Cell Biol Toxicol (2010) 26:1–20 commonly used RNA virus vectors. This large family of enveloped RNA viruses is found in all vertebrates, and they can be classified into oncoretroviruses, lentiviruses and spumaviruses (Verma and Weitzman 2005). All retroviruses consist of a lipid-enveloped virus particle (virion) that surrounds an inner core, known as the nucleocapsid. This contains two identical copies of the viral RNA genome, reverse transcriptase, integrase and protease. The nucleocap- sid is surrounded by a protein shell formed by capsid proteins enclosed by a layer of matrix proteins that interact with the lipid envelope (Fig. 2). The viral RNA forms a homodimer of linear, positive-sense, single-stranded RNA genomes con- taining three essential genes, gag, pol and env, that are flanked by long terminal repeat (LTR) sequences. These LTRs act in cis with neighbouring sequences during viral gene expression and packaging, retro- transcription and integration of the genome (Kay et al. 2001). The gag gene encodes for the core proteins, capsid matrix and nucleocapsid, which are generated by proteolytic cleavage of the gag precursor protein (Verma and Weitzman 2005). The pol gene encodes for the viral enzymes protease, reverse transcriptase and integrase. These are usually derived from the gag-pol precursor. Finally, the env gene encodes for the envelope glycoproteins, which mediate virus entry RNA genome (two copies) Fig. 2 Schematic of retrovirus. Retroviruses are RNA viruses that consist of a lipid envelope, depicted by the outer ring, which interacts with transmembrane matrix proteins and projecting receptor binding proteins. Host cell interactions are governed by these receptor binding proteins and can be altered by pseudotyping with other viral envelope proteins. The inner portion of the virion, known as the nucleocapsid, contains two identical copies of the viral genome and the viral reverse transcriptase, integrase and protease Lipid envelope Gag proteins Integrase Reverse transcriptase-RNaseH Receptor-binding protein Transmembrane protein Protease Nucleocapsid (gag) 7 (Verma and Weitzman 2005). An additional gene, the pro gene, encodes for the protease activity, responsi- ble for gag and gag-pol cleavage and the maturation of the viral particles during or after budding. Oncoretroviruses are simple viruses encoding only these three essential genes, whereas lentiviruses and spumaviruses are more complex and encode for additional viral proteins. However, all retroviruses infect cells by an interaction between specific host cell surface receptors (entry receptors) and co-receptors with viral envelope glycoproteins. The viral particle is then internalised through virus–cell membrane fusion, and the virus core is released into the cytoplasm (Verma and Weitzman 2005). The viral RNA genome is reverse- transcribed into linear double-stranded pro-viral DNA by the viral enzyme reverse transcriptase. The proviral DNA associates with viral proteins (including nucleo- capsid, reverse transcriptase and integrase) to form a pre-integration complex that is rapidly transported to the host cell nucleus. Virally encoded integrase mediates the integration of the provirus into the host cell genome, which is subsequently transcribed into RNA from the viral promoter contained in the 5′ LTR. This process is initiated by host cell transcription factors, and once transcribed, the viral RNA is trans- ported to the cytoplasm where it is translated into viral proteins. The required viral structural proteins and replication enzymes (i.e. gag-pol and gag precursors) assemble with two copies of the viral RNA to form new virion cores at the plasma membrane (Verma and Weitzman 2005). The first retroviral vectors to be developed and used in a gene therapy trial were based on the ‘simple’ moloney murine leukaemia virus (MMLV, an oncovirus; Blaese et al. 1995). Since their initial use, retroviruses have been used for stable gene transfer into mammalian cells for over 20 years (Barquinero et al. 2004). They are often the method of choice as they provide lifelong expression of the introduced gene due to integration into the host cell genome. However, simple retroviruses such as MMLV require the disruption of the nuclear membrane at mitosis to grant the pre-integration complex access to the chromatin and subsequently integrate into the host cell genome (Roe et al. 1993). Consequently, simple retroviral vectors are of limited use for gene transfer to post-mitotic neurons. Neverthe- less, MMLV has been used to deliver genes to the brain in an ex vivo manner. For example, MMLV-mediated transfer of the tyrosine hydroxylase gene to fibroblast