Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796831
Title: Peptide inhibition of herpes simplex virus type 1 DNA polymerase
Author: Owsianka, Anna Maria
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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Abstract:
Seven proteins encoded by herpes simplex virus have been shown to be essential for the replication of virus DNA and for virus growth. This study is concerned with two of these proteins: Pol and UL42. Pol is the catalytic subunit of DNA polymerase and is encoded by gene UL30; UL42 is an accessory protein which serves to increase polymerase processivity and is encoded by gene UL42. Together these two proteins form the polymerase holoenzyme, and evidence suggests that the interaction between them is an essential one and hence a potential target for antiviral drugs. To identify regions of the UL42 protein of herpes simplex virus type 1 which may affect viral DNA polymerase activity, a series of 96 overlapping pentadecapeptides spanning the entire 488 amino acid residues of the UL42 protein were synthesised and tested for their ability to inhibit polymerase activity on a primed single-stranded M13 DNA template. Two assays were used: (i) formation of full length double-stranded M13 molecules; (ii) rate of incorporation of deoxyribonucleoside triphosphates. Both of the assays were optimised to ensure that any inhibition by the peptides would be detected. Peptides from five non-contiguous regions of the UL42 protein were found to inhibit polymerase activity in both the presence and absence of the UL42 protein. The most active peptides from each region correspond to amino acid residues 23 to38 (peptide 6), 64 to 78 (peptide 14), 89 to 102 (peptide 19), 229 to 243 (peptide 47), and 279 to 293 (peptide 57). By two different methods (DNA mobility shift and DNA precipitation), peptides 14, 19, 47 and 57 were found to bind DNA; they most probably inhibit enzyme activity by this mechanism. Peptide 6 did not bind DNA and must act by some mechanism other than competing for DNA. The inhibitory peptides were also tested for activity against mammalian polymerase alpha and the Klenow fragment of Escherichia coli polymerase. Although some limited specificity was demonstrated (up to 10-fold for peptide 6), all the peptides showed significant activity against both polymerase alpha and E. coli polymerase. UL42 peptides other than those which inhibited polymerase were also found to bind DNA. Twenty-six peptides were shown to have DNA-binding properties by gel mobility shift assay. Twenty-two of these were positively charged, suggesting that non-specific electrostatic interactions were largely responsible for the observed binding. There is evidence that the carboxy-terminus of Pol is required for the formation of a functional complex with UL42. Two peptides spanning the carboxy-terminal 42 amino acid residues of Pol were tested for their ability to inhibit the processivity of the Pol-UL42 holoenzyme. They had no detectable effect.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796831  DOI: Not available
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