Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.751894
Title: Thermodynamic and structural analysis of the interactions between Epstein-Barr virus transcription factors and the host targeting factor RBP-Jkappa
Author: Simmons, Robert
ISNI:       0000 0004 7425 4142
Awarding Body: University of Sussex
Current Institution: University of Sussex
Date of Award: 2018
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Abstract:
Epstein-Barr virus (EBV) asymptomatically infects >90% of the world population but is also associated with multiple malignancies. The EBV latent nuclear proteins EBNA2, 3A, 3B and 3C orchestrate changes in host gene transcription to drive B-cell immortalisation by hijacking cellular DNA-binding transcription factors. This project set out to obtain thermodynamic and structural information on host transcription factor RBP-Jkappa, the DNA-binding mediator of the Notch pathway, and its interactions with the EBNAs. EBNA2 activates transcription by binding RBP-Jkappa through the same WΦP motif found in Notch proteins. EBNA3 proteins bind RBP-Jkappa through a distinct motif (TΦGC) and compete with EBNA2 for binding, inhibiting EBNA2-mediated gene activation, but can also use RBP-Jkappa to bind DNA independently. Interestingly, EBNA3C also contains an adjacent WΦP motif (WTP) that can bind RBP-Jkappa in vitro. We used Isothermal Titration Calorimetry (ITC) to measure binding affinities of EBNA2 and EBNA3C peptides containing these motifs to RBP-Jkappa. We found that EBNA2 peptides bound RBPJ-kappa via its WWP motif with an affinity 10-fold less than the WLP motif of Notch 2. Surprisingly, we found that EBNA3 peptides containing TΦGC motifs did not bind to RBP-Jkappa in vitro. Phosphorylation of the threonine in this motif did not lead to any detectable binding. However, EBNA3C peptides containing both the TFGC and WTP motifs bound RBP-Jkappa with a similar affinity to EBNA2 peptides, but this interaction was entirely mediated by the WTP motif. We also demonstrated that the EBNA3C TFGC peptide could not compete with EBNA2 for RBP-Jkappa binding using a fluorescent polarisation binding assay. Interestingly, we found that an EBNA3A peptide bound weakly to RBP-Jkappa via a motif with similar physiochemistry to the Notch WTP motif (FKL) and not its TLFC motif. These data indicate that TΦGC motifs in EBNA3 proteins may only direct RBPJ-kappa binding in the context of other EBNA3C residues in a properly folded full-length protein or may mediate indirect binding.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.751894  DOI: Not available
Keywords: QD0431 Proteins, peptides, amino acids, etc. ; RC0141.5 Epstein-Barr virus disease
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