
  • Nylon membrane (Hybond X‐L, Amersham‐Pharmacia)
  • RNase‐free trays
  • Shaking platform
  • Transblot apparatus (BioRad Transblot Semi‐dry)
  • UV chamber
  • Hybridization tubes with sealable caps
  • Hybridization oven
  • 85°C heating block
  • Plastic wrap
  • PhosphorImager cassettes
  • PhosphorImager or FluoroImager (depending on the probe used)
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Figures

Videos

Literature Cited

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   Panaviene, Z., Panavas, T., Serva, S., and Nagy, P.D. 2004. Purification of the cucumber necrosis virus replicase from yeast cells: Role of coexpressed viral RNA in stimulation of replicase activity. J. Virol. 78:8254‐8263.
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   Serviene, E., Shapka, N., Cheng, C.P., Panavas, T., Phuangrat, B., Baker, J., and Nagy, P.D. 2005. Genome‐wide screen identifies host genes affecting viral RNA recombination. Proc. Natl. Acad. Sci. U.S.A. 102:10545‐10550.
   Serviene, E., Jiang, Y., Cheng, C.P., Baker, J., and Nagy, P.D. 2006. Screening of the yeast yTHC collection identifies essential host factors affecting tombusvirus RNA recombination. J. Virol. 80:1231‐1241.
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Key References
   Panavas and Nagy, 2003. See above.
  This paper describes the development of yeast as a host for tombusvirus replication.
   Panavas et al., 2005b. See above.
  This paper describes the genome‐wide screen of yeast to identify host factors for tombusvirus replication.
   Serviene et al., 2005. See above.
  This paper describes a systematic genome‐wide screen of yeast for host proteins that affect RNA recombination in tombusviruses.
Internet Resources
   http://www.openbiosystems.com
  Distributor of yeast libraries for genome‐wide studies.
   http://www‐sequence.stanford.edu/group/yeast_deletion_project
  Saccharomyces genome database.
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