Vanni, Emilia (2011) Identification and functional characterisation of residues required for ICP0’s interaction with cellular E2 ubiquitin-conjugating enzymes. PhD thesis, University of Glasgow.Due to Embargo and/or Third Party Copyright restrictions, this thesis is not available in this service.
The viral infected cell protein 0 (ICP0) is required for efficient initiation of herpes simplex virus 1 (HSV-1) lytic infection and productive reactivation of viral genomes from latency. Following its entry to the nucleus, HSV-1 genomes become associated with nuclear sub-structures known as nuclear domain 10 (ND10). Individual ND10 proteins, including the major constituent protein promyelocytic leukaemia protein (PML), have been reported to repress viral gene expression and confer intrinsic antiviral resistance against viral infection, a mechanism that is counteracted by ICP0. ICP0 has been shown to target specific cellular proteins for proteasome-mediated degradation through the E3 ubiquitin ligase activity conferred by its N-terminal C3HC4 RING finger domain. The RING finger domain of ICP0 has been reported to induce poly-ubiquitin chain formation in vitro in the presence of two E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2E1. However, the residues required for this interaction remain unknown. The primary purpose of this study was to map the interaction interface between the RING finger domain of ICP0 and its cognate E2 ubiquitin-conjugating enzymes and to investigate the importance of these interactions with regard to ICP0’s function during HSV-1 infection. In this study, site-directed mutagenesis was used to mutate specific ICP0 RING finger residues corresponding to residues on other RING finger proteins previously reported to be required for interaction with their respective cognate E2 ubiquitin-conjugating enzymes. Using yeast two-hybrid analysis, we demonstrate that point mutations at specific residues within loop-1 and -2 of the RING finger domain inhibited ICP0’s ability to interact with either UBE2D1 or UBE2E1. These mutations impaired or abolished ICP0's E3 ubiquitin ligase activity in vitro, inhibited its ability to conjugate ubiquitin and induce the degradation of PML in cell culture model systems. Furthermore, RING finger mutants that were unable to interact with either UBE2D1 or UBE2E1 also failed to complement the plaque formation efficiency (PFE) defect of an ICP0 null mutant virus and induce transcription from quiescent HSV-1 genomes. Molecular homology modelling of the ICP0 RING finger domain interaction interface with UBE2D1 based upon the X-ray crystallography structure of the Casitas B-lineage lymphoma proto-oncogene (c-Cbl)-UBE2L3 complex demonstrated that these residues form a potential contact interface with UBE2D1. These findings provide unique insight into the biological importance of ICP0’s ability to interact with components of the host-cell ubiquitin pathway for the efficient initiation of viral lytic infection and the reactivation of viral genomes from latency, two fundamentally important aspects in the life cycle and replication of HSV-1.
|Item Type:||Thesis (PhD)|
|Keywords:||Virus, HSV-1, ICP0, Ubiquitin, E3 ligase, RING finger, E2 conjugating enzyme|
|Subjects:||Q Science > QR Microbiology > QR355 Virology|
|Colleges/Schools:||College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation|
|Supervisor's Name:||Boutell, Dr. Chris|
|Date of Award:||2011|
|Embargo Date:||2 November 2014|
|Depositing User:||Dr Emilia Vanni|
|Copyright:||Copyright of this thesis is held by the author.|
|Date Deposited:||17 Nov 2011|
|Last Modified:||10 Dec 2012 14:02|
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