MOLECULAR BIOLOGY OF THE RETROVIRUS
Retroviral particles typically comprise two identical copies of single-stranded, positive sense RNA as a genome, ranging between 7 – 10 kb, which is contained within a protein shell (capsid). The capsid is surrounded by a lipid membrane from which extrudes envelope glycoproteins resulting in a total particle size between 90 and 140 mm. It is the envelope proteins that confer retroviral specificity, as they are responsible for attachment to the target cell allowing for virus internalisation. The envelope-dependent tropism of retroviruses is commonly categorised into three groups; ecotropic (infects murine cells only), xenotropic (infects all cell types except those of murine origin) and amphotropic (infects cells of both murine and non-murine lineage).
Subsequent to viral entry into the cell, the RNA genome is reverse transcribed into double-stranded cDNA by the retroviral reverse transcriptase. Reverse transcribed cDNA then translocates to the nucleus and integrates into the host cell genome through the action of retroviral integrase. Therefore, retroviral-mediated transduction (the term used to describe retrovirus infection) of target cells results in stable integration of the viral genome. This is perhaps the most attractive feature of the retrovirus as a gene transfer vehicle for gene therapy, due to the requirement of permanent therapeutic transgene expression.
During the life cycle of wild-type retrovirus, production of new particles arises subsequent to the transcription of integrated viral DNA (provirus). The core proteins assemble in the cytoplasm at the plasma membrane where the RNA genome is targeted to the capsid by the packaging signal (psi). The retroviral genome is divided into three regions: gag, pol and env encoding for capsid proteins, viral enzymes and envelope proteins, respectively (figure 1A). The structural genes encoded by the gag gene (group antigen gene) are expressed in the form of a polyprotein yielding four proteins: p10 (nucleocapsid), p12, p15 (matrix protein) and p30 (capsid protein). The polymerase (pol) gene, lying downstream of the gag gene, encodes the integrase, reverse transcriptase and the protease enzymes. Finally, the envelope (env) gene, which is downstream of the pol gene, also encodes a polyprotein that is cleaved by viral protease to yield gp70 (surface glycoprotein) and p15E (transmembrane protein). The entire retroviral genome encoding these structural and enzymatic proteins is flanked by two long terminal repeats (LTRs) (see diagram one).
Diagram One: Retrovirus Genome
The LTRs are essential for the initiation of viral DNA syntheses, integration of proviral DNA and the regulation of viral gene expression. Finally, the psi located just upstream of the gag region is an essential cis element during retroviral particle production. The most commonly utilised retrovirus for gene transfer applications is that based on the Moloney Murine Leukaemia Virus (MoMLV), which has perhaps the simplest genome of all the retroviruses, thus making it ideal for modification and use as a gene transfer agent.
