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Title: The role of the HSV-1 ICP22 protein in regulation of expression of host cell genes
Author: Isa, Nur Firdaus
ISNI:       0000 0004 6498 7879
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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The eukaryotic RNA Polymerase II (Pol II) is responsible for the synthesis of all mRNA and many small regulatory RNAs. The Herpes Simplex Virus 1 (HSV-1) relies exclusively on the host Pol II to synthesise viral RNA during productive infection. A repeating structure on the carboxy terminal domain (CTD) of the Pol II largest subunit consisting of consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, has to be phosphorylated to drive Pol II-mediated transcription. The ICP22 protein of HSV-1 is capable of interacting with P-TEFb, a kinase phosphorylating the Ser2 position of the CTD. Ser2 CTD phosphorylation is a mark associated with Pol II productive elongation. ICP22 has been shown to cause loss of Ser2 CTD phosphorylation. In the first part of this thesis, I show that transient expression of ICP22 affects the level of transcribing Pol II at the beginning and end of a model gene, which mirrors the effect caused by CDK9 inhibitors. This result suggests that ICP22 possibly inhibits Pol II elongation due to inhibition of the Ser2 CTD kinase, P-TEFb. In the second part of the thesis, I describe the ICP22 interactome. I performed ICP22 pull-down assays with HeLa cell extract and the cellular protein complexes bound to ICP22 were analysed by mass spectrometry. This work confirmed the interaction between ICP22 and CDK9 and identifies novel proteins that interact, directly or indirectly, with ICP22. In the last part of the thesis, the interaction of ICP22 and P-TEFb was further characterised. Using recombinant protein of subunits of P-TEFb; CDK9 and Cyclin T1, I demonstrated that ICP22 interacts with both subunits of P-TEFb. In addition, I validated the ICP22 proteomic hits using immunoblots. Here, I reveal that ICP22 possibly interacts with transcriptional kinases other than P-TEFb. The in vitro experiments presented in this thesis involved transiently expressed ICP22 or recombinant proteins ICP22 and VP16 thus eliminating the effects of viral proteins other than ICP22 or VP16. Based on these findings, I have extended our understanding that ICP22 induces alteration of host cellular transcription by inhibiting P-TEFb and possibly other transcriptional kinases. These works serve as a platform for genome-wide analyses of the effect of ICP22 on host gene transcription and structural biology to characterise the interaction of ICP22 and P-TEFb.
Supervisor: Murphy, Shona Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available