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Title: Purification and characterisation of the herpes simplex virus type 1 DNA replication protein UL8
Author: Parry, Marc Edward
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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The genome of herpes simplex virus type 1 (HSV-1) contains seven genes whose products are directly involved in viral DNA replication. These are UL5, UL8, UL9, UL29, UL30, UL42 and UL52. I investigated the products of genes UL5, UL8, UL9, and UL52, as these were the least characterised. Peptides were synthesised with sequences corresponding to portions of the amino acid sequence of these proteins and the peptides were used to generate antisera in rabbits. The peptides produced sera reacting with two of the four proteins - UL8 and UL9. Sera were produced that reacted with both the N and C termini of UL8. The project now focused on UL8 protein which was purified to homogeneity to allow its further characterisation. An existing recombinant baculovirus/Spodoptera frugiperda expression system was used as a source of the protein. The protein was extracted from the cells in a soluble form and was stable in the extraction buffer (neither denatured nor proteolysed) for at least 4 hours at room temperature. The protein was also stable during freezing and thawing, dilution, and incubation at 4°C. A rapid and simple purification scheme was developed using DEAE sepharose and phenyl sepharose columns. Ten per cent glycerol was required in all buffers for successful chromatography. The purification produced UL8 more than 95% pure in only 3V2 hours beginning with the crude extract. The pure protein was intact and free from degradation products as determined by reaction with the N and C termini-specific sera. Gel filtration chromatography established that the molecular mass of the purified protein was 75* +/-10 kD, indicating that it is a monomer in solution. (The protein's predicted molecular mass is 80 kD.) The possible interaction of UL8 with nucleic acids was investigated using a gel band shift assay. Despite extensive investigation of different conditions this revealed no interaction of UL8 with fully or partially duplex DNA, single stranded DNA, or with a DNA/RNA hybrid. An ELISA assay was established using purified UL8 as antigen. Optimum conditions were found for detecting antibodies to UL8 so that the assay could be used to screen hybridoma cell lines for UL8-specific antibody production. A cell fusion was performed using sple nocytes from a mouse immunised with purified UL8 and this produced eighty one hybridoma lines of which 25 produced antibodies specific for UL8 in the ELISA assay. From these, nineteen ascites fluids were produced which contained antibodies specific for UL8 protein (again measured in ELISA assays). Six of these ascitic fluids reacted with UL8 in Western blotting experiments and seven immune precipitated UL8 from solution. Rabbit polyclonal sera specific for the protein were also produced. Possible future work is discussed as well as the implications of these experiments for studies of HSV-1 DNA replication.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available