The role of vaccinia virus K7R gene in evasion of innate immunity

Abstract

This thesis reports an investigation of the role of Vaccinia virus (VACV) gene K7R in evasion of innate immunity. The K7 protein is an inhibitor of intracellular signalling pathways leading to the activation of the innate immune response and was shown to act by inhibiting TBK1/IKK-ε - mediated IRF signalling by targeting DDX3, a cellular RNA helicase (Schroder et al., 2008). To characterise K7 further and study its interaction with DDX3, K7 in complex with fragments of DDX3 was expressed in bacterial cells with the aim of obtaining co-crystals. The role of K7R in the context of VACV infection was also studied. Previous in vivo work using a recombinant virus lacking K7R had shown that K7 contributes to virulence. However, virulence was retained when a mutation of the initiating methionine codon abrogated K7 protein expression while preserving the nucleic acid sequence. It has been shown here that this is likely to be due to the presence of viral miRNA(s) encoded from the K7R sequence. Computational analysis identified putative pre-miRNAs within K7R and mutated versions of the K7R ORF which contained the predicted pre-miRNA sequences but did not make protein were still able to inhibit the IFN-β, NF-κB and ISRE-dependent intracellular signalling in vitro. Several of these mutated genes were introduced into the K7R deletion VACV and their virulence will be examined in a mouse model of infection. The presence of a miRNA also needs to be formally demonstrated using northern blot and/or qPCR analysis. Finally, there is a strong interest in using attenuated VACV strains such as Modified Vaccinia Ankara (MVA) as vaccine vectors against various pathogens. To investigate whether the removal of immunomodulators from MVA, such as the orthologue of K7R, can make the virus more immunogenic, a deletion mutant and its revertant counterpart were made and characterised in vitro.