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Cellular factors restricting mouse polyomavirus infection in host cells: Studies of PML protein isoforms
Anderová, Karolína ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Promyelocytic leukaemia nuclear bodies (PML NBs) are multifunctional nuclear spherical structures formed by the PML protein shell and other interaction partners that have been described to be involved in many cellular processes and immune defences. In the antiviral immune response, PML NBs and their components act as direct restriction factors as well as in the regulation of the interferon response. On the other hand, viruses have developed antagonistic mechanisms to resist this inhibition. This work deals with the role of PML NBs in infection with model Murine polyomavirus (MPyV) and focuses on the study of PML protein isoforms. The first aim of the work was to analyse the formation of human (hPML) and mouse (mPML) NBs in a mouse embryonic fibroblast (MEF) model. Subsequently, the localization of hPML and mPML NBs during infection was determined. Close localization with viral replication centres was observed for both PML species. In the next step, the effect of infection or interferon α (IFNα) on mPML protein expression was tested. Infection and treatment with IFNα led to an increase in mPML expression at the level of both gene transcription and protein synthesis. At the same time, the data indicated the largest increase in transcription of the mPML3 isoform. The work also addressed the potential...
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Role of PML in ribosomal stress
Kremserová, Petra ; Vašicová, Pavla (advisor) ; Sztacho, Martin (referee)
PML is involved in many cellular processes. It organizes nuclear structures PML nuclear bodies (PML NBs) and it associates with nucleolus in response to ribosomal stress to form PML nucleolar associations (PNAs). The function of PNAs is unclear. To elucidate this question, one can attempt to identify proteins interacting with PML at nucleolus. The common method is co- immunoprecipitation, however, this approach cannot be used for PML due to its low solubility. To defeat this, an alternative way of proximity-dependent biotin labelling could be used. The goal of this work was to explore a suitability of biotin labelling for identification of PML nucleolar partners. For this purpose I prepared constructs of wild type or mutated PML with GFP and biotin ligase for transient and stable expression and analysed their propensity to form PML NBs and doxorubicin-induced PNAs, and biotinylate their vicinity. In transient expression, both fusion proteins formed PML NBs and only wild type but not mutated PML IV formed PNAs after doxorubicin treatment with preserved biotinylation capability. In stable expression of fusion proteins in cells with PML knockout the number and composition of PML NBs was aberrant and no PNAs were observed. However, this system was utilized for optimization of solubilisation of biotinylated...
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Role of PML in ribosomal stress
Kremserová, Petra ; Vašicová, Pavla (advisor) ; Sztacho, Martin (referee)
PML is involved in many cellular processes. It organizes nuclear structures PML nuclear bodies (PML NBs) and it associates with nucleolus in response to ribosomal stress to form PML nucleolar associations (PNAs). The function of PNAs is unclear. To elucidate this question, one can attempt to identify proteins interacting with PML at nucleolus. The common method is co- immunoprecipitation, however, this approach cannot be used for PML due to its low solubility. To defeat this, an alternative way of proximity-dependent biotin labelling could be used. The goal of this work was to explore a suitability of biotin labelling for identification of PML nucleolar partners. For this purpose I prepared constructs of wild type or mutated PML with GFP and biotin ligase for transient and stable expression and analysed their propensity to form PML NBs and doxorubicin-induced PNAs, and biotinylate their vicinity. In transient expression, both fusion proteins formed PML NBs and only wild type but not mutated PML IV formed PNAs after doxorubicin treatment with preserved biotinylation capability. In stable expression of fusion proteins in cells with PML knockout the number and composition of PML NBs was aberrant and no PNAs were observed. However, this system was utilized for optimization of solubilisation of biotinylated...
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