Retroviral Pseudotypes – From Scientific Tools to Clinical Utility Nigel J Temperton, Viral Pseudotype Unit, University of Kent, Medway, UK Edward Wright, Viral Pseudotype Unit, University of Westminster, London, UK Simon D Scott, Viral Pseudotype Unit, University of Kent, Medway, UK Based in part on the previous version of this eLS article ‘Retroviral Pseudotypes’ (2009) by Nigel J Temperton and Edward Wright. Advanced article Article Contents • Introduction • General Method • Analysis of Receptor Binding and Virus Entry and Exit • Antiviral Screening • Pseudotype Neutralisation Assays and Immunogenicity Testing • Pseudotypes as Immunogens and for Gene Therapy • Notes • Acknowledgements Online posting date: 15 th June 2015 Retroviral pseudotypes are important research and diagnostic tools for basic and clinical virol- ogy studies, facilitating the detailed investigation of individual genes, cellular receptors, antibody responses, serosurveillance and antiviral thera- pies. Importantly, pseudotypes enable the study of highly pathogenic viruses, without the need for high containment. Their use as gene therapy vectors is widely documented, but other uses, once less well known, are becoming more prominent. The substitution of envelope proteins expressed on the virion surface enables pseudotypes to be employed as surrogates for wildtype viruses in antibody neutralisation or antiviral screening assays and for the study of cell–virus receptor interactions. In addition, they are increasingly being utilised as vaccine immunogens, expressing the antigen either on the particle surface or as a transfer gene for cellular expression. These studies demonstrate the potential for using pseudotypes for both scientific and clinical applications. Introduction The standard deinition of a pseudotype is a hybrid virus particle consisting of a protein nucleocapsid (‘core’) encasing a ribonu- cleic acid (RNA) genome, with the core itself being encapsu- lated in a lipid ‘envelope’ membrane derived from the host cell. eLS subject area: Virology How to cite: Temperton, Nigel J; Wright, Edward; and Scott, Simon D (June 2015) Retroviral Pseudotypes – From Scientific Tools to Clinical Utility. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0021549.pub2 Importantly, this envelope gained when cores exit from the cell by ‘budding’ is studded with proteins derived from other viruses. Many of these heterologous envelope proteins are antigenic tar- gets for the host immune system. In pseudotypes, one or more of these envelope proteins may derive from study viruses. Many pseudotypes also carry foreign genes, called ‘transfer’ genes, engineered into their genome. When in the presence of suscep- tible cells, the envelope proteins bind to cell receptors permitting cellular entry, eventually resulting in transfer gene expression. Retroviruses have been extensively exploited as cores for pseudo- typing. The name retrovirus derives from the Latin for ‘behind’ or ‘backward’ (retro-) and this highlights their key characteristic, the ability to reverse transcribe their dimeric single-stranded RNA genome into a double-stranded deoxyribonucleic acid (dsDNA) copy, which is subsequently integrated into the cell genome via the use of viral and cellular enzymes. For retroviral pseudotypes, this usually leads to expression of the transfer/reporter gene, the latter being readily quantiiable. Thus, reporter gene expression directly correlates with eficiency of viral envelope/receptor inter- action, and conversely whether individual antibody responses or antiviral agents could interfere with the entry and replication process of the native virus. See also: Retroviral Replication; Vesicular Stomatitis Virus Current pseudotyping protocols are based on several decades of vector development, often for use in gene therapy. The irst retro- viral vectors were based on native gammaretroviruses, such as Moloney mouse leukaemia virus (MLV). To produce such retro- virus vectors, a stable cell line containing the retroviral genome minus the packaging signal needed for genome encapsidation was irst generated. These cells were then transfected with a plas- mid construct containing the packaging signal and all cis-acting sequences necessary for virus particle assembly, but in which the genes required for genome replication and nucleocapsid forma- tion had been removed (often replaced by a gene of interest – the transfer gene). Thus, all genes needed for retrovirus particle assembly were provided by the cell line in trans. However, recom- bination between newly produced retroviral vector and inte- grated helper virus sequences occasionally led to production of replication-competent viruses using these systems (Sakuma et al., 2012). In order to reduce the production of replication-competent eLS © 2015, John Wiley & Sons, Ltd. www.els.net 1