Dokument: Characterisation of interactions between the cGAS-STING pathway for type I IFN production and autophagy during vaccinia virus infection
| Titel: | Characterisation of interactions between the cGAS-STING pathway for type I IFN production and autophagy during vaccinia virus infection | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=63535 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20240919-084840-4 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Vago, Noemi [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Drexler, Ingo [Gutachter] Prof. Dr. Willbold, Dieter [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | Poxviruses are large dsDNA viruses whose replication occurs inside the cytoplasm in special compartments, which are called viral factories. There are several virulent strains (e.g., Western Reserve [WR] and Lister) expressing immunomodulatory genes, which inhibit type I and II interferon (IFN) production during infection. In addition to virulent viruses, there are also highly attenuated strains, an example of which is modified vaccinia virus Ankara (MVA). MVA is unable to complete a full replication cycle in most mammalian cells, but it induces a type I IFN response and autophagy upon infection. The dsDNA-induced cGAS-STING pathway has been established to play a crucial role in inducing type I IFN expression upon viral infection, while autophagy is responsible for cleaning infectious materials from cells. Notably, only a limited amount of information is available regarding how these two major pathways are affected during vaccinia virus (VACV) infection; therefore, the present study aimed to gain a deeper understanding of how MVA induces and WR inhibits these important innate immune effector mechanisms. The findings could lead to further improvements of MVA as a vaccine vector for fighting against tumour development and infectious diseases.
Several knockout (KO) cell lines specifically affecting the autophagy and/or cGAS-STING pathways for type I IFN production were generated and tested during VACV infection. Immunoblotting revealed that MVA induced and WR inhibited STING-dependent non-canonical autophagy, in which the presence of Beclin1 and the downstream members of the canonical autophagy pathway are essential, contrary to previously documented cGAMP-induced autophagy. Furthermore, the absence of TBK1 negatively affected the phosphorylation of STING in MEF cells; however, it had no impact on elevated LC3 lipidation, suggesting an accelerated degradation of P-STING. Moreover, p62 was proven to be critical for the removal of the P-STING:P-TBK1 complex after MVA infection, but it was not essential for STING-dependent non-canonical autophagy activation. In addition, different early- and late-stage inducers or inhibitory reagents for canonical autophagy and cGAS-STING pathways for type I IFN production, or even strategies for inactivating VACV strains chemically or non-chemically, were introduced. These approaches revealed that WR-induced inhibition of the activation of the autophagy and cGAS-STING pathways for type I IFN production were heavily dependent on uncoating and early viral gene expression, while MVA likely requires only binding to induce activation for both mechanisms. To identify the gene(s) responsible for inhibiting the activation of autophagy and cGAS-STING pathways for type I IFN production during infection with the virulent strain, a siRNA screen of 80 conserved WR genes was conducted using high-content confocal microscopy and flow cytometry. Once the results of both approaches had been analysed and summarised, five virulent virus genes (A10L, A18R, B5R, B13R, and C10L) were selected according to their levels of mismatch (presence of mutated, truncated, or deleted genes) between MVA and WR genome sequences. One additional candidate was included (B2R) in the target list due to a recent publication describing the effect of this specific poxin gene. Then, the selected genes were used to generate recombinant MVA viruses, which were reconstituted with and expressed the previously selected WR genes in their natural locus. The preliminary results indicated that recombinant MVA (recMVA) expressing the WR gene B2R (MVA-VV-B2) was able to interfere with the autophagy and cGAS-STING pathways both in the early and late phases of infection. In addition, cells infected with MVA-VV-A10 and -A18 for 4 h displayed reduced autophagy activation without influencing the cGAS-STING pathway for type I IFN production. However, cells previously infected for 24 hours with MVA-VV-A18 exhibited an even more decreased P-STING level upon AraC treatment, which was contrary to the expected results. Since A18R is an early/late gene, while A10L is a late gene, these findings could suggest that the early expressed precursor forms of late viral genes in the infected cells could have different effects on the cellular pathways compared with their actual late-expressed forms. This should be investigated further in future studies. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Medizinische Fakultät » Institute » Institut für Virologie | |||||||
| Dokument erstellt am: | 19.09.2024 | |||||||
| Dateien geändert am: | 19.09.2024 | |||||||
| Promotionsantrag am: | 29.03.2023 | |||||||
| Datum der Promotion: | 28.08.2023 |
