Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366205
Title: Characterisation of UL102, the helicase-primase associated protein of human cytomegalovirus
Author: Hamill, Pamela
ISNI:       0000 0001 3528 2296
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2001
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
Amongst the set of 11 proteins which are required for human cytomegalovirus (HCMV) origin-dependent DNA synthesis, are six which are conserved amongst the herpesvirus family and which perform the essential functions required for viral DNA synthesis. In HCMV these functions, a processive DNA polymerase, ss DNA-binding and helicase-primase activities are provided by the UL54, UL44, UL57, UL70, UL102 and UL105 proteins which are referred to as the core replication fork proteins. UL54 and UL44 comprise the catalytic and processivity subunits, respectively, of the DNA polymerase holoenzyme, UL29 functions as the ss DNA-binding protein whilst UL70, UL102 and UL105 associate to form the heterotrimeric helicase-primase complex. Current models of herpesvirus DNA synthesis are based mostly upon knowledge regarding the herpes simplex virus type 1 (HSV-1) replication fork proteins which have been more extensively studied than any of their counterparts in other herpesviruses. In HSV-1 the replication fork proteins are UL30/UL42 (DNA polymerase), UL29 (ss DNA- binding protein) and UL5/UL8/UL52 (helicase-primase complex). Multiple interactions between the HSV-1 replication fork proteins have been demonstrated and it is thought that these interactions serve to co-ordinate the functions of the HSV-1 replication proteins for efficient viral DNA synthesis. Unlike the HSV-1 replication proteins, the HCMV replication proteins are not well characterised and therefore their roles in HCMV DNA synthesis have been largely predicted by analogy with their HSV-1 counterparts. The aim of this project was to investigate the properties of UL102, one of the essential HCMV replication fork proteins which forms part of the helicase-primase complex, and compare its characteristics with that of its HSV-1 homologue, UL8, to assess whether it may perform similar functions to UL8 during HCMV DNA synthesis. The following specific properties of UL102 were investigated to allow direct comparison with HSV-1 UL8: 1) Its ability to interact with the HCMV DNA polymerase catalytic subunit, UL54. 2) Its ability to bind to DNA and DNA/RNA hybrid oligonucleotides representing the forms of nucleic acid present at the replication fork. 3) Its intracellular localisation when expressed atone in mammalian cells and also its ability to alter the intracellular localisations of the other HCMV helicase-primase subunits, UL70 and UL105, when it is co-expressed with these proteins in mammalian cells. In order to address these objectives and to enable subsequent structure-function analysis, a variety of UL102 reagents were produced. These included 1) a panel of 51 UL102-specific monoclonal antibodies 2) constructs to express, in E. coli, full-length and truncated UL102 GST-fusion proteins 3) constructs to express, in mammalian cells, full-length UL102, UL70 and UL105 proteins and truncated UL102 proteins and 4) constructs which express UL102 and UL54 that permit 2-hybrid analysis in mammalian cells. Using three different methods, ELISA, co-immunoprecipitation and mammalian-2- hybrid analysis, no evidence for a specific interaction between UL102 and UL54 could be demonstrated. In this respect UL102 may differ from its HSV-1 counterpart, UL8. In immunofluorescence studies, UL102 efficiently translocated to the nucleus of mammalian transfected cells. In this respect it differs from UL8, which localises to the cytoplasm when expressed alone. Both UL70 and UL105 displayed a cytoplasmic localisation when expressed on their own. UL102 did not influence their cytoplasmic localisations when co-expressed with either protein. Preliminary results indicate that nuclear localisation of the helicase-primase proteins occurs only when all three are coexpressed. In this respect, UL102 behaves similarly to UL8, which is required for efficient nuclear localisation of the HSV-1 helicase-primase proteins but does not influence their intracellular localisation when it is expressed with either protein individually. Nucleic acid binding experiments were performed either in the presence of 50 mM NaCI or in the absence of any salt. Under both conditions, UL102 did not detectably bind to ss DNA, ds DNA or DNA-RNA hybrids representing the structures of nucleic acid present at the replication fork. Similarly, UL8 did not detectably bind DNA. The UL102 characterisation studies presented in this thesis indicate that it is unlikely that UL102 performs the same precise set of functions which have been attributed to HSV-1 UL8. As UL102 shares some characteristics in common but also possesses differing characteristics to UL8, it is probable that it performs a subset of the functions carried out by UL8 including a likely role in nuclear translocation of the HCMV helicase-primase complex.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.366205  DOI: Not available
Keywords: Biochemistry
Share: