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Raccoon polyomavirus: example or exception of polyomavirus driven oncogenesis?
Schreiberová, Lucie ; Španielová, Hana (advisor) ; Hirsch, Ivan (referee)
Polyomaviruses (PyV) are widespread through human and animal populations and typically associated with asymptomatic persistent infection. Rarely, natural PyV infections can lead to oncogenic transformation. Virus genome is usually integrated into the host DNA of tumour tissue. Over the past few years, an increased number of very aggressive brain tumours and olfactory tumours have been observed in raccoons. These tumours are associated with the newly discovered raccoon polyomavirus, which was found as an intact episome in host cells. This bachelor thesis is therefore focused on comparison of current state of knowledge on raccoon polyomavirus with previously described mechanisms of PyV tumorogenesis. Unlike for other PyVs, the fact that primary neuronal stem cell infection is most likely to occur can play a key role in raccoon polyomavirus driven oncogenesis. Tumours also exhibit unusually high expression of virus-encoded micro RNA that can be connected with tumour induction. Similary to other tumours caused by PyV, a large amount of early viral proteins with oncogenic potential is found in tumours. Revealing unknown factors responsible for the development of tumours caused by raccoon polyomavirus may help in understanding of mechanisms of oncogenesis. Key words Polyomavirus, oncogenesis, raccoon...
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Histone modifications and methylation of polyomaviral genomes during the infection
Mrkáček, Michal ; Forstová, Jitka (advisor) ; Šmahelová, Jana (referee)
Similarly to other viruses, polyomaviruses require for their successful replication enzymes and other proteins encoded by their host cells. Additionally, because of their relatively small genome with only a few genes, polyomaviruses utilize for their efficient replication cellular regulation mechanisms. One of these regulations are posttranslational modifications of histones, which form nucleosomes together with viral DNA. The spectrum of these modifications is very wide, but in case of polyomaviruses, almost only ones studied are histone acetylations and methylations. Second possible regulation is a methylation of cytosine in CpG dinucleotides, which is associated with repression of gene expression. Current knowledge however suggest that polyomaviruses do not utilise this kind of modification. Moreover, because of a relatively small amount of CpG dinucleotides present in their genomes, they seem to avoid it. The goal of this work is to describe the individual types of these modifications and show their possible importance in the infectious cycle of polyomaviruses. Key words: polyomavirus, epigenetics, histone modification, DNA methylation, CpG dinucleotides
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Minor Structural Proteins of Polyomaviruses: Attributes and Interactions with Cellular Structures
Vinšová, Barbora ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Even though polyomaviruses have been intensively studied for more than 60 years, the role of minor structural proteins VP2 and VP3 in some important steps of viral life cycle has still not been fully elucidated, explicitly their role in viral genome delivery to the cell nucleus and their involvement in late phases of viral life cycle. This diploma thesis focuses on the study of minor proteins of Mouse polyomavirus (MPyV) and Human polyomavirus BK (BKV). Four rabbit polyclonal antibodies against minor proteins of polyomaviruses MPyV or BKV have been prepared within this diploma thesis. Two of these prepared antibodies target minor proteins of MPyV (α-MPyV VP2/3) or BKV virus (α-BKV VP2/3), other two prepared antibodies recognize C-terminal sequence common to minor proteins VP2 and VP3 of MPyV (α-MPyV C-termVP2/3) or BKV virus (α-BKV C-termVP2/3). In the second part of this diploma thesis we aimed to study toxicity of BKV virus minor proteins during individual production in mammalian cells. Obtained results suggest that minor proteins of BKV virus might not exhibit as high levels of cytotoxicity as minor proteins of MPyV virus. Third part of this diploma thesis is devoted to investigation of interactions of BKV and MPyV minor proteins with cellular proteins and within one another respectively....
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Vesicular trafficking from acidic compartments to the endoplasmic reticulum
Polidarová, Markéta ; Forstová, Jitka (advisor) ; Plocek, Vítězslav (referee)
The cell uses retrograde transport from endosomes to Golgi apparatus and further to the endoplasmic reticulum to recycle its receptors and other proteins. There are several pathways starting on different types of endosomes aimed to the trans-Golgi network and from it further to the endoplasmic reticulum. From the early and maturing endosomes the proteins are transported using the retromer complex. Rab9 GTPase is essential for transport from the late endosomes. Rab6 and Rab11 play major role in the transport form the recycling endosomes. There are two pathways going through the Golgi apparatus. The first one is mediated by COPI vesicles which are regulated by Arf1 GTPase and the pathway is sensitive to brefeldin A. The second pathway is regulated by Rab6 GTPase. Except for endogenous proteins the retrograde transport is used by protein toxins and small unenveloped DNA viruses as well. Rab6 pathway from the recycling endosomes and through the Golgi apparatus is characteristic for Shiga toxin. The retrograde transport of ricin starts on the early endosomes and is less clear. Scientists only started uncovering the transport of small unenveloped DNA viruses.
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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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Experimental system for the mouse polyomavirus life cycle study
Pergner, Jiří ; Mašek, Tomáš (referee) ; Španielová, Hana (advisor)
Experimental system for the mouse polyomavirus life cycle study Abstract: Murine polyomavirus (MPyV) is the prototype of the Polyomaviridae family. This family includes also some important human pathogens (BKV, JCV, Merkel cell polyomavirus). Due to their specific properties viruses within this family may serve as versatile vectors for gene therapy or recombinant vaccine production. New methodological approaches may help to understand some yet unknown facts about MPyV life cycle. Clarification of some processes during murine polyomavirus life cycle may be also important to fully exploit polyomaviruses for therapeutic purposes. The aim of this diploma thesis was to preparare two innovative experimental systems that extend possibilities of studying the life cycle of MPyV. The first part of the diploma thesis focusses on construction of recombinant MPyV which expresses yellow fluorescent protein (EYFP) in the early stages of infection. Such virus can be very useful for studying the infection spreading by live- cell imaging and Fluorescence-Activated Cell Sorting (FACS) and can be employed for co- localization studies of YFP-tagged LT antigen with certain cellular proteins. Second part of the diploma thesis describes preparation of a hybrid cell line prepared by fusion of mouse and monkey cells. This new cell...
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