Retroviral Pseudotypes

Nigel J Temperton, University College London, London, United Kingdom
Edward Wright, University College London, London, United Kingdom

Published online: March 2009

DOI: 10.1002/9780470015902.a0021549

Retroviral pseudotypes are a main research and diagnostic tool of basic and clinical scientists facilitating the detailed study of individual viral genes, receptors and highly pathogenic viruses, without the need to handle live virus. Their use as gene therapy vectors is widely documented, but their other uses are less well known. The switching of envelope proteins expressed on the surface enables them to be used as surrogate viruses in neutralization/antiviral assays and for the study of cell–virus receptor interactions. However, they have recently been used as vaccine immunogens, expressing the antigen either on the pseudotype surface or as the transfer gene for stable or transient expression. Only now is their full potential being realized.

Key concepts

Students will learn:

  • What are retroviral pseudotypes.
  • How they are constructed.
  • The common elements that comprise a retroviral pseudotype.
  • How retroviral pseudotypes can be used to deliver genes.
  • How they can be used as tools for the study of viral attachment, entry and receptor identification.
  • About using pseudotypes as surrogate viruses in neutralization assays.
  • The use of pseudotypes to study antiviral potency and resistance.
  • How pseudotypes can be used as experimental vaccines.

Keywords: retroviral pseudotype; lentiviral pseudotype; tropism; neutralization assays; virus neutralization; virus envelope

Figure 1. Flexible plasmid platform used to produce pseudotype particles. (a) The retroviral structural (GAG) and enzymatic (POL) proteins are expressed from a single plasmid lacking the packaging signal that has been removed to prevent incorporation into mature particles. PRO=promoter. (b) Envelope (env) gene expression is driven by a promoter specific to the envelope. (c) The transfer/marker gene (insert) is cloned between flanking LTR regions that play a role in integration and transcription of the pseudotype's RNA genome. A packaging signal () and further promoter (PRO) are incorporated upstream of the transfer gene to ensure it is packaged into pseudotype particle and regulates expression once the gene is integrated.
Figure 2. Pseudotype production and uses. Plasmids are transfected into producer cells and the resulting pseudotype particles are harvested 48 and 72 h posttransfection. These particles comprise a core encasing the RNA genome, surrounded by an outer matrix protein complex, with the cell plasma membrane, displaying the foreign envelope proteins, forming the viral membrane. These mature pseudotype particles can then be added onto target cells and the level of transduction/infection measured using the appropriate methods.
Figure 3. Reporter gene read-outs for use in measuring transduction of target cells. The detection of pseudotypes depends on the reporter-transfer gene incorporated into the particle. This can be (a) lacZ and staining directed for infection foci (X-gal) or with a colorimetric substrate such as CPRG or ONPG. (b) A fluorescent protein such as GFP. (c) The light emitted from luciferase oxidizing its substrate luciferin (Luc).
close
 References
    Adam MA, Ramesh N, Miller AD and Osborne WR (1991) Internal initiation of translation in retroviral vectors carrying picornavirus 5¢ nontranslated regions. Journal of Virology 65: 4985–4990.
    Bartosch B, Bukh J, Meunier JC et al. (2003) In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes. Proceedings of the National Academy of Sciences of the USA 100: 14199–14204.
    Buchschacher GL Jr. (2001) Introduction to retroviruses and retroviral vectors. Somatic Cell and Molecular Genetics 26: 1–11.
    book Capecchi B, Fasolo A, Alberini I et al. (2008) "Use of pseudotyped particles expressing Influenza A/Vietnam/1194/2004 hemagglutinin in neutralization assays". In: Katz JM (ed.) Options for the Control of Influenza VI, pp. 303–305. London: International Medical Press.
    Chai N, Chang HE, Nicolas E et al. (2007) Assembly of hepatitis B virus envelope proteins onto a lentivirus pseudotype that infects primary human hepatocytes. Journal of Virology 81: 10897–10904.
    Clements MO, Godfrey A, Crossley J et al. (2006) Lentiviral manipulation of gene expression in human adult and embryonic stem cells. Tissue Engineering 12: 1741–1751.
    Deng HK, Unutmaz D, KewalRamani VN and Littman DR (1997) Expression cloning of new receptors used by simian and human immunodeficiency viruses. Nature 388: 296–300.
    Dye C, Temperton N and Siddell SG (2007) Type I feline coronavirus spike glycoprotein fails to recognize aminopeptidase N as a functional receptor on feline cell lines. Journal of General Virology 88: 1753–1760.
    Hacein-Bey-Abina S, von Kalle C, Schmidt M et al. (2003) A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. New England Journal of Medicine 348: 255–256.
    Higashikawa F and Chang L (2001) Kinetic analyses of stability of simple and complex retroviral vectors. Virology 280: 124–131.
    Hofmann H, Pyrc K, van der Hoek L et al. (2005) Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry. Proceedings of the National Academy of Sciences of the USA 102: 7988–7993.
    Holmes RK, Malim MH and Bishop KN (2007) APOBEC-mediated viral restriction: not simply editing? Trends in Biochemical Sciences 32: 118–128.
    Kim YB, Lee MK, Han DP and Cho MW (2001) Development of a safe and rapid neutralization assay using murine leukemia virus pseudotyped with HIV type 1 envelope glycoprotein lacking the cytoplasmic domain. AIDS Research and Human Retroviruses 17: 1715–1724.
    Li CK, Wu H, Yan H et al. (2008) T cell responses to whole SARS coronavirus in humans. Journal of Immunology 181: 5490–5500.
    Nefkens I, Garcia JM, Ling CS et al. (2007) Hemagglutinin pseudotyped lentiviral particles: characterization of a new method for avian H5N1 influenza sero-diagnosis. Journal of Clinical Virology 39: 27–33.
    Neil SJ, Zang T and Bieniasz PD (2008) Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 451: 425–430.
    Neumann J, Stitz J, Konig R et al. (2006) Retroviral vectors for vaccine development: induction of HIV-1-specific humoral and cellular immune responses in rhesus macaques using a novel MLV(HIV-1) pseudotype vector. Journal of Biotechnology 124: 615–625.
    Parry CM, Kohli A and Pillay D (2008) Gag-protease interrelationships in drug resistance and viral fitness. HIV Medicine 9(suppl. 1): 3.
    Pizzato M, Franchin E, Calvi P et al. (1998) Production and characterization of a bicistronic Moloney-based retroviral vector expressing human interleukin 2 and herpes simplex virus thymidine kinase for gene therapy of cancer. Gene Therapy 5: 1003–1007.
    Rubin H (1965) Genetic control of cellular susceptibility to pseudotypes of Rous sarcoma virus. Virology 26: 270–276.
    Saeed MF, Kolokoltsov AA and Davey RA (2006) Novel, rapid assay for measuring entry of diverse enveloped viruses, including HIV and rabies. Journal of Virological Methods 135: 143–150.
    Sanders DA (2002) No false start for novel pseudotyped vectors. Current Opinion in Biotechnology 13: 437–442.
    Sandrin V and Cosset FL (2006) Intracellular versus cell surface assembly of retroviral pseudotypes is determined by the cellular localization of the viral glycoprotein, its capacity to interact with Gag, and the expression of the Nef protein. Journal of Biological Chemistry 281: 528–542.
    Schroder AR, Shinn P, Chen H et al. (2002) HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110: 521–529.
    Sharkey CM, North CL, Kuhn RJ and Sanders DA (2001) Ross River virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production. Journal of Virology 75: 2653–2659.
    Simmons G, Reeves JD, Rennekamp AJ et al. (2004) Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry. Proceedings of the National Academy of Sciences of the USA 101: 4240–4245.
    Su CY, Wang SY, Shie JJ et al. (2008) In vitro evaluation of neuraminidase inhibitors using the neuraminidase-dependent release assay of hemagglutinin-pseudotyped viruses. Antiviral Research 79: 199–205.
    Szecsi J, Boson B, Johnsson P et al. (2006) Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses. Virology Journal 3: 70.
    book Temperton NJ, Capecchi B, Rappuoli R et al. (2008) "Measurement of neutralizing antibody responses to influenza H5N1 clade 1 A/Viet Nam/1194/04 and clade 2 A/Indonesia/5/05 hemagglutinin using a sensitive high throughput luciferase-based retroviral pseudotype assay". In: Katz JM (ed.) Options for the Control of Influenza VI, pp. 94–96. London: International Medical Press.
    Temperton NJ, Chan PK, Simmons G et al. (2005) Longitudinally profiling neutralizing antibody response to SARS coronavirus with pseudotypes. Emerging Infectious Diseases 11: 411–416.
    Temperton NJ, Hoschler K, Major D et al. (2007) A sensitive retroviral pseudotype assay for Influenza H5N1 neutralizing antibodies. Influenza and Other Respiratory Viruses 1: 105–112.
    Towers GJ (2007) The control of viral infection by tripartite motif proteins and cyclophilin A. Retrovirology 4: 40.
    Wang P, Chen J, Zheng A et al. (2004) Expression cloning of functional receptor used by SARS coronavirus. Biochemical and Biophysical Research Communications 315: 439–444.
    book Wang SY, Su CY, Temperton NJ et al. (2008a) "HA-pseudotyped retroviral vectors for screening and evaluation of anti-Flu inhibitors". In: Katz JM (ed.) Options for the Control of Influenza VI, pp. 472–474. London: International Medical Press.
    Wang W, Butler EN, Veguilla V et al. (2008b) Establishment of retroviral pseudotypes with influenza hemagglutinins from H1, H3, and H5 subtypes for sensitive and specific detection of neutralizing antibodies. Journal of Virological Methods 153: 111–119.
    Weinberg JB, Matthews TJ, Cullen BR and Malim MH (1991) Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes. Journal of Experimental Medicine 174: 1477–1482.
    Wool-Lewis RJ and Bates P (1998) Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines. Journal of Virology 72: 3155–3160.
    Wright E, Temperton NJ, Marston DA et al. (2008) Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison. Journal of General Virology 89: 2204–2213.
 Further Reading
    book Coffin JM (ed.) (1997) Retroviruses. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
    book Machida CA (ed.) (2002) Viral Vectors for Gene Therapy: Methods and Protocols. New York: Humana Press.
    book Weiss RA (ed.) (1984) RNA Tumor Viruses: Molecular Biology of Tumor Viruses. Cold Spring Harbor: Cold Spring Harbor Laboratory.

Related Sub-Topics:

Viruses in Gene Therapy | RNA Viruses
Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

Article Title: Retroviral Pseudotypes
 
How to Cite close
Temperton, Nigel J, and Wright, Edward(Mar 2009) Retroviral Pseudotypes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021549]