Characterization of the role of ICP34.5 and ORF P in the HSV-1 lifecycle
The work carried out in this thesis comprises two main sections: i) characterisation of the role of ICP34.5 and ORF P in HSV-1 replication; and ii) identification of cellular and viral proteins which interact with ORF P. A complication in the analysis of the role of ICP34.5 in the HSV-1 lifecycle is the presence of overlapping antisense genes, ORF O/P, which are also deleted in ICP34.5 negative mutants. In 1991, MacLean, A. et al, isolated 1716, a HSV-1 17+ ICP34.5/ORF O/P deletion mutant. This mutant has been demonstrated to have specific characteristics both in vitro and in vivo. To attribute characteristics which were originally attributed solely to ICP34.5 to each of the genes, a number of HSV-1 recombinant viruses that express ICP34.5 and ORF O/P independently were constructed, purified and characterised. In all recombinant viruses an expression cassette was inserted into the 1716 UL43/UL43.5 locus containing the gene of interest under the control of the HSV-1 gD promoter and b galactosidase in the opposite orientation. Recombinants 1622 and 1625 express ICP34.5 and in both 1624 and 1624.5 ORF P is inserted in UL43 and UL43.5, respectively. Additionally, in 1625 ORF P is inserted in the non essential US5 gene. Western blotting analysis of 1622 and 1625 infected BHK cells demonstrated at least an eight-fold increase in ICP34.5 levels compared to wild type 17+. In the recombinants 1624, 1624.5 and 1625 no ORF P was detected, suggesting that these recombinants express low levels of ORF P, as the rabbit polyclonal sera used exhibited problems of detection with low amounts of ORF P from 17+ but not from the overproducing ts mutant in ICP4, tsK. Also, it is possible that the recombinants do not express ORF P. As we were not able to detect ORF P from our recombinant viruses, we proceeded to look for its RNA. A band with the size expected for ORF P RNA of about 700 bp was detected in 1624, 1624.5 and 1625. The 700 bp ICP34.5 transcript was also detected in 1622.