Characterisation of selected members of the Alphaherpesvirus ICP0 family of proteins

Dudley, Giles (2005) Characterisation of selected members of the Alphaherpesvirus ICP0 family of proteins. PhD thesis, University of Glasgow.

Abstract

The HSV-1 immediate early protein ICPO is a well-characterised regulator of gene expression, which enhances expression of all classes of genes throughout the viral genome. ICPO plays an important role in reactivation of HSV-1 from quiescent infection, and for efficient initiation of the lytic cycle. One way ICPO may favour the lytic cycle is by altering the intracellular environment by targeting cellular proteins for degradation through its RING fmger mediated E3 ubiquitin ligase activity. Amongst the proteins that have been found to be degraded in cells expressing ICPO are certain components of cellular nuclear sub-structures known as ND1O or PML nuclear bodies. These proteins may be part of a cellular repression mechanism that inhibits viral gene expression and leads to the assembly of viral genomes into a quiescent chromatin structure. Alternatively, disruption of NDIO could release transcription factors that stimulate viral gene expression. All of the aforementioned activities have been shown to be dependent of ICPO having a functional RING fmger domain, a cysteine-rich sequence that is situated near the N terminus of the protein and is structurally coordinated by the presence of two zinc atoms. ICPO and its isolated RING fmger domain have been shown to act as E3 ubiquitin ligases in vitro and cause substrate independent polyubiquitin chain formation in the presence of the E2 ubiquitin conjugating enzymes UbcH5a and UbcH6.The proteins of interest to this study are the homologues of ICPO that are expressed by other members of the alphaherpesvirus sub-family, including BICPO of BHV-1, Eg63 of EHV-1, EPO of PRV and Vg61 of VZV. The homology between these proteins is limited to the RING fmger domain and indeed mainly to the zinc coordinating and certain other structurally important residues therein. Previously, it was shown that in a similar manner to that of ICPO these related proteins to varying degrees cause the disruption of NDIO by affecting certain cellular proteins, and they induce the formation of colocalising conjugated ubiquitin. Together, this evidence suggested that these members of the ICPO family of proteins also act as E3 ubiquitin ligase enzymes. Using the techniques of protein expression, purification and in vitro ubiquitin conjugation assays, the work described in this thesis demonstrates that the isolated RING finger domains of the ICPO related proteins, to varying degrees, act as E3 ubiquitin ligase enzymes. In addition, they simulate the same E2 ubiquitin conjugating enzymes UbcH5a and UbcH6, in a manner similar to that previously shown for ICPO. It was also shown that the isolated RING finger domains of the ICPO family of proteins could form stable interactions with specific E2 partners in vitro. Transfection assays additionally demonstrated that the full-length versions of the ICPO related proteins formed foci that co-localised with certain E2 proteins in vivo. In addition, studies using truncated versions of the RING finger domains of Eg63 and BICPO identified differences between their activities in partnership with the E2 enzymes UbcH5a and UbcH6 that depended on residues on the C-terminal side of their core RING finger domains. Finally, using a transfection assay, it was shown that even though the homology of the ICPO family of protein is largely limited to their RING finger domains, to varying degrees they are able to complement ICPO-null mutant HSV-1.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Roger Everett
• Keywords: Virology
• Date of Award: 2005
• Depositing User: Enlighten Team
• Unique ID: glathesis:2005-74184
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 23 Sep 2019 15:33
• Last Modified: 23 Sep 2019 15:33
• URI: https://theses.gla.ac.uk/id/eprint/74184