Investigating the structure and function of HSV-1 tegument proteins: UL7 and UL51
| cam.restriction | thesis_access_embargoed | |
| cam.supervisor | Graham, Stephen | |
| cam.thesis.funding | false | |
| dc.contributor.author | Owen, Danielle | |
| dc.date.accessioned | 2018-08-06T11:07:34Z | |
| dc.date.available | 2018-08-06T11:07:34Z | |
| dc.date.issued | 2018-08-08 | |
| dc.date.submitted | 2017-09-28 | |
| dc.date.updated | 2018-08-04T10:43:16Z | |
| dc.description.abstract | Herpes simplex virus-1 (HSV-1) has a large double-stranded DNA genome encased within an icosahedral capsid. The capsid is surrounded by a protein-rich layer, termed tegument, and a membranous envelope containing viral glycoproteins. HSV-1 genome replication and encapsidation occurs in the nucleus, after which, DNA-loaded capsids enter the cytoplasm where they undergo tegumentation and assembly. This thesis presents a structural and functional investigation of two HSV-1 tegument proteins, UL7 and UL51 that are conserved across all herpesviruses. Deletion of UL7 or UL51 results in impaired viral replication, a small plaque phenotype and an accumulation of unenveloped capsids in the cytoplasm, the latter of which is indicative of a defect in tegumentation and/or secondary envelopment. Similar phenotypes have been observed upon deletion of homologous proteins in pseudorabies virus and human cytomegalovirus, suggesting a conserved role for these proteins. This thesis presents evidence for the formation of a UL7-UL51 complex in transfected and infected cells. Pull-down experiments using recombinant UL7 and UL51 protein purified from E. coli demonstrated that the interaction is direct, and mapped the UL7-binding region within UL51. The interaction was shown to be conserved between UL7 and UL51 homologues from murid herpesvirus, ORF42 and ORF55, respectively. The UL7-UL51 complex was purified from E.coli and, after optimisation of the purification protocol and the UL51 construct, a pure protein sample was obtained that was suitable for crystallisation trials. Two conditions were identified that produced reproducible crystals. These crystals proved to be thin and fragile, preventing their analysis by X-ray diffraction. Optimisation of the crystallisation conditions to produce more robust crystals and/or in situ diffraction measurements may yet yield X-ray diffraction data for the complex. Host-cell binding partners for UL7 and UL51 were identified by yeast-two-hybrid screen and quantitative proteomics (SILAC). An interaction between UL51 and the G-Box domain of the centriole protein CPAP was identified by Y2H screen, and validated by immunoprecipitation from transfected cells and in pull-down experiments using recombinant proteins purified from E. coli. The CPAP-binding region within UL51 was mapped and shown to resemble a motif present in the cellular protein STIL that mediates an interaction between STIL and CPAP. Two UL51 point-mutations within the putative CPAP-binding motif blocked the interaction between UL51 and the CPAP G-box domain. A mutant HSV-1 virus carrying these UL51 mutations was generated, but no difference was evident between single-step growth curves of wild-type HSV-1 and the UL51 mutant. Host-cell proteins pontin and reptin were identified as putative UL7/UL51 interaction partners in two SILAC screens. Purified GST-tagged UL7-UL51 complex was able to interact with pontin and reptin. However, it is likely that the interaction is non-specific since pontin and reptin were also found to bind a misfolded protein in a similar manner. | |
| dc.identifier.doi | 10.17863/CAM.26007 | |
| dc.identifier.uri | https://www.repository.cam.ac.uk/handle/1810/278659 | |
| dc.language.iso | en | |
| dc.publisher.department | Pathology | |
| dc.publisher.institution | University of Cambridge | |
| dc.rights | All rights reserved | |
| dc.rights | All Rights Reserved | en |
| dc.rights.uri | https://www.rioxx.net/licenses/all-rights-reserved/ | en |
| dc.subject | herpes simplex virus | |
| dc.subject | UL7-UL51 complex | |
| dc.subject | UL7 | |
| dc.subject | UL51 | |
| dc.title | Investigating the structure and function of HSV-1 tegument proteins: UL7 and UL51 | |
| dc.type | Thesis | |
| dc.type.qualificationlevel | Doctoral | |
| dc.type.qualificationname | Doctor of Philosophy (PhD) | |
| dc.type.qualificationtitle | PhD in Molecular Biology |
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