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Methylation of viral RNA
Šimonová, Anna ; Macíčková Cahová, Hana (advisor) ; Sýkora, David (referee) ; Elleder, Daniel (referee)
Viruses are the major force that shapes the evolution of both pro- and eukaryotic organisms. They have a simple inner organization and contain only a few, usually well-described RNAs. In the case of +(ss)RNA viruses, their genomic RNA serves also as mRNA. This makes them a perfect model system for searching for new mRNA modifications as well as for understanding the role of already known modifications. In this work, Human Immunodeficiency Virus type 1 (HIV-1) from the Retroviridae family was used as a model system. In the following study, four representatives from the Picornaviridae family were tested for RNA methylation profile. To get the information, a combination of two techniques was developed, liquid chromatography- mass spectrometry (LC-MS) and sequencing techniques. Results of LC-MS reveal a surprisingly high amount of 1-methyladenosine (m1 A) in RNA isolated from HIV-1. Nevertheless, the m1 A mapping sequencing technique confirm m1 A position only in co-packed tRNA. This led to the recalculation of HIV-1 virion RNA composition. In the case of Picornaviridae, LC-MS revealed m1 A and 5-methylcytidine (m5 C) in two insect viruses (Sacbrood virus, SBV and Deformed wing virus, DWV). RNA seq techniques (m1 A mapping and bisulfite sequencing) confirmed the presence of m1 A and m5 C only in tRNA....
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Host-virus interactions of mammalian endogenous retroviruses
Farkašová, Helena ; Elleder, Daniel (advisor) ; Hirsch, Ivan (referee) ; Mělková, Zora (referee)
Endogenous retroviruses (ERVs) originate by germline infection and subsequent mendelian inheritance of their exogenous counterparts. With notable exceptions, all mammalian ERVs are evolutionarily old and fixed in the population of its host species. Some groups of retroviruses were believed not to be able to form endogenous copies. We discovered an additional endogenous Lentivirus and a first endogenous Deltaretrovirus. Both of these groups were previously considered unable to form endogenous copies. Endogenous lentiviruses were discovered only recently and are still quite rare. These are still just small pieces of evidence insufficient to give a broader picture about the history of virus endogenization. We described a novel endogenous Lentivirus in the genome of Malayan colugo (Galeopterus variegatus) denoted ELVgv (endogenous Lentivirus of G. variegatus). Based on several analyses we proved that this is the oldest Lentivirus discovered up to date and confirmed its presence in the only other extant species of Dermoptera - Cynocephalus volans. Endogenous deltaretroviruses were the last group without a single endogenous member. We detected the remnants of endogenous Deltaretrovirus in the genome of Natal Long-fingered bat (Miniopterus natalensis). However, this sequence was present in the genome only in one...
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Study of endogenous retroviruses: Insight into the retroviral evolution and virus-host interactions
Hron, Tomáš ; Elleder, Daniel (advisor) ; Kejnovský, Eduard (referee) ; Hirsch, Ivan (referee)
In my doctoral project, I studied the evolution of retroviruses and long-term interactions with their hosts. Retroviruses infect a broad range of species including possibly all vertebrates. They are unique in their ability to efficiently create endogenous retroviruses (ERVs) - viral copies integrated into the host genomes and consequently inherited by successive generations as usual genomic locus. ERVs represent a significant portion of vertebrate genomes and play an important role in a variety of cellular processes and pathologies; however, their sequences are still largely unexplored. The results of my work contributed to the uncovering of ancient evolutionary history of retroviruses. In this regard, I employed the ERV sequences, as they represent "genetic fossils" of viral infections that occurred throughout entire retroviral evolution. By discovery and analysis of ancient ERV lineages, I shed light on the deep history of retroviruses and revealed how the past infections shaped the evolution of vertebrate antiviral defense. In addition to the investigation of retroviral evolution, I also studied process of ongoing endogenization and fixation of newly emerged ERVs in a mammalian host population. In this part of my work, I focused on a unique model of ERV that have been recently invading mule deer genome.
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Adaptive evolution of Toll-like receptors in birds
Velová, Hana ; Vinkler, Michal (advisor) ; Elleder, Daniel (referee) ; Novák, Karel (referee)
Adaptive evolution of Toll-like receptors in birds Hana Velová, PhD thesis 6 Abstract Toll-like receptors (TLRs) are one of the key and presumably also evolutionary most original components of animal immune system. As Pattern recognition receptors they form the first line of innate immune defence against various pathogens. The proper receptor binding of pathogenic ligands is crucial for their correct recognition and for subsequent triggering of an appropriate immune response. Because there exists a direct interaction between the receptor surface and the pathogenic ligand, host-pathogen coevolution on molecular level can be predicted. Thus, through variability of their ligands, TLRs are exposed to extensive selective pressures that may be detected on both genetic and protein levels. Surprisingly, the variability we revealed in birds is even higher than previously expected based on the reports from other vertebrates, mainly mammals. In my doctoral thesis I summarise the results of my contribution to the avian TLR research. We were the first who experimentally verify the absence of functional TLR5 in several avian species and duplication of TLR7 in others. We finally resolved the origin of duplication in TLR1 and in TLR2 family. An important part of my research project focused on the prediction of potentially...
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Regulation of transcription by proteins of the Early growth response and Myb families
Čermák, Vladimír ; Dvořák, Michal (advisor) ; Vomastek, Tomáš (referee) ; Elleder, Daniel (referee)
The regulation of transcription of tens of thousands of genes in a vertebrate organism is an enormously complex phenomenon which entails the participation of thousands of various regulatory proteins. The largest functional category of these regulators is accounted for by sequence-specific DNA-binding proteins known as transcription factors. Proteins of the EGR and Myb families of transcription factors are long-studied regulators of a variety of physiological processes including cellular proliferation and differentiation. The structural and physical aspects of their function have been well characterized. Their cell-type specific participation in complex gene-regulatory networks, on the other hand, is still incompletely understood and represents a major challenge in the respective research areas. Preliminary analysis of gene expression data from metastasizing PR9692 and non- metastasizing PR9692-E9 chicken sarcoma cell lines revealed that the transcription factor EGR1 is expressed at a higher level in metastasizing cells and can thus take part in the regulatory processes that underlie the differences between the two cell lines. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from PR9692...
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POSITIVE AND NEGATIVE HOST FACTORS OF AVIAN SARCOMA AND LEUKOSIS VIRUSES
Krchlíková, Veronika ; Elleder, Daniel (advisor) ; Růžek, Daniel (referee) ; Španielová, Hana (referee)
Identification and characterization of the host cell factors that either support or inhibit virus replication constitutes a major direction in virological research. In this work we focus on several such host factors in the context of avian cell. Chicken Tva, cell entry receptor for subgroups A and K of Avian sarcoma and leukosis virus (ASLV), was identified to be orthologous to human receptor for cellular uptake of cobalamin (Cbl). Here, we describe Cbl uptake in chicken cells and its dependency on Tva. Additionally, we characterize in vivo Tva knockout in chicken. Chicken Tvb receptor conferring susceptibility to subgroups B, D and E of ASLV was previously shown to participate in virus-induced cytopathic effects. In this work, we identify a natural ligand of Tvb and investigate its participation in apoptosis. RIG-I-like receptors (RLR) are a key family of cytosolic viral RNA sensors. The activation of these receptors leads to establishment of an antiviral state in the cell. In this study, we describe repeated evolutionary losses of RLR genes in birds: the loss of MDA5 in two avian orders and the loss of RIG-I in multiple species. Tetherin is an antiviral restriction factor blocking the release of newly formed viral particles. We identify tetherin orthologs in avian species and investigate...
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Effect of small DNA viruses on regulation of interferon production
Hofman, Tomáš ; Hirsch, Ivan (advisor) ; Elleder, Daniel (referee)
Plasmacytoid dendritic cells (pDC) represent innate immune cells capable to detect viruses in their endosomal environment via Toll-like receptors (TLRs). Viral nuclear acid recognition leads to the massive production of type I interferon (IFN I) and induction of the antiviral state in uninfected cells. Crosslinking of the surface regulatory receptors, such as BDCA-2, with monoclonal antibodies or with some viruses leads to the activation of MEK1/2- ERK signaling pathway and inhibition of IFN I production in pDC. In this study, the role of MEK1/2 kinase has been highlighted. Its inhibition reversed the inhibitory effect of BDCA-2 crosslinking and its direct activation with PMA led to the inhibition of IFN-α production. Yet an unclear role of pDC in sensing of BK polyomavirus virus (BKV) responsible for kidney transplant rejection was investigated as a major topic of this thesis. Experiments with the pDC cell line Gen2.2 and HRPTEC primary cell line showed that pDCs were not able to detect BKV particles, however, exposure of activated Gen2.2 cells to BKV inoculum dramatically upregulated production of IFN-α. Most importantly, coculture of Gen2.2 cells with BKV- infected HRPTEC cells resulted in IFN-α and TNF-α production, which was prevented by Bafilomycin. These results suggest that BKV-infected...
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Adaptive evolution of Toll-like receptors in birds
Velová, Hana ; Vinkler, Michal (advisor) ; Elleder, Daniel (referee) ; Novák, Karel (referee)
Adaptive evolution of Toll-like receptors in birds Hana Velová, PhD thesis 6 Abstract Toll-like receptors (TLRs) are one of the key and presumably also evolutionary most original components of animal immune system. As Pattern recognition receptors they form the first line of innate immune defence against various pathogens. The proper receptor binding of pathogenic ligands is crucial for their correct recognition and for subsequent triggering of an appropriate immune response. Because there exists a direct interaction between the receptor surface and the pathogenic ligand, host-pathogen coevolution on molecular level can be predicted. Thus, through variability of their ligands, TLRs are exposed to extensive selective pressures that may be detected on both genetic and protein levels. Surprisingly, the variability we revealed in birds is even higher than previously expected based on the reports from other vertebrates, mainly mammals. In my doctoral thesis I summarise the results of my contribution to the avian TLR research. We were the first who experimentally verify the absence of functional TLR5 in several avian species and duplication of TLR7 in others. We finally resolved the origin of duplication in TLR1 and in TLR2 family. An important part of my research project focused on the prediction of potentially...
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Interaction of HIV-1 infection and expresion of endogenous retroviruses
Machač, David ; Elleder, Daniel (advisor) ; Drda Morávková, Alena (referee)
The aim of this bachelor thesis is to describe the effect of HIV-1 on the expression of HERV proviruses in infected cells, how the presence of HERV transcripts and proteins can affect the replication cycle of HIV-1, the composition, structure and infectivity. These interactions may have negative but also positive effects on HIV-1 infectivity. For example, the mutual presence of HIV-1 and HERV proteins encoded by the gag genes responsible for the capsid, nucleocapsid, and virion matrix, may mix together, and form aberrant capsids that will not play a correct role in the HIV-1 replication cycle. Further, due to the phenomenon called viral pseudotyping, HERV envelope glycoproteins could be incorporated into the HIV-1 membrane. This could be a pathway, how the HIV-1 could theoreticaly extend the tropism to other cells than only CD4 T-lymphocytes, macrophages and dedritic cells. Key Words: HERV, HIV-1, virus pseudotypes, retrovirus replication
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Effect of small DNA viruses on regulation of interferon production
Hofman, Tomáš ; Hirsch, Ivan (advisor) ; Elleder, Daniel (referee)
Plasmacytoid dendritic cells (pDC) represent innate immune cells capable to detect viruses in their endosomal environment via Toll-like receptors (TLRs). Viral nuclear acid recognition leads to the massive production of type I interferon (IFN I) and induction of the antiviral state in uninfected cells. Crosslinking of the surface regulatory receptors, such as BDCA-2, with monoclonal antibodies or with some viruses leads to the activation of MEK1/2- ERK signaling pathway and inhibition of IFN I production in pDC. In this study, the role of MEK1/2 kinase has been highlighted. Its inhibition reversed the inhibitory effect of BDCA-2 crosslinking and its direct activation with PMA led to the inhibition of IFN-α production. Yet an unclear role of pDC in sensing of BK polyomavirus virus (BKV) responsible for kidney transplant rejection was investigated as a major topic of this thesis. Experiments with the pDC cell line Gen2.2 and HRPTEC primary cell line showed that pDCs were not able to detect BKV particles, however, exposure of activated Gen2.2 cells to BKV inoculum dramatically upregulated production of IFN-α. Most importantly, coculture of Gen2.2 cells with BKV- infected HRPTEC cells resulted in IFN-α and TNF-α production, which was prevented by Bafilomycin. These results suggest that BKV-infected...
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