The thesis work presented evaluated the potential for use of disabled herpes simplex virus-1 (HSV-1) as gene delivery vectors for neural precursor cells and studied the effects of delivered recombinant factors on the de novo development of dopaminergic neurons from neural precursor cells. Highly and less disabled HSV-1 has been studied with respect to gene delivery efficiency and effects on cellular integrity in primary neural progenitor cells, neural stem cells grown as neurospheres, and in endogenous neural stem cell niches in the adult rat. Data from autografts of virally transduced neurospheres into the striatum of rats were also presented. The characteristics of virally transduced neural precursor cells were compared to other viral vector systems reported in literature. With respect to the study of differentiation factors, work has concentrated on fibroblast growth factor 8b (FGF8b). This has demonstrated that FGF8b is a mitogen for neural precursor cells in vitro. The study showed that neural stem cells isolated from different regions of the developing brain can be expanded in FGF8b alone and retain their stem cell characteristics, e.g. the capacity of self-renewal and multipotentiality. Growth curves and dose responses of neural precursor cells expanded in FGF8b further confirmed these findings. The study also showed survival effects of FGF8b on dopaminergic neurons derived from mesencephalic precursor cells. Further the effects of FGF8b on proliferation and differentiation of endogenous stem cells were also investigated. Finally, the thesis work involved the construction and generation of highly and less disabled viruses expressing FGF8b, sonic hedgehog, basic fibroblast growth factor, and the transcription factor nurr1. Expression and bioactivity of the various constructs was confirmed. The effects of these factors on dopamine neuron development were then studied in vitro using neural progenitors and neural precursor cells for which gene delivery had been optimized in the first part of this thesis.

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Herpesvirus saimiri (HVS) is the prototype gamma-2-herpesvirus which has significant homology to the medically important Kaposi's sarcoma-associated herpesvirus (KSHV). HVS encodes an essential multifunctional protein, ORF57, which is conserved in all herpesviruses. ORF57 plays a pivotal role in transactivating viral gene expression by specifically mediating the nuclear export of intronless viral transcripts. To perform this function, ORF57 has the ability to shuttle between the nucleus and the cytoplasm, bind viral RNA and interact with various cellular proteins involved in nuclear import and export pathways.

The aims of the work presented in this thesis were to use a variety of mutagenesis techniques to investigate the proteins of the HSV-1 capsid. Triplexes are heterotrimers formed by two proteins in a 1:2 stoichiometry. The single-copy protein is called VP19C, and the dimeric protein is VP23. Insertional and deletional mutagenesis was carried out on VP19C and the effects of the mutations on virus growth and capsid assembly were examined. Insertional mutagenesis showed that VP19C can be divided into three regions with respect to their ability to tolerate five amino acid insertions, with two regions of approximately 100 amino acids at the N- and C-terminal regions of the protein being more tolerant of such insertions than a ~350 amino acid central region. The N-terminal ~100 amino acids of the protein, which are particularly insensitive to insertional mutagenesis, correspond to a region that is poorly conserved among herpesviruses. Some, but not all, severely disabled mutants were compromised in their ability to bind VP23 and VP5. Analysis of deletional mutants revealed the presence of an unusual nuclear localisation signal (NLS) near the N-terminus of VP19C. This was mapped to a 33 amino acid region by fusion of specific sequences to a green fluorescent protein (GFP) marker. By replacing the endogenous NLS with that from the simian virus 40 (SV40) large T antigen, we were able to show that the first 45 amino acids of VP19C were not essential for assembly of functional capsids and infectious virus particles. However, removing the first 63 amino acids resulted in the formation of aberrant capsids and prevented virus growth, suggesting that the poorly-conserved N-terminal sequences have some as-yet-unidentified function.