Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796431
Title: Replication of herpes simplex virus DNA : study of an origin binding protein
Author: Weir, Hazel Margaret
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1990
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Abstract:
The herpes simplex virus type 1 (HSV-1) genome contains two distinct origins of viral DNA replication of related sequence. One (oriL) lies close to the centre of the long unique region whilst two copies of the other (oriS) are present within the inverted repeat regions flanking the short unique region. Previous experiments identified DNA fragments of 100 bp or less which specify a functional oriS and include a 45 bp near perfect palindrome with a central AT-rich region. Moreover, DNase I footprint assays demonstrated the presence of a specific binding site for an HSV-1 encoded polypeptide which overlapped the end of the palindromic sequence. By analogy with other well-characterised origin binding proteins, the HSV-1 encoded origin binding protein was thought likely to play an important role in initiation of HSV DNA synthesis. The work presented in this thesis describes the identification of the HSV-1 gene encoding this origin binding activity and investigates the role of its interaction with oriS. A sensitive gel retardation assay was set up to allow the identification of the virus induced origin binding activity. Incubation of nuclear extract from cells infected with wt HSV-1 with a radio-labelled oriS fragment resulted in the formation of a major specific retarded complex. Experiments with synthetic oligonucleotides demonstrated the presence of two specific binding sites within the origin region, one of which (site I) corresponded to the previously described site whilst the other (site II) was located on the opposite side of the palindromic sequence. While these experiments were in progress, Challberg and colleagues identified a set of seven HSV-1 genes which were necessary and sufficient for HSV DNA synthesis. These genes encoded the viral DNA polymerase (UL30), a single-strand-specific DNA binding protein (UL29), a double-stranded DNA binding protein (UL42) and four less well understood functions (UL5, UL8, UL9 and UL52). It was considered very likely that one of these latter four genes would encode the protein which binds specifically to oriS. Several approaches were used in attempts to identify the gene encoding origin binding activity. These included analysis of origin binding activity induced by ts mutants with defects in DNA synthesis at the non-permissive temperature and transfection of tissue culture cells with fragments encoding individual replication genes. Neither of these approaches however was successful. HSV-1 tsK recombinants which express individual replication genes UL5, UL8, UL9 or UL52 at the non-permissive temperature were available in the laboratory. When these were tested, only the tsK/UL9 recombinant virus expressed origin binding activity at the non-permissive temperature. The protein-DNA complex obtained with extracts from cells infected with the tSK/UL9 recombinant virus exhibited a smeared binding pattern of lower mobility than previously seen with extracts from cells infected with wt HSV-1. Treatment of the tsK/UL9 extract with protease produced a smaller complex of similar mobility to that seen with wt HSV-1 extracts. In addition, antibody reactive with the UL9 protein further retarded the major complex observed with wt HSV-1 extracts, demonstrating that this complex contained a portion of the UL9 polypeptide. Taken together, the above results suggested that a specific domain of the UL9 protein was probably responsible for sequence-specific binding. Fragments of the UL9 gene were therefore expressed as fusion proteins in Escherichia coli and the C-terminal 317 amino acids were found to bind specifically to HSV oriS , indicating that sequence-specific recognition and binding activities resided within the C-terminal 1/3 of the protein. This result was independently confirmed by expressing the C-terminal 1/3 of the UL9 gene in an in vitro transcription and translation system. Of the two UL9 binding sites within oriS , one (site I) contains an 11 bp sequence which is also present in HSV-2 ori S, HSV oriL and varicella-zoster virus oriS, and the other (site II) includes a related sequence element which differs in two positions from the corresponding region of site I. A third 11 bp sequence (motif III) which lies adjacent to site I and differs from the site I element at only a single position was also recognised. Each of the three 11 bp sequences was deleted from within functional copies of HSV-1 oriS and the effect on origin activity and binding of the UL9 protein examined.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796431  DOI: Not available
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