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Analysis of Zika and Dengue virus proteases
Novotný, Pavel ; Konvalinka, Jan (advisor) ; Vondrášek, Jiří (referee)
in English Zika and Dengue flaviviruses are transmitted by mosquitoes in human populations living in tropical areas. They cause fevers which in the case of Dengue can lead to life threatening haemorrhagic form. There is a possible relationship between pregnant women being infected by Zika virus and higher risk of microcephaly in new-borns. The infection is currently treated mainly symptomatically. However, there is an effort to develop compounds which block viral life cycle and viral spread through organism. Viral enzymes, such as flaviviral proteases, are regarded as suitable targets for this effort. These serine proteases with chymotrypsin fold are heterodimers which consist of flaviviral non- structural proteins NS2B and NS3. NS3 domain also contains a helicase, which can be removed by gene recombination for study purposes. NS2B is a transmembrane protein, but only a hydrophilic 40 amino acid peptide is important for the interaction with NS3 domain. This peptide has a chaperon function and participates in substrate binding to the active site. In this study, six variants of recombinant proteins containing activating peptide of NS2B and protease domain of NS3 were expressed and purified. Four variants were characterized in enzymologic studies including testing of possible inhibitors. A dipeptide...
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Role of phosphorylation in nuclear import of viral proteins and complexes
Pokorná, Karolína ; Forstová, Jitka (advisor) ; Roučová, Kristina (referee)
Replication of many different viruses occurs in the nucleus of the host cell. These viruses discovered ways how to overcome the nuclear membrane and often use cell transport machinery to transport their proteins and genome into the nucleus. For many viral proteins the timing of their nuclear import in order to secure correct viral replication is important. Regulated nuclear import also allows these proteins to perform several functions depending on their localization. Nuclear import of viral proteins and complexes can be regulated by phosphorylation. Phosphorylation can, for example, modulate affinity of proteins for importins or other cellular proteins. Phosphorylation can also cause conformational change, which can lead to unmasking of localization sequence.
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The role of histone deacetylase 6 in murine polyomavirus replication cycle
Vlachová, Štěpánka ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
The replication cycle of polyomaviruses is, consistently with other viruses, fully dependent on host cells. Not only the cellular replicational and translational mechanisms are important for viruses, but also the virus infection is affected by other cellular proteins. This work is focused on the role of major cytoplasmic deacetylase, histone deacetylase 6 (HDAC6) in replication cycle of murine polyomavirus (MPyV). We showed that the presence of fully functional HDAC6 is essential for successful and productive infection. We found that HDAC6 affects not only early phase, but also late phase of infection. Cells with inhibited, or absent HDAC6 are infected with decreased effectivity and moreover lower amount of infectious viral particles is produced. On the other side, using cells with partially functional HDAC6, either in its deacetylase activity or in ubiquitin-binding activity, leads to increased ability of MPyV to infect those cells. Analysis of levels of early LT antigen and late structural protein VP1 in the infected cells showed, that viral proteins are affected by HDAC6. Our data suggest, that in the replication cycle of MPyV mainly the ubiquitin-binding domain of HDAC6 is required and the role of this domain in protein metabolism and degradation. In the second part of diploma project, we...
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Analysis of Zika and Dengue virus proteases
Novotný, Pavel ; Konvalinka, Jan (advisor) ; Vondrášek, Jiří (referee)
in English Zika and Dengue flaviviruses are transmitted by mosquitoes in human populations living in tropical areas. They cause fevers which in the case of Dengue can lead to life threatening haemorrhagic form. There is a possible relationship between pregnant women being infected by Zika virus and higher risk of microcephaly in new-borns. The infection is currently treated mainly symptomatically. However, there is an effort to develop compounds which block viral life cycle and viral spread through organism. Viral enzymes, such as flaviviral proteases, are regarded as suitable targets for this effort. These serine proteases with chymotrypsin fold are heterodimers which consist of flaviviral non- structural proteins NS2B and NS3. NS3 domain also contains a helicase, which can be removed by gene recombination for study purposes. NS2B is a transmembrane protein, but only a hydrophilic 40 amino acid peptide is important for the interaction with NS3 domain. This peptide has a chaperon function and participates in substrate binding to the active site. In this study, six variants of recombinant proteins containing activating peptide of NS2B and protease domain of NS3 were expressed and purified. Four variants were characterized in enzymologic studies including testing of possible inhibitors. A dipeptide...
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Avian polyomaviruses
Škvára, Petr ; Fraiberk, Martin (advisor) ; Forman, Martin (referee)
Avian polyomaviruses are small, nonenveloped viruses from family Polyomaviridae, which can cause acute infection with high mortality of infected birds. In contrast with other polyomaviruses, avian polyomaviruses do not share similar pathogenesis, because most of them do not cause acute diseases. The main point of this work is to summarize available information about avian polyomaviruses and to compare them to each other. This work also compares avian polyomaviruses with mammalian polyomaviruses. The primary focus is made on genome organization, structure and function of viral proteins, but also on pathogenesis and epidemiology of avian polyomaviruses. In conclusion, even if avian polyomaviruses share common signs with other polyomaviruses, they have different conserved sequences, host specificity and pathogenicity. Summary of current knowledge about avian polyomaviruses could help to understand the importance of this newly established genus in the field of polyomavirus research. Key words: avian polyomaviruses, taxonomy, genome, viral proteins, pathogenesis
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Role of phosphorylation in nuclear import of viral proteins and complexes
Pokorná, Karolína ; Forstová, Jitka (advisor) ; Roučová, Kristina (referee)
Replication of many different viruses occurs in the nucleus of the host cell. These viruses discovered ways how to overcome the nuclear membrane and often use cell transport machinery to transport their proteins and genome into the nucleus. For many viral proteins the timing of their nuclear import in order to secure correct viral replication is important. Regulated nuclear import also allows these proteins to perform several functions depending on their localization. Nuclear import of viral proteins and complexes can be regulated by phosphorylation. Phosphorylation can, for example, modulate affinity of proteins for importins or other cellular proteins. Phosphorylation can also cause conformational change, which can lead to unmasking of localization sequence.
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