Mouse Polyomavirus: Propagation, Purification, Quantification, and Storage

Lenka Horníková1, Vojtěch Žíla1, Hana Španielová1, Jitka Forstová1

1 Department of Genetics and Microbiology, Charles University in Prague, Prague
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14F.1
DOI:  10.1002/9780471729259.mc14f01s38
Online Posting Date:  August, 2015
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Abstract

Mouse polyomavirus (MPyV) is a member of the Polyomaviridae family, which comprises non‐enveloped tumorigenic viruses infecting various vertebrates including humans and causing different pathogenic responses in the infected organisms. Despite the variations in host tropism and pathogenicity, the structure of the virions of these viruses is similar. The capsid, with icosahedral symmetry (ø, 45 nm, T = 7d), is composed of a shell of 72 capsomeres of structural proteins, arranged around the nucleocore containing approximately 5‐kbp‐long circular dsDNA in complex with cellular histones. MPyV has been one of the most studied polyomaviruses and serves as a model virus for studies of the mechanisms of cell transformation and virus trafficking, and for use in nanotechnology. It can be propagated in primary mouse cells (e.g., in whole mouse embryo cells) or in mouse epithelial or fibroblast cell lines. In this unit, propagation, purification, quantification, and storage of MPyV virions are presented. © 2015 by John Wiley & Sons, Inc.

Keywords: polyomavirus; mouse polyomavirus; density gradient purification; virus quantification

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Propagation of MPyV
  • Basic Protocol 2: MPyV Isolation and Purification
  • Alternate Protocol 1: Small‐Scale MPyV Propagation, Isolation, and Purification
  • Alternate Protocol 2: MPyV Propagation from Plasmid DNA
  • Virus Quantification
  • Basic Protocol 3: Quantification of Purified MPyV Stock by Plaque Assay
  • Basic Protocol 4: Quantification of Purified MPyV Stock by Immunological Detection of Early Proteins
  • Basic Protocol 5: Quantification of Purified MPyV Stocks by Hemagglutination Assay
  • Support Protocol 1: Growth and Maintenance of Cell Lines
  • Support Protocol 2: Detection of the MPyV VP1 Capsid Protein by Dot‐Blot Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Propagation of MPyV

  Materials
  • WME cells (whole mouse embryo cells; primary culture of mouse cells) or 3T6 cells (ATCC # CCL‐96) grown to near confluence in a 100‐mm Petri dish (for more information see protocol 8)
  • Serum‐free DMEM medium (see recipe)
  • Concentrated MPyV stock (prepared from plasmid as described in protocol 4)
  • Complete DMEM with antibiotics (see recipe), prewarmed
  • 100‐mm Petri dishes
  • 13‐mm coverslips (optional)
  • Rocking platform
  • Additional reagents and equipment for growth and maintenance of WME or 3T6 cells (see protocol 8) and detection of early large antigen ( protocol 6; optional)

Basic Protocol 2: MPyV Isolation and Purification

  Materials
  • Dishes with infected cultures ( protocol 1)
  • 10 mM Tris·Cl, pH 7.4 ( )
  • Neuraminidase (Sigma‐Aldrich, cat. no. N7885)
  • Aprotinin (Sigma‐Aldrich, cat. no. A6279).
  • 10 mM Tris·Cl, pH 9 ( )
  • 20% sucrose (see recipe)
  • Buffer B (see recipe)
  • Cesium chloride (CsCl)
  • Paraffin oil (e.g., Bayol F; Serva, cat. no. 14500.02)
  • 10% sucrose (see recipe)
  • 40% sucrose (see recipe)
  • Rubber scraper
  • 50‐ and 15‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Refrigerated centrifuge
  • 10‐ml Potter‐Elvehjem tissue grinder (homogenizer) with tight pestle, prechilled on ice
  • Rocking platform
  • Abbe refractometer (e.g., Carl Zeiss)
  • 38.5‐ml polyallomer ultracentrifuge tubes for SW28 rotor (Beckman Coulter, cat. no. 326823)
  • Ultracentrifuge with Beckman SW28 rotor, SW55Ti rotor, and SW41Ti rotor (or equivalents)
  • Sonicator with a microtip probe (e.g., Hielscher UP50H or equivalent)
  • 5‐ml polyallomer ultracentrifuge tubes for SW55Ti rotor (Beckman Coulter, cat. no. 326819)
  • Fraction recovery system (Beckman, cat. no. 343890)
  • 16‐mm dialysis tubing, MWCO 12,000 to 14,000 (e.g., Servapol)
  • 13.2‐ml polyallomer ultracentrifuge tubes for SW41Ti rotor (Beckman Coulter, cat. no. 331372)
  • Linear gradient maker (e.g., Hoefer SG50 or equivalent)
  • 0.6‐ml siliconized tubes (e.g., Sigma‐Aldrich, cat. no. T4691)
  • Additional reagents and equipment for detection of polyoma capsid protein by dot‐blot analysis ( protocol 9), and dialysis (Zumstein, )

Alternate Protocol 1: Small‐Scale MPyV Propagation, Isolation, and Purification

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • 150‐mm Petri dish

Alternate Protocol 2: MPyV Propagation from Plasmid DNA

  Materials
  • pMJA3 plasmid DNA or equivalent (endotoxin‐free, isolated from bacteria with, e.g., Qiagen Endofree plasmid kit or alternative)
  • EcoRI restriction endonuclease and 10× buffer (Thermo Scientific or equivalent)
  • 3 M sodium acetate, pH 5.2 ( )
  • 96% (v/v) ethanol
  • 70% (v/v) ethanol
  • T4 DNA ligase and 10× buffer (Thermo Scientific or equivalent)
  • Nuclease‐free water
  • 1% agarose gel and running buffer (Voytas, )
  • Amaxa Cell Line Nucleofector Kit V (Lonza, cat. no. VCA‐1003)
  • RPMI 1640 medium (Sigma‐Aldrich, cat. no. R0883)
  • Complete DMEM with antibiotics (see recipe)
  • 3T6 cells (ATCC # CCL‐96)
  • Heat block or water bath
  • Refrigerated high‐speed table‐top centrifuge (e.g., Beckman Microcentrifuge R or equivalent)
  • NanoDrop spectrometer (or equivalent)
  • 50‐ml conical centrifuge tubes
  • Refrigerated table‐top centrifuge with swinging‐bucket rotor (e.g., Beckman GS‐15R or equivalent)
  • Nucleofector II electroporator (Lonza)
  • 150‐mm Petri dishes
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ) and immunofluorescent staining of cells for MPyV large T antigen (see protocol 6; optional)

Basic Protocol 3: Quantification of Purified MPyV Stock by Plaque Assay

  Materials
  • MEF cells grown to near confluence in a 100‐mm tissue culture Petri dish (see protocol 8)
  • Complete DMEM medium (see recipe)
  • Serum‐free DMEM medium (see recipe)
  • Complete DMEM medium with 0.8% agarose (see recipe)
  • MPyV virus stock ( protocol 2)
  • Staining solution (see recipe)
  • 6‐well tissue plates
  • Rocking platform
  • 0.6‐ml siliconized tubes (e.g., Sigma‐Aldrich, cat. no. T4691)
NOTE: MEF cells can be prepared as described by Hogan et al. ( ) or obtained from Lonza (M‐FB‐481) or American Type Culture Collection (ATCC #CCL‐2752)

Basic Protocol 4: Quantification of Purified MPyV Stock by Immunological Detection of Early Proteins

  Material
  • 3T6 cells grown to near confluence in a 60‐mm tissue culture Petri dish ( protocol 8)
  • Complete DMEM (no antibiotics; see recipe)
  • Complete DMEM medium with antibiotics (see recipe)
  • Serum‐free DMEM medium (see recipe)
  • MPyV virus stock ( protocol 2)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Fixing solution (see recipe)
  • Permeabilizing solution (see recipe)
  • Blocking solution (see recipe)
  • Primary antibody: rat monoclonal anti‐MPyV LT antibody (e.g., Abcam, cat. no. ab18187 or equivalent)
  • Secondary antibody: goat anti‐rat IgG conjugated with Alexa Fluor 488 (e.g., Life Technologies, cat. no. A‐11006 or equivalent)
  • Mounting medium (50% glycerol; see recipe)
  • 24‐well tissue culture plates
  • 13‐mm coverslips
  • Microscope slides
  • Fluorescent microscope with appropriate filters

Basic Protocol 5: Quantification of Purified MPyV Stocks by Hemagglutination Assay

  Materials
  • Red blood cells from guinea pig in Alsever's solution (Sigma‐Aldrich, cat. no. A3551)
  • Hemagglutination buffer (see recipe)
  • MPyV virus stock ( protocol 2)
  • Buffer B (see recipe)
  • Capillaries with surface treated with heparin (Hirschmann Inc., cat. no. 9100160)
  • Refrigerated centrifuge
  • Burner
  • Centrifuge with rotor for hematocrit tubes
  • 96‐well U‐bottom plate
  • 8‐channel repeating pipettor

Support Protocol 1: Growth and Maintenance of Cell Lines

  Materials
  • 3T6 cells (ATCC #CCL‐96), MEF cells, or WME cells grown to near confluence in a 100‐mm Petri dish
  • 0.02% EDTA (see recipe)
  • 0.25% trypsin (see recipe)
  • Complete DMEM medium (see recipe)
  • 100‐mm Petri dish
  • Inverted tissue culture microscope
  • 37°C water bath

Support Protocol 2: Detection of the MPyV VP1 Capsid Protein by Dot‐Blot Analysis

  Materials
  • Virus fractions ( protocol 2, step 20, or optionally also step 8)
  • 5% nonfat milk solution (see recipe)
  • Primary antibody: mouse monoclonal anti‐MPyV VP1 antibody (Forstová et al., 1993) or equivalent
  • PBS (see recipe)
  • Secondary antibody: HRP‐conjugated goat anti mouse antibody (e.g., BioRad, cat. no. 172‐1011) or equivalent
  • Reagents for signal visualization, e.g., Pierce ECL Western Blotting Substrate (Thermo Scientific)
  • Nitrocellulose membrane, pore size 0.45 μm (e.g., Serva or equivalent)
  • Trays for membrane washing
  • X‐ray film and equipment for development in the dark room
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Figures

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Literature Cited

Literature Cited
   Bauer, P.H. , Cui, C. , Stehle, T. , Harrison, S.C. , DeCaprio, J.A. , and Benjamin, T.L. , 1999. Discrimination between sialic acid‐containing receptors and pseudoreceptors regulates polyomavirus spread in the mouse. J. Virol. 73:5826‐5832.
   Bauer, P.H. , Bronson, R.T. , Fung, S.C. , Freund, R. , Stehle, T. , Harrison, S.C. , and Benjamin, T.L. , 1995. Genetic and structural analysis of a virulence determinant in polyomavirus VP1. J. Virol. 69:7925‐7931.
   Berke, Z. and Dalianis, T. 2000. Studies on polyomavirus persistence and polyomavirus‐induced tumor development in relation to the immune system. Adv. Cancer Res. 79:249‐276.
   Bolen, J.B. , Fisher, S.E. , Chowdhury, K. , Shan, T.C. , Williams, J.E. , Dawe, C.J. , and Israel, M.A. 1985. A determinant of polyomavirus virulence enhances virus growth in cells of renal origin. J. Virol. 53:335‐339.
   Brockman, W.W. and Nathans, D. 1974. The isolation of simian virus 40 variants with specifically altered genomes. Proc. Natl. Acad. Sci. U.S.A. 71:942‐946.
   Burnett, L. C. , Lunn, G. , and Coico, R. 2009. Biosafety: Guidelines for working with pathogenic and infectious microorganisms. Curr. Protoc. Microbiol. 13:1A.1.1‐1A.1.14.
   Chen, L. and Fluck, M. 2001. Kinetic analysis of the steps of the polyomavirus lytic cycle. J. Virol. 75:8368‐8379.
   Dawe, C.J. , Freund, R. , Mandel, G. , Ballmer‐Hofer, K. , Talmage, D.A. , and Benjamin, T.L. 1987. Variations in polyoma virus genotype in relation to tumor induction in mice. Characterization of wild type strains with widely differing tumor profiles. Am. J. Pathol. 127:243‐261.
   Eddy, B.E. and Stewart, S.E. 1959. Characteristics of the SE Polyoma Virus. Am. J. Public Health Nations Health 49:1486‐1492.
   Feng, H. , Shuda, M. , Chang, Y. , and Moore, P. S. 2008. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 319:1096‐1100.
   Freund, R. , Garcea, R.L. , Sahli, R. , and Benjamin, T.L. 1991. A single‐amino‐acid substitution in polyomavirus VP1 correlates with plaque size and hemagglutination behavior. J. Virol. 65:350‐355.
   Gross, L. 1953. A filterable agent, recovered from Ak leukemic extracts, causing salivary gland carcinomas in C3H mice. Proc. Soc. Exp. Biol. Med. 83:414‐421.
   Hogan, B. , Beddington, R. , Costantini, F. , and Lacy, E. 1994. Manipulating the Mouse Embryo: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press.
   Huang, A.S. 1973 Defective interfering viruses. Annu. Rev. Microbiol. 27:101‐117.
   Kohse, L.M. , McGrath, L. , and Hudson, J.B. 1971. The recovery of polyoma virus from infected mouse cells: Relevance to virus purification. Can. J. Microbiol. 17:775‐781.
   Krauzewicz, N. , Streuli, C.H. , Stuart‐Smith, N. , Jones, M.D. , Wallace, S. , and Griffin, B.E. 1990. Myristylated polyomavirus VP2: Role in the life cycle of the virus. J. Virol. 64:4414‐4420.
   Nakanishi, A. , Chapellier, B. , Maekawa, N. , Hiramoto, M. , Kuge, T. , Takahashi, R. , Handa, H. , and Imai, T. 2008. SV40 vectors carrying minimal sequence of viral origin with exchangeable capsids. Virology 379:110‐117.
   Orcutt, R. 1994. Polyoma virus. In Manual of Microbiologic Monitoring of Laboratory Animals ( K. Waggie , ed.). Diane Publishing, Darby, Pa.
   Parker, J.C. , Tennant, R.W. , and Ward, T.G. 1966. Prevalence of viruses in mouse colonies. Natl. Cancer Inst. Monogr. 20:25‐45.
   Pastrana, D.V. , Tolstov, Y.L. , Becker, J.C. , Moore, P.S. , Chang, Y. , and Buck, C.B. 2009. Quantitation of human seroresponsiveness to merkel cell polyomavirus. PLoS Pathog. 5:e1000578. doi:10.1371/journal.ppat.1000578.
   Rochford, R. , Moreno, J.P. , Peake, M.L. , and Villarreal, L.P. 1992. Enhancer dependence of polyomavirus persistence in mouse kidneys. J. Virol. 66:3287‐3297.
   Stehle, T. and Harrison, S.C. 1996. Crystal structures of murine polyomavirus in complex with straight‐chain and branched‐chain sialyloligosaccharide receptor fragments. Structure 4:183‐194.
   Stehle, T. , Yan, Y. , Benjamin, T.L. , and Harrison, S.C. 1994. Structure of murine polyomavirus complexed with an oligosaccharide receptor fragment. Nature 369:160‐163.
   Thorne, H.V. 1973. Cyclic variation in susceptibility of balb‐c 3T3 cells to polyoma virus. J. Gen. Virol. 18:163‐169.
   Türler, H. and Beard, P. 1985. Simian virus 40 and polyoma virus: Growth, titration, transformation and purification of viral components. In Virology: A Practical Approach ( B.W.J. Mahy , ed.) pp. 169‐192. IRL Press, Oxford.
   Türler, H. and Salomon, C. 1985. Small and middle T antigens contribute to lytic and abortive polyomavirus infection. J. Virol. 53:579‐586.
   Twigg, A.J. and Sherratt, D. 1980. Trans‐complementable copy‐number mutants of plasmid ColEl. Nature (London) 283:216‐218.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   Weil, R. , May, E. , May, P. , and Türler, H. 1974. A study on polyoma and SV40‐induced chromosome replication and mitosis. In Molecular Studies in Viral Neoplasia (25th Annual Symposium on Fundamental Cancer Research) pp. 397‐419. The University of Texas, Houston, Texas.
   White, M.K. , Gordon, J. , and Khalili, K. 2013. The rapidly expanding family of human polyomaviruses: Recent developments in understanding their life cycle and role in human pathology. PLoS Pathog. 9: doi: 10.1371/journal.ppat.1003206.
   Zumstein, L. 1998. Dialysis and ultrafiltration. Curr. Protoc. Mol. Biol. 41:A.3C.1‐A.3C.7.
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