Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320841
Title: Mutational analysis of the herpes simple virus type 1 UL8 protein
Author: Barnard, Eleanor Claire
ISNI:       0000 0001 3445 5941
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1996
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Abstract:
Seven genes encoded by herpes simplex virus type 1 (HSV-1) are essential and sufficient for viral origin-dependent DNA synthesis in transfected tissue culture cells. The products of three of these - UL5, UL8 and UL52 - form a heterotrimeric complex exhibiting DNA helicase and primase activities. Studies using temperature-sensitive HSV-1 recombinants have shown that co-expression of all three subunits is essential for efficient nuclear localisation of any one of them. Since a sub-assembly consisting of UL5 and UL52 is enzymatically active but remains in the cytoplasm, it was suggested that UL8 may play an important role in facilitating nuclear uptake of the trimeric complex. Biochemical assays utilising recombinant baculovirus-expressed DNA replication proteins subsequently demonstrated that UL8 stimulates the synthesis of primers by UL5/UL52 and/or their utilisation in DNA synthesis, although the mechanisms by which this occurs remain unclear. Since homologues of UL8 have not been identified outside the herpesvirus family, little information about the functions of UL8 has been gained from its amino acid sequence. The aims of the work presented in this thesis were therefore to perform a mutational analysis of the UL8 protein, in an attempt to define important regions in UL8 and to relate these to the known functions of the protein. A random set of N- and C-terminally truncated versions of the 750 amino acid UL8 protein were created and expressed from plasmids in transfected tissue culture cells under the control of a strong constitutive promoter (the human cytomegalovirus major immediate-early promoter). An additional mutant containing an internal deletion spanning amino acids 78-339 of UL8 was also generated. To investigate the effect of the mutated UL8 proteins on the intracellular location of UL5 and UL52, without the requirement for constructing temperature-sensitive HSV-1 recombinants, a plasmid-based assay for intracellular localisation of the helicase-primase complex was developed. This assay, coupled with the availability of monoclonal antibodies to UL8, permitted the previous findings using recombinant viruses to be confirmed and extended. The results demonstrated that co-expression of the three components of the helicase-primase complex in the absence of all other viral proteins is sufficient for their efficient nuclear localisation, and that UL8 can apparently enter the nucleus in the absence of UL5 and UL52. The mutated UL8 proteins were screened for ability to facilitate nuclear localisation of UL5 and UL52 in the plasmid-based assay, and for ability to replace wild- type UL8 in transient transfection assays of HSV-1 origin-dependent DNA synthesis. In addition, selected mutants were used to generate recombinant baculoviruses, resulting in expression of sufficient quantities of the mutated UL8 proteins to permit examination of their ability to co-immunoprecipitate with UL5 and/or UL52 from extracts of infected insect cells. The results demonstrated that a deletion of 5 amino acids from the N-terminus of UL8 can be tolerated without affecting its ability to support HSV-1 origin-dependent DNA synthesis or facilitate nuclear localisation of the helicase-primase complex, whereas larger deletions of 23 to 165 amino acids abolish both of these activities. When expressed by a recombinant baculovirus, the protein in which the N-terminal 165 amino acids had been deleted,coprecipitated with both UL5 and UL52 from extracts of appropriately infected insect cells. Removal of the 4 most C-terminal amino acids from UL8 did not affect its ability to facilitate efficient nuclear localisation of the helicase-primase complex, but the protein supported less than wild-type levels of HSV-1 origin-dependent DNA synthesis in the transfection assay. Larger C-terminal deletions of 33 to 497 amino acids, and deletion of residues 78-339 of UL8, abolished both transient replicative ability and efficient nuclear localisation of the helicase-primase complex in transfected cells. When expressed by recombinant baculoviruses, mutated UL8 proteins with deletions spanning amino acids 78-339, 471-750 and 718-750 were each capable of coprecipitating both UL5 and UL52 from extracts of infected insect cells. The ability of the mutated UL8 proteins to support origin-dependent DNA synthesis therefore correlates well with their ability to facilitate efficient nuclear localisation of UL5 and UL52. Moreover, most of the mutants can, or are predicted to be able to, interact with UL5 and UL52. The possibility therefore arises that the UL8 mutants fail to support DNA synthesis in the transient assay solely due to their failure to facilitate efficient nuclear localisation of the helicase-primase complex. It is possible that complexes incorporating mutant UL8 in place of the wild-type protein may be sufficiently altered in conformation as to fail to present the appropriate signals for nuclear uptake and retention. Although known functions of UL8 can not as yet be associated with particular stretches of amino acids, it is clear from these studies that residues close to each terminus (amino acids 6-23 and 718-746) of UL8 are indispensable for UL8 function. These studies provide the basis for a number of future experiments aimed at identifying functional domains within UL8.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.320841  DOI: Not available
Keywords: Genetics
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