The Effect of Orf Virus Infection on the IFN Stimulated JAK/STAT Signalling Pathway

Orf Virus (ORFV) is a large, 134 kbp dsDNA virus that is the type species of the Parapoxvirus genus. ORFV infections are epitheliotropic resulting in lesions of the skin. The natural host range of the virus consists of sheep and goats, although zoonotic infection of humans is common. ORFV encodes several immuno-modulating factors that allow this virus to manipulate a range of critical host antiviral defence mechanisms. One of the most important mammalian signalling pathways is the JAK/STAT pathway, stimulated by IFN-α/β and IFN-γ. Stimulation of this pathway results in the upregulation of hundreds of genes involved in the innate and adaptive antiviral immune response. Sequence analysis of the ORFV genome, strain NZ2, revealed a gene OV057, which encodes a highly conserved phosphatase motif, and amino acid sequence similarity to the well-characterized VH1 dual-specific phosphatase of Vaccinia virus that is known to inhibit the JAK/STAT pathway by dephosphorylating Signal Transducer and Activator of Transcription 1 (STAT1). The aim of this thesis was to investigate whether ORFV had the ability to affect the JAK/STAT signalling pathway and if so was this due to OV057. Initial findings showed that ORFV was able to rapidly dephosphorylate STAT1 in the human HeLa cell line stimulated with IFN-α or IFN-γ when infected at moderate multiplicities of infection (MOI 10), while no STAT1 protein degradation was observed. It was then demonstrated that the viral mediated dephosphorylation of STAT1 at tyrosine 701 during the early stages of ORFV infection, could be inhibited by pretreating cells with the tyrosine phosphatase inhibitor sodium vanadate. Interestingly ORFV was found not to possess the ability to dephosphorylate the serine 727 residue of STAT1. ORFV protein synthesis was inhibited through the pretreatment of cells with adenosine N1-oxide. Results showed that ORFV was still able to dephosphorylate STAT1 at the same MOI as observed in the presence of adenosine N1-oxide, suggesting that the viral encoded tyrosine phosphatase is of a structural origin, as no viral protein synthesis was required for STAT1 dephosphorylation. To investigate whether OV057 was able to dephosphorylate STAT1, a eukaryotic expression vector expressing an EGFP-057 fusion protein was developed. Recombinant plasmid transfected cells stimulated with IFN-γ showed a 22% decrease in phosphorylated STAT1 levels when compared with an empty vector control (expressing only EGFP), confirming that OV057 is able to dephosphorylate STAT1 in the absence of other viral factors. This study has provided insight into how ORFV is able to disrupt the signalling mechanism of the most important innate antiviral immune response in the host cell