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Mouse polyomavirus: The role of cell cytoskeleton in virus endosomal trafficking and properties of the minor capsid proteins
Žíla, Vojtěch ; Forstová, Jitka (advisor) ; Hozák, Pavel (referee) ; Rumlová, Michaela (referee)
Mouse polyomavirus (MPyV) is a non-enveloped DNA tumor virus, which replicates in the host cell nucleus. MPyV enters cells by receptor-mediated endocytosis and its subsequent transport towards the nucleus requires acidic environment of endosomes and intact microtubules, which are important for virus delivery to endoplasmic reticulum (ER). In ER, capsid disassembly and uncoating of viral genome take place. The mechanism of subsequent translocation of viral genome from ER into nucleoplasm is still only poorly understood process with predicted involvement of cellular factors and viral minor capsid proteins VP2 and VP3. Once the genome appears in the nucleus, early viral antigens are produced and mediate suitable environment for replication of viral genomes. After replication of viral DNA and morphogenesis of virions, virus progeny is released from the cells during its lysis. The research presented in the first part of thesis focused on intracellular transport of MPyV and involvement of cytoskeletal networks during virus delivery to the ER. In particular, we investigated still unclear role of microtubules during virus trafficking in endosomes, and involvement of microtubular motors. We found that MPyV trafficking leading to productive infection does not require the function of kinesin-1 and kinesin-2,...
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Chemically modified Murine Polyomavirus-like particles and their interaction with Prostate-Specific Membrane Antigen (PSMA)
Blažková, Kristýna ; Konvalinka, Jan (advisor) ; Horníková, Lenka (referee)
Prostate cancer is one of the most abundant types of cancer among men and the demand for a specific treatment is very high. In this thesis, I have focused on using Glutamate Carboxypepti- dase II (GCPII), as a target for a proof-of-principle delivery system. GCPII is a transmembrane protein that internalizes after a binding of a ligand and is overexpressed in prostate cancer. Virus-like particles from Murine polyomavirus (VLPs) are a suitable nanocarrier for the delivery of imaging agents and drugs. Here I describe modifying these VLPs with inhibitors of GCPII and fluorescent dyes and characterize their binding to GCPII on surface plasmon resonance and to cells expressing GCPII on confocal microscopy. VLPs carrying a GCPII inhibitor show specific binding to GCPII on surface plasmon reso- nance, however they bind non-specifically to cells that don't express GCPII. Several approaches have been tried to avoid that. The substitution of BC loop on the exterior surface of VLPs that is partially responsible for the binding of sialic acid did not seem to affect specificity on cells. Another approach tested was coating of the wild-type VLPs with large polymer carrying a flu- orescent label and a GCPII inhibitor. After the conjugation of the polymer to the VLP, specific binding and internalization in GCPII-positive...
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Studies of properties of the minor structural proteins of the Murine polyomavirus
Bílková, Eva ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Murine polyomavirus (MPyV) is a member of the Polyomaviridae family. Its capsid is composed of the major capsid protein, VP1, and the minor proteins, VP2 and VP3. The minor capsid proteins probably assure delivery of the viral genome through the endoplasmic reticulum membrane to the nucleus during early phase of infection. However, precise mechanism is not known. Expression plasmids encoding mutated VP2 or VP3 fused with EGFP have been constructed to study the interaction of VP2 and VP3 with membranes. The mutated proteins have deletions in the predicted hydrophobic domains. In this thesis, cell localisation of mutated proteins was followed. The study revealed that the hydrophobic domain 2 is the most important for association of VP2 and VP3 with membranes, while domains 1 and 3 are rather expendable. Further, nature of VP2 and VP3 isoforms has been studied. Isoforms with different electrophoretic mobility were separated on SDS-PAGE. Consequent mass spectrometry analysis showed that they differ in deamidation of asparagine, present at both minor proteins (position 253 of VP2 and 137 of VP3). Previously, acetylation of VP3 N-terminal alanine has been identified. To elucidate the function of these modifications, mutated viruses were constructed with substitution of these amino acids. Pilot...
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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 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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Development of the experimental system based on Cre/loxP recombination for polyomavirus mutant production.
Hron, Tomáš ; Španielová, Hana (advisor) ; Šroller, Vojtěch (referee)
Murine polyomavirus is an important member of Polyomaviridae family offering potential applications in gene therapy and immunotherapy. Viral mutant analysis is crucial for study of the virus, however, commonly used methods of its production are laborious and give low yields. This thesis involves development of the new experimental system that can produce intact viral genome from recombinant plasmid in vivo using Cre/loxP-mediated recombination. One loxP site is unavoidably introduced into newly generated viral genome during recombination. Two variants of production plasmids generating wild type viral genome with incorporation of loxP between the poly(A) signal sites of early and late genes or into the intronic region of early genes were prepared. LoxP insertion between the poly(A) signal sites has a dramatic effect on viral gene expression and leads to complete loss of virus infectivity. Conversely, the infectious virus was obtained from the viral genome containing loxP site in the early intronic region. To ensure expression of Cre recombinase I also prepared stably transfected cell lines which can simplify the virus production. This thesis shows that newly designed system gives satisfactory yield of the virus, solves restrictions connected with commonly used methods and can be used for low infectious viral...
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Construction of mouse polyomavirus chimeric VLP bearing melanoma epitopes
Kojzarová, Martina ; Drda Morávková, Alena (advisor) ; Tachezy, Ruth (referee)
Major capside protein of Polyomaviridae family viruses is able to selfassemble into virus-like particle (VLP) even without the presence of minor proteins, bind exogenous DNA non-specifically and recognise the receptor on the cellular surface. These characteristics determine its use as vector in gene therapy or immunotherapy. It was discovered before that MPyV VLPs significantly stimulate immune system and have strong adjuvant effect. Chimeric VLP derived from mouse polyomavirus carrying exogenous antigene or epitop is supposed to elicit specifically targeted immune response after immunisation. The main obstacle is choice of immunogene that is strong enough to cause adequate immune response. The goal of this thesis was to construct chimeric particles carrying epitop of malignant melanoma, one of the most immunogenic tumours, on their surface, using methods of genetic engineering. For future research of particle's immunogenic properties three types of particles were developed - particles with human and mouse melanoma epitopes, respectively and control particles with ovalbumine epitop. For the purpose of production of chimeric protein was used baculovirus expression system. It was verified then, with the use of electron microscopy, that introduction of tumour antigen into one of surface loops of VP1...
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Major capsid protein of mouse polyomavirus: interaction with cellular structures
Horníková, Lenka ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee) ; Mělková, Zora (referee)
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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Targetting prostate tumor cells by polyomavirus virus-like particles
Suchanová, Jiřina ; Španielová, Hana (advisor) ; Němečková, Šárka (referee)
The aim of this thesis is to investigate the targeting potential of mouse polyomavirus (MPyV) based virus-like particles (VLPs) as vectors for directed cell delivery of therapeutic or diagnostic compounds. Major capsid protein VP1 of MPyV is able to selfassemble into the noninfectious VLPs. Our main goal is to retarget these VLPs from its native receptor to the prostatic cancer cells by changing the receptor binding site in the surface-exposed loop of VP1. We introduced a peptide ligand CTITSKRTC, which binds prostate-specific membrane antigen (PSMA), by insertion or substitution into BC loop of VP1. These modifications did not change the stability of the particles and genetic substitution prevented the native receptor binding. PSMA-specific binding of modified VLPs was tested by pull-down assay and surface plasmon resonance. In order to further utilize these VLPs, we tested several approaches for preparation of VLPs as vehicles for compounds delivery into eukaryotic cells. Although the method for encapsidation of the DNA into the VLPs in cellular nuclear extracts, which mimic the in vivo conditions, did not enabled us to produce pseudocapsids, we successfully optimized procedure for dissassembly and reassembly of purified particles. This method will be use for encapsidation of molecules into the...
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Studies of minor capsid proteins of the mouse polyomavirus
Vít, Ondřej ; Němečková, Šárka (referee) ; Forstová, Jitka (advisor)
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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