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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...
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
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...
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...
Studies of properties of gene products of the Merkel cell carcinoma polyomavirus: Antibody preparation and expression vector construction.
Sauerová, Pavla ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Merkel cell polyomavirus (MCPyV) is a recently discovered human virus, having it's genome often integrated in a genome of Merkel carcinoma cells. Although this type of carcinoma is not so usual, it is very aggressive and it's incidence has been rising in last few years. It is not surprising that this virus is nowadays in the centre of scientific interest, as well as other pathogens and mechanisms affecting human life. Because the virus was discovered not so long ago, its research has been at the whole beginning. This diploma thesisaims to contribute to the study of this virus from the molecular-virology point of view. A neutralizing monoclonal antibody, type IgG2a, targeted against the main capsid protein of MCPyV, VP1, and recognizing its conformational epitote was prepared. This antibody was then used for a pilot study of VP1 VLPs MCPyV movement in mammalian cells. Results showed that the studied virus, at least particularly, utilizes caveolin-1-carrying vesicles for its movement in cells (colocalisation of VP1 VLPs and caveolin-1 was observedColocalisation with EEA1 marker of early endosomes, LamP2 marker of endolysosomal compartments or with BiP marker of endoplasmic reticulum was sporadic but significant. These preliminary results suggest that MCPyV might utilise an endocytic pathway leading...
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...
New chimeric antigen receptor (CAR) for therapy of human cytomegalovirus (HCMV) infection
Kroutilová, Marie ; Němečková, Šárka (advisor) ; Forstová, Jitka (referee)
Human cytomegalovirus (HCMV, Herpesviridae) can cause severe complications in the infected individuals undergoing hematopoietic stem cell transplantation. Nowadays, these patients are treated using antivirotics or HCMV-specific T cells derived from the seropositive graft donor. This study explored the possibility of redirecting HCMV-non-specific T cells from a seronegative donor towards HCMV-infected cells via chimeric antigen receptor (CAR), i.e. artificially designed T cell receptor. Viral glycoprotein B (gB) has been selected as a target for this receptor. Published sequence of a single chain variable fragment of a human antibody was used for the design of the CAR against gB (gBCAR). After the verification of production and surface localization in cell lines, gBCAR was being introduced into human T cells via lentiviral vectors. Human fetal lung fibroblasts (LEP) infected with HCMV were used as target cells after the expression of gB at their surface was demonstrated. gBCAR functionality was evaluated by the incubation of modified T cells with infected cells and subsequent analysis of media for IFNγ concentration, which was significantly higher in the setting of gBCAR T cells incubated with HCMV-LEP than in the control incubations. The results obtained show the specificity of gBCAR against...
Roles of cytoskeleton in mouse polyomavirus trafficking
Klímová, Lucie ; Forstová, Jitka (advisor) ; Šmahel, Michal (referee)
6 Roles of cytoskeleton in mouse polyomavirus trafficking ABSTRACT: Mouse polyomavirus (mPyV) is small non-enveloped DNA virus. Its endocytic pathway is studied for a potential utilisation of polyomaviral virus-like particles in gene therapy and/or immunotherapy. mPyV enter cells by internalisation into smooth monopinocytic vesicles. During it's journey through the cell, it pass through early endosomes, and at the time 3 hours post infection, it is localised in endoplasmic reticulum and recycling endosomes. Many aspects of mPyV trafficking and nuclear entry are not clear yet. Time-lapse live imaging fluorescence confocal microscopy was used to describe the mouse polyomavirus intracellular movements. For these studies, we utilised mPyV fluorophore-labeled virions and cells expressing GFP-tagged g-actin or alpha-tubulin. Some virion-loaded vesicles were seen to move with actin organised into dynamic structures. Some of these structures resembled actin comets created by Listeria or vaccinia virus. At the same time post infection (40-60 min post infection), movement of the virion loaded vesicles along mirotubules was observed suggesting the simultaneous involvement of actin and tubulin during mPyV trafficking. Dynamitin, a dominant negative inhibitor of dynein-dynactin function reduced mPyV infection. Taken...
Viruses and cytosketelon of the cell nucleus
Cibulka, Jakub ; Forstová, Jitka (advisor) ; Šťovíček, Vratislav (referee)
The nuclear cytoskeleton (the nucleoskeleton) provides a structural integrity to the nucleus and is involved in number of key processes including transcription, chromatin remodelling and mRNA transport. The nucleoskeleton consists of nuclear lamins, nuclear actin and other proteins. Some viruses, which replicate themselves in the nucleus, use nuclear cytoskeleton in their life-cycle. On the other hand the nucleosketon may also represent a barrier for viral infection. Herpesviruses need nuclear actin for capsid assembly and transport, but they have to desintegrate the nuclear lamina in order to escape the nucleus. Nuclear actin also participates in the morphogenesis and probably nuclear export of baculovirus capsids. Some retroviruses transport their unspliced RNAs from the nucleus using nuclear actin and there is also some evidence of retrovirus-induced nuclear lamina disruption. In this work, I focus on the interactions of above-mentioned viruses with the nuclear cytoskeleton (namely nuclear actin and lamins).

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2 Forstová, Jana
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