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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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Negative regulation of the IFI16 and cGAS DNA sensing pathways
Muhič, Samra ; Huerfano Meneses, Sandra (advisor) ; Kanwal, Madiha (referee)
DNA sensors are molecules with the ability to sense DNA constituting an important tool of innate immunity. They are initiators of various signalling pathways, one of them being the production of interferons, which induce not only an anti-viral cell state but also protect cells of treats not related to pathogens. At least, fourteen DNA sensors have been described so far, among them - IFI16 and cGAS. Both sensors signal via an adaptor protein STING resulting in the production of type I IFN. All three of these molecules (cGAS, IFI16, STING) are strictly regulated either by host-cells in order to prevent immune over-activation or by viruses for the immune evasion. This work focuses on the mechanisms of negative regulation of the three molecules: post-translational modifications such as phosphorylation, ubiquitination, SUMOylation, acetylation and methylation; protein-protein interactions; degradation by the proteasomal system or by autophagy. Not surprisingly, viruses encode proteins able to down-regulate IFN responses for example, some proteins of herpes viruses interact with cGAS, IFI16 or STING preventing their activation or leading to their degradation. Other proteins of herpes viruses cause the degradation of the mRNA of the sensors or the adaptor. Dengue protease factor NS2B degrades cGAS or the...
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Interplay between small DNA viruses and the PML Nuclear Bodies
Lovtsov, Alexey ; Huerfano Meneses, Sandra (advisor) ; Kadlečková, Dominika (referee)
This work focuses on interactions between small DNA viruses and PML NBs. PML NBs are membrane- free nuclear bodies that contain, permanently or transiently, more than 170 proteins. The permanent ones include, e.g., the PML, Daxx, and Sp100 proteins. Many PML NB proteins are subjected to SUMOylation. In addition to functions in regulating apoptosis and gene expression, chromatin remodelling, and DNA damage responses, they are also involved in antiviral cell defence. On the other hand, viruses have developed strategies to thwart the restrictive effects of PML NBs, or even to misuse some of their components to support the infection. Apparently, PML NBs play a significant role in some viral infections, but models of such processes are still incomplete and sometimes controversial. Some adenovirus proteins interact with PML NB components to prevent the negative effects of PML NBs on transcription and replication. In contrast, papillomavirus genomes can be protected by PML NBs from degradation upon entry into the nucleus and during transcription. For polyomaviruses, both promoting and restriction functions have been published. In Chicken anaemia virus infection, the interaction of the viral apoptin and PML led to the induction of cell apoptosis, but the results were questioned. In patients with chronic...
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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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The role of the Smc5/6 complex in DNA viral infection
Protivová, Eliška ; Huerfano Meneses, Sandra (advisor) ; Pokorná, Karolína (referee)
The Smc5/6 complex is an eukaryotic protein complex that, together with Smc1/3 cohesin and Smc2/4 condensin, is involved in ensuring genome stability. It contributes to this by participating in the organization and maintenance of chromosomal structures as well as in the response to DNA damage. In addition, the Smc5/6 complex has been shown to play an important role in viral infection. This thesis focuses on the mechanisms of interaction of the Smc5/6 complex with viral DNA genomes, DNA intermediate genomes and viral proteins. In the case of HBV of the Hepadnaviridae family, Smc5/6 acts as a restriction factor. The same is true for HSV-1 of the Herpesviridae family, viruses of the Papillomaviridae family and HIV-1 of the Retroviridae family. An interaction of the Smc5/6 complex with the JC virus of the Polyomaviridae family has also been discovered. Nevertheless, the meaning of this interaction remains elusive. Some of the above-mentioned viruses can prevent this restriction. In detail, HBx protein of HBV mediates proteasomal degradation of the Smc5/6 complex or Vpr protein of HIV-1 induces degradation of the SLF2 protein, which is responsible for the Smc5/6 localization on HIV-1 DNA intermediate genomes. Keywords: Smc5/6 complex, DNA repair, ATPase, sumoylation, DNA viruses, viruses with a DNA...
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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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Studies of the Mouse polyomavirus: properties of the minor structural proteins, requirements for virion productive trafficking to the nucleus and observed side effects of DNA transfection.
Huérfano- Meneses, Sandra ; Forstová, Jitka (advisor) ; Mělková, Zora (referee) ; Reiniš, Milan (referee)
In this thesis, we aimed to investigate the role of Mouse polyomavirus (MPyV) minor structural proteins, VP2 and VP3 in the viral life cycle as well as to study specific aspects of the productive trafficking of the virus. The thesis is divided into 3 chapters as follows: chapter 1 addresses questions about the interaction of the minor structural proteins, VP2 and VP3, of MPyV with host cells and their role during late stages of virus infection. In the chapter 2, aspects concerning the role of late endosomes, recycling endosomes and actin cytoskeleton in MPyV productive infection are addressed and in the chapter 3, results of studies of interferon (IFN) responses induced after DNA nucleofection are presented. The study in chapter 3 was preceded by a microarray analysis primary performed to reveal cell responses to the presence of the minor proteins within host cells. We showed that both MPyV minor structural proteins, VP2 and VP3, when produced individually (without VP1) are highly cytotoxic, inducing fast, caspase dependent apoptosis. Immuno-electron and confocal microscopy revealed affinity of both proteins to endocellular membranes. Both VP2 and VP3 were found to be bound to damaged ER and mitochondrial membranes. Interaction of the proteins with membranes causing physical damage of organelles...
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