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Negative regulation of the IFI16 and cGAS DNA sensing pathways
Muhič, Samra ; Huerfano Meneses, Sandra (advisor) ; Kanwal, Madiha (referee)
DNA sensors are molecules with the ability to sense DNA constituting an important tool of innate immunity. They are initiators of various signalling pathways, one of them being the production of interferons, which induce not only an anti-viral cell state but also protect cells of treats not related to pathogens. At least, fourteen DNA sensors have been described so far, among them - IFI16 and cGAS. Both sensors signal via an adaptor protein STING resulting in the production of type I IFN. All three of these molecules (cGAS, IFI16, STING) are strictly regulated either by host-cells in order to prevent immune over-activation or by viruses for the immune evasion. This work focuses on the mechanisms of negative regulation of the three molecules: post-translational modifications such as phosphorylation, ubiquitination, SUMOylation, acetylation and methylation; protein-protein interactions; degradation by the proteasomal system or by autophagy. Not surprisingly, viruses encode proteins able to down-regulate IFN responses for example, some proteins of herpes viruses interact with cGAS, IFI16 or STING preventing their activation or leading to their degradation. Other proteins of herpes viruses cause the degradation of the mRNA of the sensors or the adaptor. Dengue protease factor NS2B degrades cGAS or the...
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Mouse polyomavirus:The way of virus translocation to the cell nucleus and sensing of viral genomes by sensors of innate immunity
Soldatova, Irina ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee) ; Pichová, Iva (referee)
To understand molecular mechanisms of individual steps of virus infection is a prerequisite for successful design of specific and effective antiviral drugs. Polyomaviruses, replicating in the cell nucleus, travel from plasma membrane to the endoplasmic reticulum (ER) in endosomes. However, it is not clear how they deliver their DNA genomes from ER to the nucleus. In this thesis, we found that partially disassembled virions of the Murine polyomavirus (MPyV) interact with importin β1 at around 6 hours post infection. Mutational disruption of the nuclear localization signal (NLS) of the major capsid protein, VP1, and/or common NLS sequence of the minor capsid proteins VP2 and VP3 did not affect the structure and composition of virions, but it resulted in decreased viral infectivity (up to 80%). Virions are thus released from ER to cytosol and translocate to the nucleus via nucleopores. Mutation analyses of NLSs of individual capsid proteins showed that MPyV virions can utilize VP1 and VP2/VP3 NLSs in concert. However, one functional NLS, either that of VP1 or VP2/3 seems to be sufficient for the delivery of VP1-VP2/3 complexes into the nucleus, although none of these proteins is delivered into the nucleus separately. Thus, the conformation of NLS regions given by the presence of all three capsid...
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Sensing of MPyV infection by innate immunity sensors
Rjabčenko, Boris ; Forstová, Jitka (advisor) ; Anděra, Ladislav (referee) ; Mělková, Zora (referee)
Host sensors that recognize pathogen associated molecular patterns and the mechanisms of innate immune response to mouse polyomavirus (MPyV) infection were the main topics of current work. We found that MPyV did not induce interferon (IFN) production during early events of infection, but induced interleukin-6 (IL-6) and other cytokine production without inhibiting virus multiplication. Cytokine microenvironment changed the phenotype of adjacent non infected fibroblasts toward the cancer-associated fibroblast (CAF)-like phenotype. We identified Toll-like receptor 4, a sensor of the innate immunity system, to be responsible for infection dependent IL-6 production. In an effort to determine whether and where virions are released from endosomal compartments into the cytosol, we found that the hydrophobic domains of minor capsid proteins, exposed on the surface of virions after their partial disassembly in the ER, play an important role in effective escape of virions from the lumen part of endoplasmic reticulum into the cytosol, Although naked, partially disassembled virions appear before translocation to the nucleus in the cytosol, viral DNA is not recognized by cytosolic sensors at this phase of infection Sensing of MPyV resulting in IFN production occurs first during viral replication. Mutant virus,...
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