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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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Mouse polyomavirus:The way of virus translocation to the cell nucleus and sensing of viral genomes by sensors of innate immunity
Soldatova, Irina ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee) ; Pichová, Iva (referee)
To understand molecular mechanisms of individual steps of virus infection is a prerequisite for successful design of specific and effective antiviral drugs. Polyomaviruses, replicating in the cell nucleus, travel from plasma membrane to the endoplasmic reticulum (ER) in endosomes. However, it is not clear how they deliver their DNA genomes from ER to the nucleus. In this thesis, we found that partially disassembled virions of the Murine polyomavirus (MPyV) interact with importin β1 at around 6 hours post infection. Mutational disruption of the nuclear localization signal (NLS) of the major capsid protein, VP1, and/or common NLS sequence of the minor capsid proteins VP2 and VP3 did not affect the structure and composition of virions, but it resulted in decreased viral infectivity (up to 80%). Virions are thus released from ER to cytosol and translocate to the nucleus via nucleopores. Mutation analyses of NLSs of individual capsid proteins showed that MPyV virions can utilize VP1 and VP2/VP3 NLSs in concert. However, one functional NLS, either that of VP1 or VP2/3 seems to be sufficient for the delivery of VP1-VP2/3 complexes into the nucleus, although none of these proteins is delivered into the nucleus separately. Thus, the conformation of NLS regions given by the presence of all three capsid...
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Major capsid protein of mouse polyomavirus: interaction with cellular structures
Horníková, Lenka
Mouse polyomavirus (MPyV) is small non-enveloped DNA virus. Although this virus has been studied for almost 60 years, it still remains unclear, how can virus transport its genetic information to the cell nucleus. Also, the mechanism of virion morphogenesis is not well understood. First part of this work is focused on endocytic pathway which is used by MPyV for trafficking toward the cell nucleus. Using dominant negative mutant of caveolin-1 we showed that caveolin-1dependent endocytic pathway, described for SV40, is not used by MPyV for productive infection. MPyV is transported to early endosomes. Acidic milieu of endosomes is indispensable for productive infection. Preventing virus localisation into early endosomes (dominant negative mutant of Rab 5 GTPase) or endosomes alkalisation (by ammonium chloride or bafilomycin A1) led to dramatic decrease of virus infectivity. Alkalisation of endosomes entailed retention of MPyV in early endosomes. It indicates that virus is further transported to late endosomes. Finally, we confirmed by FRET that MPyV is in perinuclear space localized into recycling endosomes. Another poor characterized process is virion morphogenesis. To characterize the participation of cellular proteins in virion precursor complexes, nuclear as well as whole-cell lysates of infected cells or...
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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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Targeting of Viral Nanoparticles to CD44 via Hyaluronic Acid
Hustedová, Anna ; Španielová, Hana (advisor) ; Hubálek Kalbáčová, Marie (referee)
Hyaluronic acid (HA) is widely studied as a targeting moiety to CD44 overexpressing cancer cells. Various types of nanoparticles (NPs) were modified by HA. Virus-like particles (VLPs) derived from mouse polyomavirus are an interesting class of NPs that can be modified by various targeting agents to increase their potential as gene or drug delivery vehicles for e.g. theragnostic purposes. HA has not been tested as a targeting moiety on VLPs, hence this was the focus of the current study. HA (~14 kDa) was attached to the VLPs via a bispecific Bodipy-derived fluorescent probe. To test the targeting potential of HA on comparable non-viral NPs, nanodiamonds were prepared in a similar manner. NPs functionalized with HA, together with Bodipy-labeled control variants, were tested on interactions with MDA-MB-231 cells overexpressing CD44. The NP-cell interaction via CD44 was assessed by a competitive cell-binding assay, where non-labeled HA competed for HA-binding sites at CD44 with the NPs. CD44 specific cell interactions were detected in studies with HA functionalized nanodiamonds, whereas VLP-HA* associated with cells in a less specific manner. Control VLPs with polyethylene glycol (PEG) did not interact with the cells. Results indicate that the HA targeting strategy for the VLPs requires optimization to...
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Analysis of posttranslational modifications of proteins by mass spectrometry
Musil, Dominik ; Šulc, Miroslav (advisor) ; Kukačka, Zdeněk (referee)
Viruses, etiological agents of many infectious diseases, are small noncellular particles that run their replication process solely inside host cells. It is generally assumed that the posttranslational modifications of viral capsid proteins are responsible for their infectivity (e.g. phosphorylation catalysed by kinases of host cells). The appropriate model for study of the viral phosphorylation "profile" relation with its infectivity is mouse polyomavirus of the Polyomaviridae family. By comparison of virions, as well as the major capsid protein VP1 of wild type mouse polyomavirus with viral mutant created by deletion of the part of the genome coding regulatory proteins of virus and produced in two different cell lines WOP and 3T6 was found by mass spectrometry a major phosphorylation in the three specific amino acids of VP1. These are considered important for proper morphogenesis of virions and their ability to infect host cells. The qualitative representation was not affected by the cell line selection. Furthermore, in case of VP1 "dimerization" detected on SDS-PAGE electrophoretogram the double phosphorylation of VP1 pThr63, pSer66 was confirmed in our experimental in vivo approach. Therefore, posttranslational modifications, specifically phosphorylation, could probably affect structural...
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Major structural protein of Polyomaviruses: Interactions with host cell structures
Mrkáček, Michal ; Horníková, Lenka (advisor) ; Němečková, Šárka (referee)
The main structural protein VP1 is the product of late polyomaviral genes and it is the largest and the most abundant protein of the whole polyomaviral capsid. Because of the low coding capacity of the polyomaviral genomes, it is considered that in addition to its structural role the VP1 protein might have some additional functions in the late phase of the infectious cycle. This diploma thesis is exactly on these additional functions. In the case of the VP1 protein of mouse polyomavirus, it was observed that the protein is capable of binding to the structure of cellular microtubules. The first objective of this work was to test whether pentamers of the VP1 protein are able of this binding without the participation of other cellular (or viral) proteins. Based on an in vitro experiment, we showed that protein VP1 binds to the structure of microtubules very inefficiently. The second objective of this work was to prepare a detection system that would allow an identification of potential interaction partners of BK polyomavirus VP1 protein. Therefore, expression plasmids producing the N and C-terminally tagged VP1 protein were prepared. These tagged proteins had the property of being biotinylated whilst being produced in the transfected cells. By using affinity chromatography, the entire protein complexes...
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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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Interactions of polyomavirus structures in the endoplasmic reticulum and on the path to the nucleus
Svobodová, Terezie ; Huerfano Meneses, Sandra (advisor) ; Weber, Jan (referee)
Mouse polyomavirus is a member and model virus of Polyomaviridae family. In order to infect cells and produce viral progeny, the viral chromosome must be transported to the nucleus. Several studies suggest that virions are transporeted to the endoplasmic reticulum, from which they are transferred to the cytosol with assistace of host proteins. Two of these proteins are the chaperon, BiP (binding immunoglobulin protein) and the cochaperone, DNAJ B14. Polyomaviruses probably enter the nucleus through nuclear pores with the assistence of importins. These processes were mainly studied with SV40. In this work, we show that MPyV infection induces a change in distribution of the DNAJ B14 protein, which became clustered into foci, where it co-localizes with the viral capsid protein, VP1. The occurrence of foci varies during infection. With use of proximity ligation assay, we have shown that during an early fase of MPyV infection, DNAJ B14 and BiP get in the close proximity with VP1. It is suggested that negatively charged amino acids at the N-terminus of the minor capsid protein, VP2, are required for targeting virions to translocon and proteins associated with ERAD. We created MPyV with VP2 mutated in these amino acids. The negatively charged amino acid at position 17 is not necessary for successful...
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