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Title: Functional analysis of conserved motifs within herpes simplex virus regulatory protein ICP0
Author: Pheasant, Kathleen
ISNI:       0000 0004 5921 9322
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2015
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Herpes simplex virus type 1 (HSV-1) Immediate-early protein ICP0 is important for regulating the balance between lytic and latent infections. The RING finger domain of ICP0 acts as an E3 ubiquitin ligase, binding to E2-ubiquitin conjugating enzymes and target proteins, promoting their polyubiquitination and subsequent degradation. ICP0 localises to cellular nuclear sub-structures known as ND10 at early stages of infection, and the RING finger domain induces the degradation and dispersal of ND10 proteins, which form part of the cell's intrinsic antiviral defence mechanism. The RING finger domain of ICP0 consists of an alpha-helix, and this and the loop regions are involved in interactions with the E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2E1. Previous work using an ICP0-inducible cell line system found a mutation (N151D) in ICP0's alpha-helix, which allowed complementation of an ICP0-null mutant virus plaque formation but caused a substantial defect in the induction of reactivation of quiescent HSV-1. This raised the possibility that the mechanisms controlling lytic infection and reactivation may be separable. The main focus of this study was to investigate the N151D mutation and other mutations located within the alpha-helix (including K144E and K144E/N151D) using an inducible ICP0 expression cell system and virus infection studies. The phenotypes of these alpha-helix mutants were characterised to investigate if complementation/lytic infection and reactivation involve differential activities of ICP0. Additionally, these alpha-helix mutants were analysed for their in vitro E3 ligase ability and ability to interact with components of the ubiquitin pathway, focusing on E2 ubiquitin-conjugating enzymes. The results from this study using the inducible ICP0 cell system confirmed previous results. The alpha-helix mutants had a greater defect during reactivation than complementing the plaque forming defect of an ICP0-null mutant virus, and this was more noticeable for the K144E mutant. The virus infection studies showed a greater correlation between the effects of the mutations on the degradation of ND10 proteins, plaque formation and replication at low multiplicity of infection. The defect of the K144E mutation was more profound than N151D in all the assays, and the activity of a double mutant including mutations at both K144 and N151 (KE/ND) was reduced to levels comparable with the RING finger deletion mutant. Infection with mutants K144E and KE/ND greatly reduced the efficiency of reactivation of quiescent HSV-1 even at multiplicities of infection where their lytic infection was not severely affected, whereas N151D showed an intermediate phenotype. Furthermore, this study showed that ICP0 has the potential to interact with multiple E2 ubiquitin-conjugating enzymes, and the alpha-helix mutations may affect these interactions. Further investigation will be required to examine the roles that these E2 ubiquitin-conjugating enzymes play during HSV-1 infection. The data in this study indicate that there is no strong evidence to suggest that ICP0 utilises differential activities of its RING finger to mediate reactivation and the stimulation of lytic infection, despite the likely dissimilar nature of the viral chromatin structure in the two situations. These findings, however, provide an insight into the biological importance of the RING finger alpha-helix of ICP0 during the course HSV-1 infection and especially during reactivation from quiescence. In addition, motifs present within ICP0 that may be involved in interactions with other cellular proteins were analysed. These included the PPEYPTAP motif present within retroviral Gag proteins, the SIAH-1 interaction motif and residue T67 (which are all involved in interactions with cellular E3 ubiquitin ligases), and the CoREST binding region. Furthermore, a region of homology between alphaherpesvirus ICP0 proteins downstream of the RING finger domain, which had not previously been investigated in detail, was studied. These studies indicated that the region downstream of the RING finger domain (residues 211-222) may contribute to ICP0's activity, but no major role was detected for the other motifs studied.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology ; QR355 Virology