Newcastle Disease Virus‐Like Particles: Preparation, Purification, Quantification, and Incorporation of Foreign Glycoproteins

Lori W. McGinnes1, Trudy G. Morrison1

1 University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 18.2
DOI:  10.1002/9780471729259.mc1802s30
Online Posting Date:  October, 2013
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Abstract

Virus‐like particles (VLPs) are large particles, the size of viruses, composed of repeating structures that mimic those of infectious virus. Since their structures are similar to that of viruses, they have been used to study the mechanisms of virus assembly. They are also in development for delivery of molecules to cells and in studies of the immunogenicity of particle‐associated antigens. However, they have been most widely used for development of vaccines and vaccine candidates. VLPs can form upon the expression of the structural proteins of many different viruses. This chapter describes the generation and purification of VLPs formed with the structural proteins, M, NP, F, and HN proteins, of Newcastle disease virus (NDV). Newcastle disease virus‐like particles (ND VLPs) have also been developed as a platform for assembly into VLPs of glycoproteins from other viruses. This chapter describes the methods for this use of ND VLPs. Curr. Protoc. Microbiol. 30:18.2.1‐18.2.21. © 2013 by John Wiley & Sons, Inc.

Keywords: virus‐like particles; Newcastle disease virus; paramyxovirus; chimera glycoproteins

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Large‐Scale Generation of ND VLPs
  • Alternate Protocol 1: Small‐Scale VLP Generation
  • Support Protocol 1: Radioactive Labeling of VLPs
  • Basic Protocol 2: Large‐Scale Purification of VLPs
  • Alternate Protocol 2: Small‐Scale VLP Purification
  • Basic Protocol 3: Quantification of Protein Content of VLPs
  • Alternate Protocol 3: Use of Western Blotting for Characterization of VLP Protein Content
  • Alternate Protocol 4: Use of Autoradiography for Assessment of Protein Incorporation into VLPs
  • Basic Protocol 4: Incorporation of Foreign Glycoproteins into ND VLPs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Large‐Scale Generation of ND VLPs

  Materials
  • Tissue culture cells (ELL‐0 are preferable but COS7, CHO, or 293T cell lines are acceptable; all are available at http://www.atcc.org)
  • Cell culture medium such as DMEM with appropriate supplementation (see recipe) for the cells to be used (see instructions from ATCC)
  • Plasmids containing cDNAs encoding NDV NP, M, F, and HN proteins (prepared using Qiagen Endo‐free kit or the equivalent; use of the pCAGGS vector as the expression vector is highly recommended—this plasmid may be obtained from the Belgian Coordinated Collection of Microorganisms, BCCM/LMBP; bccm.lmbp@dmbr.UGent.be)
  • Lipofectamine (Life Technologies) or similar transfection reagent
  • Fetal bovine serum (heat inactivated at 56°C for 30 min; see appendix 2A)
  • 150‐cm2 (T‐150) tissue culture flasks
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)

Alternate Protocol 1: Small‐Scale VLP Generation

  Additional Materials (also see protocol 1)
  • 6‐well tissue culture plates (or individual 35‐mm plates)

Support Protocol 1: Radioactive Labeling of VLPs

  Additional Materials (also see protocol 1 and protocol 2)
  • DMEM without methionine and cysteine (Life Technologies)
  • Dialyzed fetal bovine serum (dialyzed FBS; Life Technologies)
  • [35S]cysteine/methionine (PerkinElmer Express 35S Protein Labeling Mix, or equivalent)

Basic Protocol 2: Large‐Scale Purification of VLPs

  Materials
  • Supernatants containing large‐scale amounts of VLPs ( protocol 1)
  • DMEM medium with FBS
  • TNE buffer (see recipe)
  • 10%, 20%, 25%, 35%, 45%, 50%, 55%, 65%, 80% (w/v) sucrose solutions in TNE buffer (see recipe for buffer)
  • Adaptors and 250‐ml sterile/disposable conical centrifuge bottles (or comparable)
  • Beckman GS‐6R centrifuge (or comparable)
  • Beckman Type 19 rotor and Type 19 bottles and Deldrin Cap Assemblies (or comparable)
  • Glass Dounce homogenizer (10 ml) with a tight‐fitting pestle, pre‐chilled on ice (optional)
  • Beckman SW28.1, SW41, SW50.1 rotors, buckets, and tubes (30‐ml, 17‐ml, 11‐ml, and 5‐ml tubes) or equivalent
  • Beckman ultracentrifuge (or comparable)
  • 150‐cm2 (T‐150) tissue culture flasks
  • 2‐ml vials stable at −80°C

Alternate Protocol 2: Small‐Scale VLP Purification

  Additional Materials (also see protocol 4)
  • Supernatants containing small‐scale amounts of VLPs ( protocol 2)
  • TNE buffer (see recipe) or gel sample buffer (see recipe)
  • Beckman GS‐6R centrifuge (or comparable)
  • 20% and 65% sucrose solutions dissolved in TNE buffer (weight/volume)
  • Beckman ultracentrifuge (or comparable)
  • SW50.1 rotor and buckets or comparable
  • SW50.1 tubes

Basic Protocol 3: Quantification of Protein Content of VLPs

  Materials
  • Protein standard (e.g., BSA standards from BioRad Quick Start Protein Assay)
  • Large‐scale purified VLPs ( protocol 4)
  • Silver stain kit (Thermo Scientific) or Coomassie stain kit (Life Technologies or BioRad or comparable; also see appendix 3M)
  • Rotator (platform shaker)
  • Gel scanner (Syngene G Box or comparable, http://www.syngene.com/)
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis ( appendix 3M)

Alternate Protocol 3: Use of Western Blotting for Characterization of VLP Protein Content

  Additional Materials (also see protocol 6)
  • Specific protein standard (same as protein of interest)
  • Large‐scale purified VLPs ( protocol 4) or small‐scale purified VLPs ( protocol 5)
  • Western blot transfer system (iBlot gel transfer system from Invitrogen, or comparable)
  • Western blot chemiluminescence detection reagents (ECL from GE Health Care, or comparable)
  • PBS‐Tween: phosphate‐buffered saline (PBS; see recipe) containing 0.5% (v/v) Tween 20
  • Nonfat powdered milk
  • Protein‐specific antibodies
  • Rotator
  • X‐ray film (Thermo Scientific CL‐XPosure Film) or comparable or instrument designed to detect western blots as, for example, Syngene G box)
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis ( appendix 3M) and western blotting (immunoblotting; see unit 14.2, Basic Protocol 4 and Gallagher et al., )

Alternate Protocol 4: Use of Autoradiography for Assessment of Protein Incorporation into VLPs

  Materials
  • Radioactively labeled VLPs prepared in protocol 3 and purified by protocol 5
  • Gel dryer (BioRad or comparable)
  • X‐ray film for detection of radioactively labeled proteins (Kodak BioMax XAR Film from Carestream Health, http://www.carestream.com/, 5 × 7 in.; cat. no. 05‐728‐36, or comparable)
  • X‐ray film processor
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis ( appendix 3M)

Basic Protocol 4: Incorporation of Foreign Glycoproteins into ND VLPs

  Additional Materials (also see Alternate Protocols protocol 21 and protocol 52)
  • Restriction enzymes
  • Tissue culture cells (ELL‐0 are preferable but COS7, CHO, or 293T cell lines are acceptable; all are available at http://www.atcc.org)
  • PBS‐CM (see recipe)
  • Sulfo‐NHS‐SS‐biotin (Pierce)
  • Complete cell culture medium containing serum
  • Cell lysis buffer (see recipe)
  • Neutravidin agarose resin (Pierce)
  • Tween 20
  • 10% sodium dodecyl sulfate (SDS)
  • Gel sample buffer (see recipe)
  • DNA synthesis facility (e.g., GeneWiz, Gene Art, Celtek Genes)
  • 35‐mm tissue culture plates
  • Rotator
  • Additional reagents and equipment for basic molecular biological techniques (primer design, PCR, restriction digestion, ligation; Ausubel et al., ), small‐scale VLP generation ( protocol 2), polyacrylamide gel electrophoresis ( appendix 3M), and western blotting (immunoblotting; see protocol 7; unit 14.2, Basic Protocol 4; and Gallagher et al., ), generation of VLPs containing chimera proteins ( protocol 5), and assessment of efficiency of incorporation of chimera protein into ND VLPs ( protocol 7 or 4)
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Figures

Videos

Literature Cited

Literature Cited
  Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.). 2013. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
  Ciancanelli, M.J. and Basler, C.F. 2006. Mutation of the YMXL in the Nipah virus matrix protein abrogates budding and alters subcellular localization. Virol. J. 80:12070‐12078.
  Collins, P.L. and Crowe, J.E. 2007. Respiratory Syncytial Virus and Metapneumovirus, 5th ed. Lippincott Williams and Wilkins, Philadelphia.
  Coronel, E.C., Murti, K.G., Takimoto, T., and Portner, A. 1999. Human parainfluenza virus type I matrix and nucleocapsid genes transiently expressed in mammalian cells induce release of virus‐like particles containing nucleocapsid structures. J. Virol. 73:7035‐7038.
  Deml, L., Speth, C., Dierich, M.P., Wolf, H., and Wagner, R. 2005. Recombinant HIV‐1 Pr55gag virus‐like particles: Potent stimulators of innate and acquired immune responses. Mol. Immunol. 42:259‐277.
  Fu, T.‐M., Grimm, K.M., Citron, M.P., Freed, D.C., Fan, J., Keller, P.M., Shiver, J.W., Liang, X., and Joyce, J.G. 2009. Comparative immunogenicity evaluations of influenza A virus M2 peptide as recombinant virus like particle or conjugate vaccines in mice and monkeys. Vaccine 27:1440‐1447.
  Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. 2008. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 83:10.8.1‐10.8.28.
  Grgacic, E.V.L. and Anderson, D.A. 2006. Virus‐like particles: Passport to immune recognition. Methods 40:60‐65.
  Halsey, R.J., Tanzer, F.L., Meyers, A., Pillay, S., Lynch, A., Shephard, E., Williamson, A.‐L., and Rybicki, E.P. 2008. Chimaeric HIV‐1 subtype C Gag molecules with large in‐frame C‐terminal polypeptide fusions form virus‐like particles. Virus Res. 133:259‐268.
  Harper, D.M., Franco, E.L., Wheeler, C.M., Moscicki, A.‐B., Romanowski, B., Roteli‐Martins, C.M., Jenkins, D., Schuind, A., Clemens, S.A.C., and Dubin, G. 2006. Sustained efficacy up to 4‐5 years of a bivalent L1 virus‐like particle vaccine against human papillomavirus types 16 and 18: Follow‐up from a randomised trial. Lancet 367:1247‐1255.
  Haynes, J.R., Dokken, L., Wiley, J.A., Cawthon, A.G., Bigger, J., Harmsen, A.G., and Richardson, C. 2009. Influenza‐pseudotyped Gag virus‐like particle vaccines provide broad protection against highly pathogenic avian influenza challenge. Vaccine 27:530‐541.
  Jennings, G.T. and Bachmann, M.F. 2008. The coming of age of virus‐like particle vaccines. Biol. Chem. 389:521‐536.
  Kaczmarczyk, S.J., Sitaraman, K., Young, H.A., Hughes, S.H., and Chatterjee, D.K. 2012. Protein delivery using engineered virus‐like particles. Proc. Natl. Acad. Sci. U.S.A. 108:16998‐17003.
  Kang, S.‐M., Song, J.‐M., Quan, F.‐S., and Compans, R.W. 2009. Influenza vaccines based on virus‐like particles. Virus Res. 143:140‐146.
  Karron, R.A. and Collins, P.L. 2007. Parainfluenza viruses. In Fields Virology, vol. 1 (D.M. Knipe and P.M. Howley, eds.) pp. 1497‐1526. Lippincott Williams & Wilkins, Philadelphia.
  Lamb, R.A. and Parks, G.D. 2007. Paramyxoviridae: The viruses and their replication. In Fields Virology, Fifth Edition, vol 1. (D.M. Knipe, P.M. Howley, D.E. Griffin, R.A. Lamb, M.A. Martin, B. Roizman, and S.E. Strauss, eds.) pp 1450‐1496. Lippincott Williams & Wilkins, Philadelphia.
  Li, M., Schmitt, P.T., Li, Z., McCrory, T.S., He, B., and Schmitt, A.P. 2009. Mumps virus matrix, fusion, and nucleocapsid proteins cooperate for efficient production of virus‐like particles. J. Virol. 83:7261‐7272.
  Ludwig, C. and Wagner, R. 2007. Virus‐like particles―universal molecular toolboxes. Curr. Opin. Biotechnol. 18:537‐545.
  McGinnes, L.W., Pantua, H., Laliberte, J.P., Gravel, K.A., Jain, S., and Morrison, T.G. 2010. Assembly and biological and immunological properties of Newcastle disease virus‐like particles. J. Virol. 84:4513‐4523.
  McGinnes, L.W., Gravel, K.A., Finberg, R.W., Kurt‐Jones, E.A., Massare, M.J., Smith, G., Schmidt, M.R., and Morrison, T.G. 2011. Assembly and immunological properties of Newcastle disease virus‐like particles containing the respiratory syncytial virus F and G proteins. J. Virol. 85:366‐377.
  Morrison, T.G. 2010. Newcastle disease virus‐like particles as a platform for the development of vaccines for human and agricultural pathogens. Future Virol. 5:545‐554.
  Murawski, M.R., McGinnes, L.W., Finberg, R.W., Kurt‐Jones, E.A., Massare, M., Smith, G., Heaton, P.M., Fraire, A., and Morrison, T.G. 2010 Newcastle disease virus‐like particles containing respiratory syncytial virus G protein induced protection in BALB/c mice with no evidence of immunopathology. J. Virol. 84:1110‐1123.
  Noad, R. and Roy, P. 2003. Virus‐like particles as immunogens. Trends Microbiol. 11:438‐444.
  Pantua, H.D., McGinnes, L.W., Peeples, M.E., and Morrison, T.G. 2006. Requirements for the assembly and release of Newcastle disease virus‐like particles. J. Virol. 80:11062‐11073.
  Patch, J.R., Crameri, G., Wang, L.F., Eaton, B.T., and Broder, C.C. 2007. Quantitative analysis of Nipah virus proteins released as virus‐like particles reveals a central role for the matrix protein. Virol. J. 4:1‐12.
  Quan, F.‐S., Kim, Y., Lee, S., Yi, H., Kang, S.‐M., Bozja, J., Moore, M.L., and Compans, R.W. 2011. Virus‐like particle vaccine induces protection against respiratory syncytial virus infection in mice. J. Infect. Dis. 204:987‐995.
  Sadeyen, J.R., Tourne, S., Shkreli, M., Sizaret, P.‐Y., and Coursaget, P. 2003. Insertion of a foreign sequence on capsid surface loops of human papillomavirus type 16 virus‐like particles reduces their capacity to induce neutralizing antibodies and delineates a conformational neutralizing epitope. Virology 309:32‐40.
  Saini, M. and Vrati, S. 2003. A Japanese encephalitis virus peptide present on Johnson grass mosaic virus‐like particles induces virus‐neutralizing antibodies and protects mice against lethal challenge. J. Virol. 77:3487‐3494.
  Schmidt, M.R., McGinnes, L.W., Kenward, S.A., Willems, K.N., Woodland, R.T., and Morrison, T.G. 2012. Long term and memory immune responses in mice against Newcastle disease virus‐like particles containing respiratory syncytial virus glycoprotein ectodomains. J. Virol. 86:11654‐11662.
  Schmitt, A.P., Leser, G.P., Waning, D.L., and Lamb, R.A. 2002. Requirements for budding of paramyxovirus simian virus 5 virus‐like particles. J. Virol. 76:3952‐3964.
  Sugahara, F., Uchiyama, T., Watanabe, H., Shimazu, Y., Kuwayama, M., Fujii, Y., Kiyotani, K., Adachi, A., Kohno, N., Yoshida, T., and Sakaguchi, T. 2004. Paramyxovirus Sendai virus‐like particle formation by expression of multiple viral proteins and acceleration of its release by C protein. Virology 325:1‐10.
  Takimoto, T., Bousse, T., Coronel, E.C., Scroggs, R.A., and Portner, A. 1998. Cytoplasmic domain of Sendai virus HN protein contains a specific sequence required for its incorporation into virions. J. Virol. 72:9747‐9754.
  Takimoto, T., Murti, K.G., Bousse, T., Scroggs, R.A., and Portner, A. 2001. Role of matrix and fusion proteins in budding of Sendai virus. J. Virol. 75:11384‐11391.
  Ye, L., Lin, J., Sun, Y., Bennouna, S., Lo, M., Wu, Q., Bu, Z., Pulendran, B., Compans, R.W., and Yang, C. 2006. Ebola virus‐like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies. Virology 351:260‐270.
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