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Title: Vaccinia virus protein B14, an inhibitor of NF-κB activation, and its counterpart in MVA
Author: McCoy, Laura Ellen
ISNI:       0000 0004 2682 9159
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2010
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Vaccinia virus (VACV) encodes multiple proteins which modulate NF-[kappa]B activation to evade the host immune response. One example is B14, a virulence factor that reduces NF-[kappa]B activation via interaction with IKK [Beta]. B14 has orthologues in many VACV strains, including modified virus Ankara (MVA), which lacks many of the immunomodulators present in other VACV strains. Here, the MVA counterpart of B14, protein 183, was characterised. 183R was both removed from MVA and inserted into the B14R locus of a VACV strain Western Reserve (WR) lacking B14R, but in each case there was no change of phenotype. Protein 183 shares 95 % amino acid identity with B14 but, unlike B14, was unstable in eukaryotic cells, unless protein degradation was inhibited. Protein 183 did not inhibit NF-[kappa] B activation in response to cytokine stimulation, as does B14, nor did it restore the virulence phenotype of WR lacking B14R back to wild type. Therefore, the mutations incurred by 183 during the derivation of MVA have rendered the protein non-functional. However, other MVA immunomodulators remain to be characterised and this thesis describes a bacterial artificial chromosome (BAC) system that may facilitate the improvement of MVA in its role as a vaccine vector. To further characterise VACV modulation of NF-[kappa] B, a VACV WR strain was constructed lacking B14 and another VACV modulator of NF-[kappa]B, WR A49. Initial characterisation showed no change with this mutant virus in replication or spread. The effect of the B14 on NF-[kappa]B was further characterised by studying the B14-IKK [Beta] interaction using IKK[Beta] and B14 mutants. Residues 300 to 480 of IKK [Beta] were shown to be required for the interaction and a mutant encompassing this region co-purified to a degree with B14 when expressed in E. coli. In addition, mutagenesis showed that B14 residue F130 is required for the interaction of the two proteins.
Supervisor: Smith, Geoffrey Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral