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
