Vaccinia virus protein C4 is an inhibitor of NF-κB activation and contributes to virulence

Vaccinia virus (VACV) expresses many proteins that subvert the host innate immune response and this thesis describes the characterisation of C4, a VACV protein unstudied hitherto, which was hypothesised to have such a role. C4 is conserved in six orthopoxvirus species and shares 43.7% amino acid identity to VACV protein C16, a known virulence factor. C4 is an intracellular protein that is expressed early during infection and localises to both the cytoplasm and nucleus. Functional screens revealed C4 is an inhibitor of IFNβ promoter activation via the inhibition of NF-κB activation. Mechanistically, this inhibition is attributable to the prevention of IκBα phosphorylation. Consistent with this mode of action, C4 coprecipitated with the canonical IKK proteins, IKKβ and NEMO. Protein C16 and the icIL-1Ra were also found to inhibit NF-κB activation. Furthermore, C4 is an inhibitor of cytoplasmic DNA sensing in MEFs and interacts with Ku70 and Ku80 (Ku), two components of the DNA sensor, DNA-PK. Another novel discovery is that VACV infection leads to the stabilisation and translocation of HIF-1α. This process is largely dependent on C16, however C4 is also required for maximal HIF-1α stabilisation and transactivation. The role of HIF-1α during infection and the mechanism used by C4 to contribute to its stabilisation process require further investigation. Whilst C4 is non-essential for virus replication and spread in cell culture, its deletion does result in reduced virus virulence in a murine intranasal model of infection. Finally, the deletion of both C16 and C4 lead to a synergistic attenuation of virus virulence in both the intranasal and intradermal models of infection