Viral DNA sensing by the non-homologous end joining machinery

Context and challenge
The ability of human cells to sense and respond to nucleic acids is essential for antiviral immunity, but also underlies a broad range of diseases. DNA is a potent inflammatory trigger, ubiquitous inside nuclei and mitochondria, and present in pathogens such as DNA viruses and bacteria. Human cells can sense microbial DNA and self-DNA that has leaked from damaged organelles into the cytoplasm, causing an inflammatory response. Intracellular pattern recognition receptors (PRRs) bind foreign or mis-localised DNA and drive multiple signalling outputs required to restore tissue homeostasis. Dysregulation of these intracellular DNA sensing mechanisms therefore results in susceptibility either to infection or to autoinflammatory and autoimmune diseases. Indeed, there is an extensive and growing list of inflammatory pathologies and interferonopathies where DNA sensing PRRs are implicated, including Aicardi-Goutieres syndrome (AGS), Systemic Lupus Erythematosus (SLE), Sjögren's syndrome and neurodegenerative diseases.
The DNA sensing PRRs cyclic GMP-AMP synthase (cGAS), DNA-dependent protein kinase (DNA-PK) and interferon gamma inducible protein 16 (IFI16) all bind cytosolic DNA and are required for the initiation of antiviral immune responses. It is not clear, however, how these proteins biochemically and functionally interact in different contexts to ensure appropriate signalling following infection or tissue damage. Viral genomic DNA and damaged self-DNA from the nucleus or mitochondria activate these cytoplasmic DNA sensing PRRs, but how these different triggers result in the generation of different signals is not well defined. For example, DNA-PK is indispensable in antiviral responses to poxviruses while IFI16 is essential for sensing herpesvirus infections and nuclear DNA damage. Further, we have now shown that different DNA structures can differentially stabilise DNA PRR co-complexes. As such we hypothesise that DNA sensing is context specific so we will define the specific molecular complexes that determine cGAS/STING activation and signalling outputs in response to DNA virus infection and DNA damage. To fully explore this hypothesis requires an integrated structural, biochemical and cellular approach to define the nature of these protein complexes in vitro, in cells, and in the context of virus infection.
Aims and Objectives
Aim 1: Define the molecular structures of DNA PRR complexes and their interactions with different DNA substrates
Aim 2: Determine the context-dependent mechanisms of intracellular DNA sensing in human cells
Aim 3: Define how human disease-associated variants in DNA-PK modify intracellular DNA sensing responses
Potential applications and benefits
There are broad therapeutic applications for pharmaceutical modulation of DNA sensing PRRs either for enhancing antiviral immunity or reducing the excessive auto-inflammation they can cause. Activation enhances adaptive immune responses and so these PRRs are key targets for vaccine adjuvants and neoadjuvant cancer therapies. Understanding how different triggers are linked to different signalling outputs will help to understand the disparate clinical phenotypes caused by inborn errors of immunity (IEIs) in different components of DNA sensing pathways. Specifically, in this study, we will define the impact of novel SLE-associated mutations in DNA-PK. Therefore, we will make key fundamental advancements in antiviral immunity, in keeping with the UKRI’s strategic priority ‘Tackling Infections’, whilst identifying specific mechanisms underlying SLE.