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Regulation of STING function during murine polyomavirus infection
Šnejdarová, Aneta ; Horníková, Lenka (advisor) ; Pimková Polidarová, Markéta (referee)
Stimulator of Interferon Genes (STING) is the adapter protein of an innate immunity signalling pathway, involved in detection of double-stranded DNA (dsDNA) in the cell cytoplasm, which leads to the expression of pro-inflammatory genes, including the production of type I interferon. Eventhough during the infection with a dsDNA virus, murine polyomavirus (MPyV), the STING protein is activated, the resulting interferon production is moderate. Therefore, it can be assumed that the function of the STING protein is regulated in MPyV-infected cells. The aim of this thesis was to investigate three mechanisms by which the regulation can occur, namely through protein interaction partners, post- translational modifications, or changes in the subcellular localization of the STING protein. A cell-line of mouse fibroblasts stably expressing the STING protein fused with the HA-tag was established to facilitate the research. Furthermore, two plasmids were prepared, that encode the STING protein fused with the green fluorescent protein, facilitating the monitoring of the localization of the protein in the cell, or with a composite tag containing an in vivo biotinylated BioEaseTM -tag enabling effective isolation of the STING protein. The results of colocalization observations and coimmunoprecipitation suggest that...
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Extracellular vesicles and middle T antigen of mouse polyomavirus
Kropáček, Václav ; Šroller, Vojtěch (advisor) ; Brázdová, Andrea (referee)
This study is focused on middle tumor antigen (MT Ag) of mouse polyomavirus (MPyV), potential consequences of it's secretion via extracellular vesicles (EVs) and it's effect on cellular signaling. MT Ag is membrane bound protein able to induce cellular transformation thanks to it's ability to interfere with cellular signal transduction. Mainly due to aberrant activation of MAP kinase pathway. Firstly we followed up previous observations of our group concerning ability of MT Ag to be secreted from cells via extracellular vesicles. We were interested if MT Ag could contribute to malignant transformation in recipient cells. We performed 2 types of EVs isolation from cell lines stably expressing middle T antigen (3T6MT). We confirmed presence of MT Ag in isolated EVs. Then we characterized isolated EVs by detection of exosomal markers and cryo-electron microscopy. In next step we exposed recipient cell line (3T6) to isolated EVs and with use of flow cytometry tried to detect internalization of MT Ag. Simultaneously we tried asses levels of Erk phosphorylation in 3T6 cells exposed to EVs. Secondly we tried to confirm and analyse previous unpublished observations of elevated levels of NF-kB phosphorylation in cells stably expressing MT Ag. We used western blot and detection of NF-kB dependent secreted...
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Polyomavirus minichromosomes: interactions with components of innate imunity
Satratzemis, Christos ; Forstová, Jitka (advisor) ; Trejbalová, Kateřina (referee)
The genome of polyomaviruses is a circular dsDNA (double-stranded DNA) which is associated with cellular histones within virions and during the entire viral replication cycle. Given the structural similarity to eukaryotic chromatin, the complex of polyomaviral DNA with histones is called minichromosome. The chromatin state of minichromosomes influences viral transcription and replication which could be exploited by the host innate immune response. One of the components of innate immunity, that affects viral chromatin, is the non-canonical histone H3.3, its chaperone DAXX- ATRX (death domain associated protein 6-alpha-thalassemia, mental retardation X-linked syndrome) and protein complexes called PML (promyelocytic leukemia protein). In order to trigger the innate immune response, foreign and/or stress molecules have to detected. During mouse polyomavirus (MPyV) infection, the innate immune response is initiated via the DNA sensor cGAS (cyclic GMP- AMP synthase). In this master's thesis, the distribution of histone H3.3, its chaperone DAXX-ATRX and the PML protein was analyzed during infection with MPyV. Using mass spectrometry, the histone was detected within viral chromatin. The data suggest that the chaperone complex and PML are involved in the regulation of H3.3 incorporation into the chromatin...
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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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Studies of properties of viral capsid proteins and development of recombinant vaccines and diagnostic components based on artificial viral structures
Fraiberk, Martin ; Forstová, Jitka (advisor) ; Hubálek Kalbáčová, Marie (referee) ; Hejnar, Jiří (referee)
The aim of this study was to develop a system for easy production of different veterinary chimeric vaccines based on stable mouse polyomavirus (MPyV) structures. The system is designed for antigens that are problematic in production or stability. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers based on the major capsid protein VP1 were designed to be exploited as vaccines against other pathogens. The different strategies used in this study are based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by inserting them into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, thus forming giant pentamers of a chimeric protein. Candidate vaccine antigens against porcine circovirus 2 (PCV2), the causative agent of porcine circovirus 2 systemic diseases (PCV2-SD) which causes significant economic losses in swine breeding, were prepared using the constructed vectors. All candidate vaccines induced the production of antibodies against the capsid protein of PCV2 after immunization of mice. The candidate vaccine Var C based on fusion of MPyV and PCV2 capsid proteins, is able to induce production of antibodies with...
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Preparation and characterization of modified viral particles derived from mouse polyomavirus for the transport of genes to increase the efficiency of transduction
Škvára, Petr ; Španielová, Hana (advisor) ; Sýkora, Michal (referee)
Viral particles derived from mouse polyomavirus can be potentially used as a delivery system for therapeutic genes and drugs into target cells. This thesis focuses on preparation and characterization of polyomaviral particles that are modified with cell-penetrating peptides in order to increase efficiency of transduction of reporter genes into human cells. Viral particles that are composed of major capsid protein VP1 in combination with minor capsid protein VP2 and minor capsid protein VP3 that is modified with octaarginine, LAH4 peptide or with transduction domain of adenoviral protein VI are analysed in transduction assays. The thesis also provides information about the effect of the modification on encapsidation of heterologous DNA. The results of transduction assays performed with modified particles containing encapsidated luciferase gene revealed that efficiency of transduction did not increase but decreased in comparison with unmodified particles. These findings help to elucidate the role of polyomaviral minor capsid proteins in gene transfer mediated by viral particles and contribute to the design of new strategies for modifications of viral particles derived from mouse polyomavirus for their successful application in nanomedicine. Key words: mouse polyomavirus, pseudovirions, virus-like...
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Studies of polyomavirus trafficking from late endosomes towards the cell nucleus
Štach, Martin ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Mouse polyomavirus (MPyV) is a model virus of the Polyomaviridae family. Polyomaviruses are small non-enveloped DNA viruses. They cause severe problems to immunocompromised patients. Their oncogenic potential is known in animals and humans. Trafficking of MPyV within the cell is not clear yet. The virus enters via smooth monopinocytic vesicles and continues to early and late endosomes. From there, the virus is transported to the ER by unknown mechanism. It bypasses Golgi aparatus (GA). One possible pathway is from late endosomes to trans-Golgi network (TGN) facilitated by Rab9 GTPase and then in COPI vesicles to the ER. In this thesis, the effect of inhibitors of retrograde transport (Brefeldin A, Golgicide A) on MPyV infection was evaluated. Brefeldin A is not completely specific; it has effect on whole endosomal system. Golgicide A causes specific disruption of transport via TGN and GA. Both inhibitors suppressed infection of MPyV. Confocal microscopy revealed colocalization of some MPyV virions with markers of TGN and COPI vesicles. MPyV didn't colocalize with cis-Golgi marker. Unfortunately, the effect of overexpression of Rab9 dominant negative mutant couldn't been evaluated due to its high cytotoxicity. However, overexpression of wild type Rab9 slightly increased infectivity. The results...
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Preparation of expression vectors and virus mutants for studies of the minor structural proteins of polyomaviruses.
Cibulka, Jakub ; Forstová, Jitka (advisor) ; Šroller, Vojtěch (referee)
Polyomaviruses are small non-enveloped DNA viruses infecting birds and mammals, including human. Their capsid consists of the major capsid protein, VP1, and two minor capsid proteins, VP2 and VP3. The VP2 and VP3 proteins are supposed to have an important function in the transport of viral genome into the cell nucleus, which is a key step to facilitate viral replication. VP2 and VP3 proteins of mouse polyomavirus and SV40 have an ability to bind and disrupt cellular membranes. This feature is believed to be involved in the transport of viral genome into the nucleus. Plasmids carrying genes of the minor capsid proteins of Merkel cell polyomavirus were prepared in order to produce and visualize these proteins in mammalian cells. These proteins are known to have very unusual sequences compared to other human polyomaviruses or related mouse polyomavirus. When produced alone, the minor capsid proteins of Merkel cell polyomavirus did not significantly interact with cellular membranes, unlike the minor proteins of the mouse polyomavirus. The second goal of this work was to prepare mouse polyomavirus mutants with deletion in hydrophobic domains of VP2 and VP3 proteins. These domains are likely responsible for the mentioned membrane interactions. Prepared mutants were non-infectious. The loss of infectivity was not...
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Minor Structural Proteins of Polyomaviruses: Attributes and Interactions with Cellular Structures
Vinšová, Barbora ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Even though polyomaviruses have been intensively studied for more than 60 years, the role of minor structural proteins VP2 and VP3 in some important steps of viral life cycle has still not been fully elucidated, explicitly their role in viral genome delivery to the cell nucleus and their involvement in late phases of viral life cycle. This diploma thesis focuses on the study of minor proteins of Mouse polyomavirus (MPyV) and Human polyomavirus BK (BKV). Four rabbit polyclonal antibodies against minor proteins of polyomaviruses MPyV or BKV have been prepared within this diploma thesis. Two of these prepared antibodies target minor proteins of MPyV (α-MPyV VP2/3) or BKV virus (α-BKV VP2/3), other two prepared antibodies recognize C-terminal sequence common to minor proteins VP2 and VP3 of MPyV (α-MPyV C-termVP2/3) or BKV virus (α-BKV C-termVP2/3). In the second part of this diploma thesis we aimed to study toxicity of BKV virus minor proteins during individual production in mammalian cells. Obtained results suggest that minor proteins of BKV virus might not exhibit as high levels of cytotoxicity as minor proteins of MPyV virus. Third part of this diploma thesis is devoted to investigation of interactions of BKV and MPyV minor proteins with cellular proteins and within one another respectively....
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Studies of minor capsid proteins of the mouse polyomavirus
Vít, Ondřej ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Mouse polyomavirus (MPyV) is a small non-enveloped virus. Its capsid consists of 72 pentamers of the major capsid protein VP1. The central cavity of each VP1 pentamer contains one minor capsid protein, either VP2, or VP3. The minor capsid proteins are dispensable for capsid formation, but their presence is required for infection of the host cell, presumably because of their anticipated functions during virus entry. After internalization, MPyV virions traffic to endoplasmic reticulum (ER). VP2 and VP3 have been proposed to function as factors responsible for penetration of ER membranes, which is required for subsequent delivery of the viral DNA into the nucleus, a key step of the early phase of MPyV infection. Three hydrophobic domains were predicted in the sequence of VP2 and VP3. First in the unique Nterminal part of VP2, second and third in the common part of VP2 and VP3. The third domain corresponds to C-terminal VP1binding alpha-helix. It has been previously found in our laboratory, that VP2 and VP3 fused to N-terminus of EGFP, when expressed in mammalian cells, display properties similiar to the wild-type VP2 and VP3, namely affinity to intracellular membranes and high cytotoxicity. Expression plasmids carrying mutated VP2 and VP3 fused to Nterminus of EGFP were prepared to determine the hydrophobic...
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