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Replication of Merkel cell polyomavirus (MCPyV) in human cell lines
Bučková, Alžbeta ; Saláková, Martina (advisor) ; Huerfano Meneses, Sandra (referee)
The Merkel cell polyomavirus (MCPyV) is the only human polyomavirus classified as probably carcinogenic to humans and is the causal factor of a rare aggressive skin malignancy the Merkel cell carcinoma (MCC) in around 80% of cases. Nevertheless, in addition to skin the virus was detected in various body tissues. In spite of that an ideal in vitro model system is still lacking. Three MCPyV isolates from healthy human skin are studied in this diploma thesis. The isolates harbour mutations, one has mutations is its LT coding gene, one has a rearranged non-coding control region (NCCR), and one is identical to the reference MPCyV isolate R17b. In this diploma thesis the replication capacities of the isolates were studied with the emphasis on the impact of a duplication within the NCCR and mutations on MCPyV genome replication in vitro after transfection. First, the results show that the NCCR rearrangement and the mutations in the viral protein coding genes impact the MCPyV replication in the 293 cell line. Preliminary data suggest the NCCR rearrangement negatively influences the MCPyV replication but certain mutations in protein coding genes could increase the replication. Moreover, none of the isolates replicated in the 293T cell line. The next part of the thesis focused on the replication of the MCPyV...
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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 model systems to study small DNA viral infection
Bučková, Alžbeta ; Saláková, Martina (advisor) ; Horníková, Lenka (referee)
Merkel cell polyomavirus (MCPyV) and Human papillomavirus 16 (HPV 16) are members of small tumour DNA viruses Polyomaviridae and Papillomaviridae, which represent increasing risk for humans resulting from their oncogenic potential. After the acquisition HPV 16 and MCPyV are able to persist for long term in a form of asymptomatic infection, while the aggressive disease is mostly being cleared by the host immune system. Integration of viral genome into the host DNA causes cell transformation resulting in rare but fatal skin carcinomas and epithelial lesions of anogenital tract, head and oropharynx, that may progress into malignant tumours. Their mechanisms of immune system evasion and complete life cycles are not fully understood to this day which highlights some of the reasons why continuing research in this field is of importance. The aim of this thesis is to review model systems used to study infection of MCPyV and HPV 16 in vitro and in vivo. Key words: Papillomaviruses, polyomaviruses, virus-like particles, pseudoparticles, animal models, cell culture, human papillomavirus 16, Merkel cell polyomavirus, HPV 16, MCPyV
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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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Preparation of Monoclonal Antibodies Against VP2 Protein of Human Polyomaviruses
Vochyánová, Klára ; Drda Morávková, Alena (advisor) ; Růžičková, Šárka (referee)
Aim of this diploma thesis was to prepare two protein antigens and two monoclonal antibodies, all based on VP2 minor protein of human polyomaviruses BK virus and Merkel Cell Polyomavirus. One monoclonal antibody was being prepared against unique part of VP2 protein (N-terminal epitope, not present in VP3 protein). A cell line producing such monoclonal antibody has never been established before due to low immunogenicity of the epitope. Our approach was successful in terms of mouse immunization, however, serious problems with hybridoma line stability appeared later during the preparation process. Preparation of antibody targeted to the sequence of VP2 protein of Merkel Cell Polyomavirus was another aim of this thesis. Mouse immunization and hybridoma fusion were performed successfully. After four rounds of cloning in order to purify an established clone, nine clones were cultivated in larger scale. This cultivation probably led to diminished antibody specificity and loss of production ability in most of the hybridoma cells. One more cloning should give rise to an established clone with sufficient production. Two preparations of protein antigens were performed in two expression systems. DNA encoding C-terminally truncated protein VP2 of BK virus fused with His-tag was cloned into a vector suitable for...
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Human polyomavirus isolated from Merkel cell carcinoma
Vochozková, Petra ; Šroller, Vojtěch (advisor) ; Španielová, Hana (referee)
Human polyomaviruses belong to the Polyomaviridae family. Until now, five human polyomaviruses (BK, JC, KI, WU and MCV) have been discovered. There is described the course of polyomavirus infection in the first part of the thesis. These small nonenveloped viruses penetrate into the host cell by receptor-mediated endocytosis and then travel through the ER pathway to get into the nucleus where the virus replicates and expresses viral proteins. The infection occurs during early childhood and the virus remains asymptomatic in healthy individuals. However, the virus is able to reactivate in the immunosuppressed patients and can cause some diseases. The second part of thesis is focused on the MCV. MCV genom was detected in Merkel cell carcinoma (MCC) two years ago. MCV infects the Merkel cells and its DNA integrates into host cell genome. In most cases, the MCV is detected in cancer cells using PCR. Viral sequences encoding the large and small T antigen were found in the MCC using the same method; moreover, there was expressed a significant amount of oncoproteins. These factors point out an important role of MCV in the tumor progress. The study of MCV may help to discover new approaches for the treatment of MCC and other biologically similar tumors.
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