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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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Interactions of polyomavirus structures with components of cell innate immunity
Portychová, Tereza ; Forstová, Jitka (advisor) ; Schreiberová, Lucie (referee)
The topic of this thesis are the interactions of polyomavirus structures with components of innate immunity in infected cells. This review is focused on model SV40 and MPyV polyomaviruses and human BKPyV, JCPyV and MCPyV. The research of the interplay of innate immunity response and polyomaviruses is in its infancy. Infection with all studied polyomaviruses induces, via their LT antigens, DNA damage response (DDR), necessary for their efficient replication. DDR can activate both the canonical and the non-canonical pathway of interferon induction leading to an antiviral state. Polyomaviruses are recognized by the immune system first during replication of their genomes. Interferon induction by polyomaviruses can be initiated by the DNA sensor, cGAS, followed by STING activation, but also by recognition of the viral RNA by the RIG-1 sensor. The virus early LT and st antigens and the late agnoprotein of some polyomaviruses have demonstrated the potential to regulate innate immune responses and thus contribute to the establishment of polyomavirus persistence. Keywords: polyomaviruses, innate cell immunity, large T antigen, small t antigen, agnoprotein, interferon-stimulated genes
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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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Interference of selected DNA viruses with apoptotic processes
Sauerová, Pavla ; Forstová, Jitka (advisor) ; Štěpánek, Luděk (referee)
This work is focused on selected DNA viruses and some of their mechanisms used for inhibition or induction of the apoptotic processes. The selected DNA viruses are Hepatitis B virus, polyomaviruses, papillomaviruses and herpesviruses. Viruses developed different strategies for fighting the host defense mechanism during their evolution. One of the host defense mechanisms that reacts against virus infection is apoptosis. In case of viruses we can observe the phenomenon of inhibition or induction of apoptosis (which both depend on the life cycle phase of the virus). The purpose of these "fighting" strategies is to ensure successful replication, virus releasing from the cell and finally to let it spread in an organism or among them. Some "fighting" strategies are similar e.g. targeting and manipulation on p53 oncosupresor level or production of Bcl-2 homologs; other strategies are very specific. Certain viruses have mechanisms which allow them to survive in a host organism for a long time.
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Properties and function of middle T antigen of the murine polyomavirus
Fabiánová, Anna ; Forstová, Jitka (advisor) ; Čáp, Michal (referee)
Polyomaviruses are small DNA viruses, which are able to induce a broad variety of tumors. The main oncoprotein of the mouse polyomavirus (MPyV) is middle T antigen (MT antigen) which is able to transform cells. MT antigen has not an enzymatic activity of its own. It is able to activate signal transduction of host cells through its interactions with certain cellular proteins. These proteins include protein phosphatase 2A (PP2A), Src kinase, phosphatidylinositol 3 kinase (PI3K), Shc protein, 14-3-3 protein and phospholipase Cγ1 (PLCγ1). This work is focused on interaction between MT antigen and cellular proteins and on the impact of this interaction on cell transformation. Since MT antigen is a potent oncogene, the work also deals with the character of transformed cells and tumor development in mouse mammary epithelium. Keywords: polyomaviruses, MT antigen, PP2A, PI3K, PLCγ1, Shc protein, 14-3-3 protein
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MicroRNAs encoded by polyomaviruses.
Zachovalová, Veronika ; Bruštíková, Kateřina (advisor) ; Malík, Radek (referee)
MicroRNAs are small regulating molecules of RNA that are encoded by orgamism's genome. Biogenesis of microRNA takes place partly in the nucleus and partly in the cytoplasm. Result of this biogenesis is a 22 nt long microRNA molecule. They are able to silence the genes thanks to sequence- specific degradation of a target mRNA or thanks to the repression of translation of target, complementary mRNA. In mammalian cells the mechanism of translational repression is more common. During this mechanism the microRNA molecule is not entirely complementary to 3'UTR of its target mRNA. Polyomaviruses are small, non-enveloped dsDNA viruses with a circular genome and icosahedral capsid composed of VP1 protein pentamers. These viruses belong in a group called onkoviruses, which can transform infected cells and contribute to development of serious illnesses such as Merkell cell carcinoma. Their genome encodes regulating proteins called T antigens, structural capsid proteins and also microRNAs. My main focus in this thesis will be SV40, MPyV, MCPyV, BKPyV and JCPyV encoded microRNA molecules. Key words: polyomaviruses, small interfering RNA, microRNA, siRNA, RNA interference, mouse polyomavirus, BK virus, JC virus, SV40
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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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Analysis of antibody response during BK virus infection
Tomanová, Tereza ; Španielová, Hana (advisor) ; Saláková, Martina (referee)
BK virus is a human polyomavirus which is highly prevalent in the population. The virus is usually not very dangerous to its host, but it may cause complicati- ons in immunosuppressed patients. These complications commonly appear after kidney transplantation because BK virus persists in kidney epithelial cells. There are four subtypes of BK virus and it might be clinically important to screen for the identity of subtypes in matched pairs of donors and recipients of the kidney. This determination of the subtype specific antibodies by simple test could help to manage complications after the surgery. During previous project the ELISA test that could serologically differentiate between two main BK virus subtypes (I and IV) was designed, but its development is complicated by the fact that there is a strong cross-reactivity between the BK virus subtypes and antibodies. The modification of antigen towards better specificity might be required to succeed. Consequently, the main aim of this diploma thesis was to map important spots of major capsid protein VP1 of BK virus, particulary in EF and DE loops, which could participate in binding of antibodies. This aim was addressed by targeted mutagenesis of the gene coding VP1 protein in the region of the respective loop. Nucleotides coding two surface aminoacids...
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Biological treatment and its influence on the course of latent viral infections in patients with psoriasis
Laurin, Josef ; Šmahelová, Jana (advisor) ; Janovec, Václav (referee)
There are more than 80 identified autoimmune diseases. One of the most prevalent ones is psoriasis. Its prevalence is around 2-5 % worldwide. The treatment of this inflammatory skin disease can be divided as follows: in cases of low severity, topical therapies are used for local treatment and in the cases of insufficient effect, stronger therapies are used. Phototherapy is used for moderate severity, and systemic therapy is used in moderate to severe disease. Systemic agents include cytostatic methotrexate, immunosuppressant cyclosporin, or retinoids (vitamin A analogues). However, even systemic therapies may not yield the desired effects or may have adverse effects on the overall condition of the patient. In those cases, biological therapy comes to use. Biological therapy is usually conducted using antibodies and fusion proteins, which are made using recombinant technologies. Tumour necrosis factor α (TNF-α) and interleukin 12, 17 and 23 (IL-12, IL-17 and IL-23) inhibitors are the most commonly used in the treatment of psoriasis. During the inhibition of the immune system, it has been confirmed that a reactivation of viral infections can occur. These reactivations may subsequently lead to the development of various diseases caused by latent viral infections.
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Analysis of antibody response during BK virus infection
Tomanová, Tereza ; Španielová, Hana (advisor) ; Saláková, Martina (referee)
BK virus is a human polyomavirus which is highly prevalent in the population. The virus is usually not very dangerous to its host, but it may cause complicati- ons in immunosuppressed patients. These complications commonly appear after kidney transplantation because BK virus persists in kidney epithelial cells. There are four subtypes of BK virus and it might be clinically important to screen for the identity of subtypes in matched pairs of donors and recipients of the kidney. This determination of the subtype specific antibodies by simple test could help to manage complications after the surgery. During previous project the ELISA test that could serologically differentiate between two main BK virus subtypes (I and IV) was designed, but its development is complicated by the fact that there is a strong cross-reactivity between the BK virus subtypes and antibodies. The modification of antigen towards better specificity might be required to succeed. Consequently, the main aim of this diploma thesis was to map important spots of major capsid protein VP1 of BK virus, particulary in EF and DE loops, which could participate in binding of antibodies. This aim was addressed by targeted mutagenesis of the gene coding VP1 protein in the region of the respective loop. Nucleotides coding two surface aminoacids...
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