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Sensing Foreign DNA: The Role of DNA-PKcs in Human Anti-viral Innate Immunity 10.17863/CAM.88011 Hristova, Dayana Ferguson, Brian Host cell pattern recognition receptors (PRRs) are a first line of defence against pathogens and function to generate a productive innate immune response. PRRs sense pathogen-associated molecular patterns (PAMPs), such as viral genomic DNA, which is a major PAMP during DNA virus infection. Viral DNA sensing leads to the activation of the STING-TBK1-IRF3 signalling axis and the production of type I interferon. Previously, our work identified the non-homologous end-joining protein DNA-PKcs, part of the DNA-dependent protein kinase (DNA-PK) complex, as an intracellular PRR for cytoplasmic viral DNA in murine cells. After screening several human cell lines, we established a robust system to dissect the DNA sensing pathway in human fibroblasts. In these human cells DNA-PKcs was found to be essential for the production of type I interferon via the STING pathway in response to DNA and DNA virus infection and we found that the kinase activity of DNA-PKcs was not required for this response. Many DNA viruses evade the immune response by inhibiting the pathway. We make use of attenuated Herpes simplex virus 1 (HSV- 1) and Vaccinia virus (VACV) that lack immunomodulatory proteins and drive type I interferon production. DNA-PKcs-/- cells have a defective immune response after infection with attenuated HSV and VACV. Furthermore, primary patient fibroblasts harbouring a mutation in DNA-PKcs showed a gain-of-function effect and an enhanced immune signaling to DNA and DNA virus infection. DNA-PKcs has also been linked to cell death during retrovirus integration although this has not been studied extensively. We carried out some preliminary work in this study, showing that DNA-PKcs-/- cells are more prone to cell death during HSV-1 infection and have reduced yields of virion production. This work demonstrates the role of DNA-PKcs as a viral DNA sensor in human cells and adds to the knowledge of the DNA sensing processes that are essential for anti-viral innate immunity. University of Cambridge 2021-10-01 Thesis Doctoral Doctor of Philosophy (PhD) eng Cambridge Philosophical Society https://www.repository.cam.ac.uk/handle/1810/340572 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2eac6a86-df97-48dc-8df0-0e7543930c9d/download 5a6423f54c1330cae36d1e1466dec23c https://creativecommons.org/licenses/by-nc/4.0/ innate immunity nucleic acid sensing pattern recognition receptors viral infection