O'Hare, Craig (2017) An examination of the role of IFI16 in detecting viral DNA in human immortalised keratinocytes. PhD thesis, UNSPECIFIED.
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Abstract
The presence of exogenous DNA in the cytosol results in the activation of the DNA sensor cyclic GMP-AMP synthase (cGAS). cGAS produces the second messenger cyclic GMP-AMP (cGAMP) which binds to and activates the endoplasmic reticulum adapter STimulator of INterferon Genes (STING). Activated STING initiates transcription of the anti-viral cytokine interferon-b, and by extension, induces activation of the anti-viral immune response. Other DNA sensors have been proposed to recognise exogenous DNA via STING but their relevance to the cGAS-STING pathway is unclear. Interferon-g inducible protein 16 (IFI16) is a putative DNA sensor that has also been proposed to induce interferon-b transcription via STING. This thesis demonstrates that both IFI16 and cGAS are required for full activation of immune responses to DNA and DNA virus infection in human immortalised keratinocytes (HaCaT). The cGAS-STING pathway was examined in IFI16(-/-) HaCaT cell lines to conclusively determine the contribution of IFI16 to DNA sensing. IFI16(-/-) HaCaT cell lines produced drastically reduced levels of interferon-b, interferon-stimulated genes and pro-inflammatory cytokine mRNAs following stimulation with exogenous DNA due to reduced activation of the STING pathway. IFI16 was observed not influence cGAS activity as DNA-induced cGAMP levels were comparable between Wild type and IFI16(-/-) cell lines. IFI16 was required for STING signalling following cGAMP stimulation. IFI16 was found to interact with STING promoting STING phosphorylation and translocation to peri-nuclear foci. Additionally, in preliminary experiments we observe that IFI16 may be required for STING palmitoylation. Thus, we propose that IFI16 and cGAS co-operate for the full activation of DNA sensing in HaCaT cells.