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Interaction of polyomaviruses with proteasomal system of host cell
Verdánová, Martina ; Drda Morávková, Alena (advisor) ; Horák, Martin (referee)
Interaction of polyomaviruses with proteasomal system of host cells Abstract: Viral family Polyomaviridae includes besides model organisms - mouse polyomavirus and SV40 virus, also human pathogens, for example, BK virus. Polyomaviruses are small non- enveloped viruses with double-stranded DNA. Understanding of their life cycle is important for their use in gene therapy and immunotherapy as well as for prevention and treatment of complications caused by these viruses. This thesis is focused on early phases of MPyV and SV40 infection studying, mainly on delivery of viral genome to nucleus and role of proteasomal system in this stage of infection. It was found out that inhibition of proteasomes by specific inhibitor leads to increase of early non-structural protein LT expression, which was chosen as marker for viral entry to the nucleus and successful viral expression. Relative localization of proteasomes and VP1 protein of MPyV and SV40 was monitored and it showed 10% colocalization of mentioned structures. Further, it was found out that proteasomal inhibitor MG-132 negatively influences the replication of both viral and cellular DNA. Next aim of this diploma thesis was to prepare antigen - unique part of VP2 protein of BKV - for producing antibody. Expression vector with inserted fragment of unique part of...
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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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Minor Structural Proteins of Polyomaviruses: Attributes and Interactions with Cellular Structures
Vinšová, Barbora ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Even though polyomaviruses have been intensively studied for more than 60 years, the role of minor structural proteins VP2 and VP3 in some important steps of viral life cycle has still not been fully elucidated, explicitly their role in viral genome delivery to the cell nucleus and their involvement in late phases of viral life cycle. This diploma thesis focuses on the study of minor proteins of Mouse polyomavirus (MPyV) and Human polyomavirus BK (BKV). Four rabbit polyclonal antibodies against minor proteins of polyomaviruses MPyV or BKV have been prepared within this diploma thesis. Two of these prepared antibodies target minor proteins of MPyV (α-MPyV VP2/3) or BKV virus (α-BKV VP2/3), other two prepared antibodies recognize C-terminal sequence common to minor proteins VP2 and VP3 of MPyV (α-MPyV C-termVP2/3) or BKV virus (α-BKV C-termVP2/3). In the second part of this diploma thesis we aimed to study toxicity of BKV virus minor proteins during individual production in mammalian cells. Obtained results suggest that minor proteins of BKV virus might not exhibit as high levels of cytotoxicity as minor proteins of MPyV virus. Third part of this diploma thesis is devoted to investigation of interactions of BKV and MPyV minor proteins with cellular proteins and within one another respectively....
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Studies of minor capsid proteins of the mouse polyomavirus
Vít, Ondřej ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Mouse polyomavirus (MPyV) is a small non-enveloped virus. Its capsid consists of 72 pentamers of the major capsid protein VP1. The central cavity of each VP1 pentamer contains one minor capsid protein, either VP2, or VP3. The minor capsid proteins are dispensable for capsid formation, but their presence is required for infection of the host cell, presumably because of their anticipated functions during virus entry. After internalization, MPyV virions traffic to endoplasmic reticulum (ER). VP2 and VP3 have been proposed to function as factors responsible for penetration of ER membranes, which is required for subsequent delivery of the viral DNA into the nucleus, a key step of the early phase of MPyV infection. Three hydrophobic domains were predicted in the sequence of VP2 and VP3. First in the unique Nterminal part of VP2, second and third in the common part of VP2 and VP3. The third domain corresponds to C-terminal VP1binding alpha-helix. It has been previously found in our laboratory, that VP2 and VP3 fused to N-terminus of EGFP, when expressed in mammalian cells, display properties similiar to the wild-type VP2 and VP3, namely affinity to intracellular membranes and high cytotoxicity. Expression plasmids carrying mutated VP2 and VP3 fused to Nterminus of EGFP were prepared to determine the hydrophobic...
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The role of posttranslational modifications of minor proteins and acetylation of microtubules in mouse polyomavirus infection
Mariničová, Zuzana ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Mouse polyomavirus (MPyV) capsid is composed of the main capsid protein VP1 and minor capsid proteins VP2 and VP3. Minor proteins are not essential capsid assembly, but they are key for efficient viral infection. The first part of this thesis studies the modifications of VP2 and VP3, the deamidation of Asn at 253 of VP2 (137 of VP3) and N-terminal acetylation of Ala of VP3, which could be the cause of double bands for VP2 and VP3 on SDS-PAGE. Mutated genomes of MPyV N253D (Asn to Asp) and N253E (Asn to Glu) simulating deamidation and A117V (Ala to Val) with reduced acetylation were prepared previously. We prepared three isolations of the mutant viruses and we confirmed that the deamidation is the cause of the double bands. Mutant viruses were compared to the wild type in terms of efficiency of infection, but the role of deamidation could not be proven. Virus A117V is noninfectious either due to lowered acetylation or the substitution of amino acid at this position. This thesis also studies the role of -tubulin acetylation in the infection of MPyV. The role of -tubulin acetylation in viral infection is being investigated to find new antiviral strategies. Acetylation rises after MPyV infection, but this is not due to a change in mRNA expression of tubulin acetylating (TAT1) or deacetylating enzyme...
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Minor Structural Proteins of Polyomaviruses: Attributes and Interactions with Cellular Structures
Vinšová, Barbora ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Even though polyomaviruses have been intensively studied for more than 60 years, the role of minor structural proteins VP2 and VP3 in some important steps of viral life cycle has still not been fully elucidated, explicitly their role in viral genome delivery to the cell nucleus and their involvement in late phases of viral life cycle. This diploma thesis focuses on the study of minor proteins of Mouse polyomavirus (MPyV) and Human polyomavirus BK (BKV). Four rabbit polyclonal antibodies against minor proteins of polyomaviruses MPyV or BKV have been prepared within this diploma thesis. Two of these prepared antibodies target minor proteins of MPyV (α-MPyV VP2/3) or BKV virus (α-BKV VP2/3), other two prepared antibodies recognize C-terminal sequence common to minor proteins VP2 and VP3 of MPyV (α-MPyV C-termVP2/3) or BKV virus (α-BKV C-termVP2/3). In the second part of this diploma thesis we aimed to study toxicity of BKV virus minor proteins during individual production in mammalian cells. Obtained results suggest that minor proteins of BKV virus might not exhibit as high levels of cytotoxicity as minor proteins of MPyV virus. Third part of this diploma thesis is devoted to investigation of interactions of BKV and MPyV minor proteins with cellular proteins and within one another respectively....
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Studies of properties of the minor structural proteins of the Murine polyomavirus
Bílková, Eva ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Murine polyomavirus (MPyV) is a member of the Polyomaviridae family. Its capsid is composed of the major capsid protein, VP1, and the minor proteins, VP2 and VP3. The minor capsid proteins probably assure delivery of the viral genome through the endoplasmic reticulum membrane to the nucleus during early phase of infection. However, precise mechanism is not known. Expression plasmids encoding mutated VP2 or VP3 fused with EGFP have been constructed to study the interaction of VP2 and VP3 with membranes. The mutated proteins have deletions in the predicted hydrophobic domains. In this thesis, cell localisation of mutated proteins was followed. The study revealed that the hydrophobic domain 2 is the most important for association of VP2 and VP3 with membranes, while domains 1 and 3 are rather expendable. Further, nature of VP2 and VP3 isoforms has been studied. Isoforms with different electrophoretic mobility were separated on SDS-PAGE. Consequent mass spectrometry analysis showed that they differ in deamidation of asparagine, present at both minor proteins (position 253 of VP2 and 137 of VP3). Previously, acetylation of VP3 N-terminal alanine has been identified. To elucidate the function of these modifications, mutated viruses were constructed with substitution of these amino acids. Pilot...
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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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Interaction of polyomaviruses with proteasomal system of host cell
Verdánová, Martina ; Drda Morávková, Alena (advisor) ; Horák, Martin (referee)
Interaction of polyomaviruses with proteasomal system of host cells Abstract: Viral family Polyomaviridae includes besides model organisms - mouse polyomavirus and SV40 virus, also human pathogens, for example, BK virus. Polyomaviruses are small non- enveloped viruses with double-stranded DNA. Understanding of their life cycle is important for their use in gene therapy and immunotherapy as well as for prevention and treatment of complications caused by these viruses. This thesis is focused on early phases of MPyV and SV40 infection studying, mainly on delivery of viral genome to nucleus and role of proteasomal system in this stage of infection. It was found out that inhibition of proteasomes by specific inhibitor leads to increase of early non-structural protein LT expression, which was chosen as marker for viral entry to the nucleus and successful viral expression. Relative localization of proteasomes and VP1 protein of MPyV and SV40 was monitored and it showed 10% colocalization of mentioned structures. Further, it was found out that proteasomal inhibitor MG-132 negatively influences the replication of both viral and cellular DNA. Next aim of this diploma thesis was to prepare antigen - unique part of VP2 protein of BKV - for producing antibody. Expression vector with inserted fragment of unique part of...
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