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Role of nuclear lamins and Nup358 in BK polyomavirus infection
Išler, Lukáš ; Bruštíková, Kateřina (advisor) ; Němečková, Šárka (referee)
BK polyomavirus (BKPyV) has been causing serious health complication for several decades, especially in immunocompromised patients. The aim of this work was to investigate the progress of BKPyV replication during infection of human cells, as well as the influence of BKPyV infection on the nuclear lamina and nuclear pore proteins (NPC; Nuclear Pore Complex). During characterization and comparison of BKPyV infection in RPTEC/hTERT1 and MRC-5 cells we showed that BKPyV replicates better in RPTEC/hTERT1 cells as the productive infection results in six times higher viral titer. Using confocal microscopy we did not observe any nuclear lamina disruption nor VP1 accumulation under nuclear lamina that was previuosly observed in mouse polyomavirus infection. We verified previous observations of cytoplasmic deposits of NPC colocalizing with VP1 protein 24 hours post infection (hpi) with BKPyV and we showed that Nup358, protein of NPC, is a component of NPC deposits colocalizing with VP1. Neither transient expression from vectors encoding late region of BKPyV genome (pEF- BKV-late) or VP1 alone (pIaW), nor LT antigen expression analysis did not suggest any conection of observed phenomena to the productive infection. However, pseudoinfection of RPTEC/hTERT1 cells with VLPs derived from BKPyV induced VP1...
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Major capsid protein of polyomaviruses and its interactions with nuclear lamins
Žáčková, Sandra ; Horníková, Lenka (advisor) ; Šroller, Vojtěch (referee)
In this study, we focused on interactions of structural proteins of mouse polyomavirus (MPyV) and BK virus (BKV) with the nuclear lamina. Our goal was to examine whether and how can the virus, hence viral structural proteins, interact with the nuclear lamina and how would these interactions affect its properties. We supposed, that the expression of viral proteins would induce disintegration of the structure of nuclear lamina, thus enabling nuclear egress of virions in the late phase of infection. Viral structural proteins were expressed transiently in cells transfected with an expression vector pMPyV LATE. In these cells, VP1 was localized in a likewise manner as it shows in infected cells - mostly in a perinuclear area. Concurrently, defects in staining of nuclear lamina were observed in these cells, similarly to infected cells. Also, another expression vector was used in our experiments, the pMPyV mut3 VP1 encoding for a mutated protein VP1. When transiently expressed in cells, the mutated VP1 protein showed mostly diffuse nuclear localization. However, we observed significant morphological deformations and defective staining of the nuclear lamina. These observations imply an important role of VP1 in mechanical and biochemical properties alterations of the nuclear lamina in transfected and...
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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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The role of proteins acetylation in life cycle of Polyomaviruses
Dostalík, Pavel ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Capsid of mouse polyomavirus (MPyV) is composed from three structural proteins: major structural protein VP1 and minor structural proteins VP2 and VP3. Posttranslational modifications may affect functions of proteins. This work deals with acetylation of MPyV structural proteins and its impact on the viral replication cycle. First part of the thesis is focused on acetylation of VP1. We showed that the VP1 protein is acetylated in viral particles and that interaction of VP1 with minor proteins supports VP1 acetylation. Further, we showed that cytoplasmatic deacetylase, histone deacetylase 6 (HDAC6), is important for virus infectivity. Overexpression of HDAC6 decreased MPyV infectivity, also decreased infectivity was exhibited by virus isolated from HDAC6 knock out cells. In addition, VP1 protein of virus from HDAC6 knock out cells was more acetylated in comparison with virus from parental cell line. These data suggest that VP1 is substrate for HDAC6. Second part of the thesis is focused on the characterization of N-terminal acetylation of VP3 minor structural protein. It has been previously shown that VP3 protein is N-terminally acetylated and MyPV with mutated (unacetylated) form of VP3 protein is non-infectious. The main aim of this part is to prove the hypothesis that N-terminal acetylation is...
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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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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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Analysis of BK virus life cycle
Bakardjieva - Mihaylova, Violeta ; Drda Morávková, Alena (advisor) ; Mindlová, Martina (referee)
Polyomaviruses are small unenvelope DNA viruses, whose replication take place in cell nucleus. Despite its small genome size, these viruses can cause significant changes in the host cell, one of the most significant is cell transformation. Most studies of human pathogens from this family is the focus of clinical research, but do not provide enough information about the individual events of the life cycle of viruses. This thesis mainly aims to determining the exact time when the creation of the individual viral products and generate a timeline of events during natural infection in cells that are targets for BKV in the human body. It was found that the time course of the life cycle of BKV is very similar to those for model polyomaviruses MPyV and SV40 and in permissive cells takes about 40 - 50 hours.
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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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