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Title: Characterisation of the single-stranded DNA binding protein encoded by Kaposi's sarcoma herpesvirus
Author: Dodd, Isabel
ISNI:       0000 0001 3426 8839
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2005
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The work presented in this thesis is on the characterisation of the single stranded DNA (ssDNA) binding protein of the human herpesvirus, Kaposi's sarcoma herpesvirus (KSHV), encoded by the ORF6 gene. There is a core set of six proteins conserved throughout the herpesvirus family that are required for viral DNA replication and are active at the replication fork. These proteins function as a DNA polymerase and its processivity factor, a trimeric helicase-primase complex and a single-stranded DNA binding protein. In KSHV these functions are carried out by the proteins expressed by the following genes: 0RF9 (DNA polymerase), ORF59 (processivity factor), ORF56 (helicase), ORF44 (primase), ORF40/41 (helicase-primase associated factor) and ORF6 (single-stranded DNA binding protein). Current models of herpesvirus DNA synthesis are based mostly upon knowledge of the herpes simplex virus type 1 (HSV-1) replication fork proteins, which have been more extensively studied than any of their homologues in other herpesviruses. The DNA replication proteins in KSHV have not been well studied and therefore their roles in KSHV DNA synthesis have been largely predicted by analogy with their HSV-1 counterparts. To date there has been no published characterisation of the KSHV ssDNA binding protein (pORF6). The work completed for this thesis describes the over-expression and purification of pORF6 and characterisation of its binding to ssDNA. A recombinant baculovirus expressing pORF6 was constructed and used to infect Spodoptera frugiperda cells. The protein was purified from these cells using heparin sulphate and mono-Q columns. Following successful purification of pORF6 it was established that it could bind to ssDNA, using an electrophoretic mobility shift assay (EMSA) and surface plasmon resonance measurements on a BIAcore instrument. The number of nucleotides required for pORF6 to bind to ssDNA and the effect of NaCl on binding were also investigated, revealing that optimal binding occurred at 150 mM NaCl. Fourteen nucleotides were required for pORF6 to bind, however, binding became more efficient as the length was increased to twenty. ICP8, the HSV-1 homologue of pORF6 has been shown to bind to ssDNA in a cooperative manner. The binding mechanism of pORF6 to a ssDNA of thirty-two nucleotides was investigated, again using an EMSA and surface plasmon resonance. This revealed that pORF6 may also bind to single-stranded DNA cooperatively. A comparison of ICP8 and pORF6 binding to ssDNA using the BIAcore indicated that they bind to single-stranded DNA with a similar affinity. A panel of monoclonal antibodies was generated against pORF6 and was tested by western blotting, immune-fluorescence and immune-precipitation, using Spodoptera frugiperda cells infected with a recombinant baculovirus expressing pORF6. Human B-cells infected with KSHV that had been induced into their lytic cycle were also used in immune-fluorescence assays, which revealed pORF6 to be present in globular areas within the cell, reminiscent of herpesvirus DNA replication compartments.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available