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Interaction of retroviral env glycoproteins with receptors and virus entry into the cell
Přikryl, David ; Pečenka, Vladimír (advisor) ; Horníková, Lenka (referee)
Humankind has adjusted many animals, plants and bacteria to serve their needs. Now the time has come to exploit also viruses. The most significant applications for them seem to lie in the sphere of human health. Particularly the family of retroviruses represent a perfekt vectors for gene delivery to cure genetic diseases or to destroy undesirable, e.g. cancer cells. In order to efficiently use these vehicles, a series of problems must be overcome and some segments of retroviral life cycle must be more deeply explored. This work describes some of them along with possible ways to solve them. The first described area is nonspecific adsorption of virions on the cell surface, the second is securing effective interaction between envelope glycoproteins and receptors highly specific for the targeted cell type and finally driven fusion of membranes. Key words: retrovirus, adsorption, env, receptor, phusion
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Epigenetic regulation of HLA class II genes and their role in autoimmune diseases.
Čepek, Pavel ; Kotrbová - Kozak, Anna Katarzyna (advisor) ; Horníková, Lenka (referee)
Abstract Background: Type 1 diabetes (T1D) is a multifactorial autoimmune disease. Its incidence in Europe is continuously rising. The highest T1D risk is associated with HLA (human leukocyte antigen) class II genes. HLA class II molecules play a key role in regulation of immune response. They contribute to the selection of T cell repertoire by presenting antigenic peptides to the CD4+ T lymphocytes. HLA class II expression is controlled by regulatory module that is situated 150 - 300 base pairs upstream of the transcription- initiation site in all HLA class II genes. Polymorphisms in this region are linked to some autoimmune diseases. There were identified several promoter alleles (named QAP) in the HLA DQA1 gene promoter region. Most of the polymorphisms appear to be conserved within haplotype. Individual QAP alleles may have a different promoter strength by which they influence expression of HLA DQA1 gene alleles. Promoter strength can be modulated by DNA methylation. Aims:Our aim was to define methylation profile of HLA DQA1 promoters and determine the mRNA expression of individual alleles of HLA DQA1 gene in T1D patients. The mRNA expression level of HLA DQA1 gene alleles was determined using quantitative PCR. Methods: 30 diabetic pacients (age range 21 to 76 years), were included in this pilot...
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Impact of plant alkaloids on viral infection
Šnejdarová, Aneta ; Horníková, Lenka (advisor) ; Váňová, Jana (referee)
Plant products have been used to treat various diseases since ancient times thanks to the many active substances they contain. One such group of substances are alkaloids. Alkaloids are biologically active substances which, in addition to antimicrobial, anti-inflammatory, antioxidant and many other properties, also possess antiviral properties. As a result, they can help treat viral infections, which are still a major medical problem today. Alkaloids affect all steps of virus replication, both viral components but especially cellular processes, without which a successful progress of viral cycle is not possible. In connection with the treatment of viral infections, the tropane, troponol, purine and isoquinoline alkaloids have been best investigated, which are also the subject of this work. The tropane alkaloid atropine acts mainly on enveloped viruses due to its ability to change the properties of biological membranes. Purine alkaloid caffeine, thanks to its ability to inhibit the cellular enzyme phosphodiestrase, causes an increase in intracellular cAMP levels and it has an impact on viral replication. Its antioxidant and immunomodulatory properties are also beneficial for the treatment. The anti-inflammatory effects of the tropane alkaloid colchicine stem from its ability to inhibit the dynamics of...
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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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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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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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Helicase DHX9 and its role in viral life cycles
Kertisová, Anna ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Helicases are proteins that provide strands-unwinding of polystranded conformations of nucleic acids. DHX9 is a representative of helicases with a DExD/H-box motif. It accepts molecules of RNA, DNA and hybrid strucutures as a substrate. Inside the nucleus of the eukaryotic cell, DHX9 participates in the formation of the replication fork or it acts in a complex of trancriptional factors and it connects other proteins with RNA polymerase II. Subsequently DHX9 regulates post-transcritional RNA processing. After a shuttle to the cytoplasm it assists in a correct course of translation of structurated mRNAs. Since DHX9 is involved in nucleic acids-related processes, its role in viral life cycles is investigated. Also it is a part of antiviral signaling pathways of the cell. DHX9 provides an advantage for RNA viruses and retroviruses in their replicative cycle in various manners. The binding of DHX9 on secondary structures of viral RNAs and their eventually remodelation, which causes promotion of viral replication, transcription and translation, is significant. In contrast to that, DNA viruses use DHX9 to regulate host protein expression and minimize its antiviral fuction. Understanding of relationship could lead to more effective aiming of the therapeutic drugs against viral infections. Key words:...
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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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Major structural protein of Polyomaviruses: Interactions with host cell structures
Mrkáček, Michal ; Horníková, Lenka (advisor) ; Němečková, Šárka (referee)
The main structural protein VP1 is the product of late polyomaviral genes and it is the largest and the most abundant protein of the whole polyomaviral capsid. Because of the low coding capacity of the polyomaviral genomes, it is considered that in addition to its structural role the VP1 protein might have some additional functions in the late phase of the infectious cycle. This diploma thesis is exactly on these additional functions. In the case of the VP1 protein of mouse polyomavirus, it was observed that the protein is capable of binding to the structure of cellular microtubules. The first objective of this work was to test whether pentamers of the VP1 protein are able of this binding without the participation of other cellular (or viral) proteins. Based on an in vitro experiment, we showed that protein VP1 binds to the structure of microtubules very inefficiently. The second objective of this work was to prepare a detection system that would allow an identification of potential interaction partners of BK polyomavirus VP1 protein. Therefore, expression plasmids producing the N and C-terminally tagged VP1 protein were prepared. These tagged proteins had the property of being biotinylated whilst being produced in the transfected cells. By using affinity chromatography, the entire protein complexes...
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