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Title: Inhibition of innate immune signalling pathways by cellular Cullin-RING ubiquitin ligases and poxviruses
Author: Georgana, Iliana
ISNI:       0000 0004 7960 9117
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2019
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Inflammation is a defence mechanism that higher organisms have evolved, in order to be protected from pathogenic infections and injury, and also to promote the healing process. Key players of the inflammatory responses are signalling pathways, which are tightly controlled to allow fast activation and efficient termination. Dysregulation of the fine-tune mechanisms that regulate these responses results in a failure to clear infection or, on the contrary, in the establishment of excessive, chronic inflammation. Ubiquitylation has emerged as a pivotal process in the regulation of innate immunity, by controlling both, the activation and termination of inflammatory responses. This thesis has examined mechanisms of inhibition of innate immune signalling by the ubiquitin system. A commercially available RNAi screen for Cullin-RING (CLR) complexes, revealed four novel inhibitors of the NF-κB pathway, SPSB1, FBXO8, FBXO42 and FBXL21, although due to time constrains only the characterisation of the first two proteins has taken place. Data presented in this thesis has revealed that SPSB1, a SOCS-box-containing substrate receptor protein, member of the Cullin-5 complex, controls only the NF-κB pathway, but not the IRF-3, AP-1 or JAK/STAT pathways, whereas its inhibitory activity on NF-κB lies at the level or downstream of the p50/p65 heterodimer, by a yet unknown mechanism. Furthermore, the ability of SPSB1 to inhibit the inflammatory responses driven by NF-κB was examined in the context of respiratory syncytial virus (RSV) infection. On the other hand, FBXO8, an F-box-containing substrate receptor protein, member of the Cullin-1 complex, has been shown here to negatively regulate NF-κB by acting at the level or upstream of the IKK complex, while its activity extents also on the AP-1 signalling cascade. In addition, a proteomic approach has revealed Cullin-4 as a binding partner of FBXO8, which has been successfully confirmed by conventional immunoprecipitations (IPs). The exact mechanism by which FBXO8 acts as an inhibitor of NF-κB and AP-1 signalling pathways has not been revealed and remains as focus of future studies. Opposingly to that discussed above, pathogens have evolved to escape, or antagonise the regulatory mechanisms mentioned above to their advantage. Poxviruses are known to express numerous proteins that counteract the host defence, and vaccinia virus (VACV) has been the main focus of research, due to its use as a vaccine vector. Poxviruses are the only DNA viruses known to replicate exclusively in the cytosol, which makes them susceptible to cytosolic DNA sensing, a pivotal cellular mechanism for pathogen recognition upon infection. Among the identified DNA sensors, cGAS is one of the most essential, which activates STING via the cGAMP production. Activation of STING results in its phosphorylation, dimerisation and translocation to the Golgi, where it mediates the IRF-3 activation. Data presented in this thesis revealed that infection with the attenuated VACV strain MVA, activated IRF-3 via the cGAS-STING axis and accordingly, STING dimerised and was phosphorylated. Conversely, STING dimerisation and phosphorylation was inhibited during infections with VACV strains Copenhagen (COP) and Western Reserve (WR) and in response to transfected DNA and cGAMP, thus demonstrating the efficient suppression of DNA sensing and IRF-3 activation by these strains. Infection with vv811 strain, a VACV deletion mutant lacking all the identified IRF-3 inhibitors, together with the inhibitors of DNA-PK DNA sensor, retained the ability of blocking STING activation, indicating that STING inhibition is independent of their activity. A similar inhibition of DNA-induced STING activation was also observed during cowpox and mousepox viral infections, suggesting that this mechanism might be conserved among the species. This thesis has generated a unique knowledge about the poxvirus-host interactions, as this is the first proof of poxviruses targeting DNA sensing directly at the level of cGAS-STING axis.
Supervisor: Maluquer de Motes, Carlos Sponsor: FHMS
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral