Molecular Biology of the HSV
Herpes viruses are large, enveloped DNA viruses comprising a genome of double-stranded DNA 150 kb in length located within the viral capsid and encoding some 100-200 genes (see diagram one). Typically, glycoprotein C mediates viral entry by interacting with heparin sulphate, thus initiating a series of interactions ultimately resulting in entry into the cell. Once in the cytoplasm, the viral capsid is transported to nuclear membrane pores where it injects its genome through its capsid portal. Subsequent to nuclear entry, the immediate-early, early and late genes are transcribed in a sequential order resulting in viral DNA replication and a lytic infection, where new viral particles are produced and released from the cell. Conversely, a latent infection may also take place, where the virus persists in a quiescent but persistent form. During the latent infection, the virus expresses the latency-associated transcripts, which regulate the host cell genome, preventing the induction of cell death. Herpes viruses have been explored as gene transfer vectors due to their capacity to carry long sequences of exogenous DNA and their natural ability to target the nervous system.
Diagram One: The HSV Genome
Recombinant HSV Vectors
Two approaches have been used in the production of Herpes viral vectors (see diagram). In the Amplicon approach, a plasmid containing OriS (the HSV-1 origin of replication), HSV-1 packaging signal and an expression cassette, encoding the therapeutic gene and selection marker under control a viral immediate early promoter, is co-transfected into producer cells with helper virus. The helper virus is a temperature sensitive mutant HSV that encodes all the structural genes. Selection pressure is then applied to the producer cells and viral particles encoding replicon or helper are then produced. The recipient then receives both amplicon and helper particles (see diagram two).
Diagram Two: HSV Amplicons
In the Recombinant approach, HSV is made replication-defective by the deletion of one or more immediate-early genes, e.g. ICP4, which is then provided in trans by a complementing cell line. A gene expression cassette is then introduced into the disabled HSV and used to drive epxression of therapeutic transgene. This second approach is less toxic and can mediate long-term expression of transgene at low concentrations in the brains of mice; however, toxicity in cell culture systems remains a drawback when using recombinant HSV vectors in vitro.