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Experimental system for the mouse polyomavirus life cycle study
Pergner, Jiří ; Španielová, Hana (advisor) ; Mašek, Tomáš (referee)
Experimental system for the mouse polyomavirus life cycle study Abstract: Murine polyomavirus (MPyV) is the prototype of the Polyomaviridae family. This family includes also some important human pathogens (BKV, JCV, Merkel cell polyomavirus). Due to their specific properties viruses within this family may serve as versatile vectors for gene therapy or recombinant vaccine production. New methodological approaches may help to understand some yet unknown facts about MPyV life cycle. Clarification of some processes during murine polyomavirus life cycle may be also important to fully exploit polyomaviruses for therapeutic purposes. The aim of this diploma thesis was to preparare two innovative experimental systems that extend possibilities of studying the life cycle of MPyV. The first part of the diploma thesis focusses on construction of recombinant MPyV which expresses yellow fluorescent protein (EYFP) in the early stages of infection. Such virus can be very useful for studying the infection spreading by live- cell imaging and Fluorescence-Activated Cell Sorting (FACS) and can be employed for co- localization studies of YFP-tagged LT antigen with certain cellular proteins. Second part of the diploma thesis describes preparation of a hybrid cell line prepared by fusion of mouse and monkey cells. This new cell...
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Interaction of transmembrane proteins ASCT1 and ASCT2 with retroviral envelope glycoproteins
Trávníček, Martin ; Trejbalová, Kateřina (advisor) ; Mělková, Zora (referee)
Transmembrane proteins ASCT1 and ASCT2 are ubiquitous neutral amino acid transporters. Apart from their transporter function in metabolically active cells, they also serve as receptors for a wide group of retroviruses. All retroviruses recognizing the transmembrane receptor ASCT2/ASCT1 share a similar env gene, encoding the envelope glycoprotein. Syncytin-1 is the envelope glycoprotein, encoded by human endogenous retrovirus type W, produced in placental cytotrophoblasts of primates, including human. Interaction of receptor binding domain of Syncytin-1 and specific extracellular region of ASCT2 is responsible for fusion of neighbouring cells and formation of multinucleated syncytiotrophoblast. The importance of syncytiotrophoblast lies in higher efficiency of feto-maternal exchange of nutrients and simultaneously in modulation of immune response of mother towards fetus. Defect in syncytiotrophoblast differentiation often leads to complications during pregnancy and impairs the proper development of embryo. Characterization of protein domains responsible for the interaction between Syncytin-1 and its receptors is important to uncover genetic causes of these pathologies. Furthermore, understanding the interaction helps us to clarify the mechanism of cell entry and explains the molecular basis of host...
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Rous sarcoma virus replication blocks in mammalian cells
Koslová, Anna ; Hejnar, Jiří (advisor) ; Ruml, Tomáš (referee) ; Weber, Jan (referee)
One of the important tasks of virology and immunology is to explore the species- and cell-barriers preventing virus horizontal transmission and reveal the ways how viruses overcome these barriers and "adapt" to different species. This work is based on a well- established retroviral model - avian Rous sarcoma virus (RSV) and studies virus replication blocks in mammalian cells at both pre- and post-integration level. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor mediates virus entry into cells. Although mammalian orthologues of specific chicken receptors do not support RSV entry, it was observed that some RSV strains are able to enter mammalian cells. Several RSV-transformed rodent cells lines were described and analysis of provirus H20- RSV in one these cells lines (hamster H-20 tumor cell line) showed multiple mutations including two crucial amino acid substitutions in different regions of Env. Substitutions D32G and L378S confer virus transmission to hamster, human and also chicken cells lacking the appropriate receptor. Altered conformation of H20-RSV Env is similar to a receptor-primed (activated) state of Env. This observation indicates that virus can circumvent the need of original cell receptor because of spontaneous Env activation caused by single...
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Molecular mechanisms of cellular nonpermissiveness against Rous sarcoma virus
Štafl, Kryštof ; Hejnar, Jiří (advisor) ; Hirsch, Ivan (referee)
Most viruses can infect only a reduced range of organisms and an effective replication is possible only in selected hosts. These hosts are called permissive for the virus. Molecular principles of a nonpermissiveness and viral mechanisms of overcoming replication obstacles are still not clearly elucidated. This thesis discusses the molecular causes of the cellular nonpermissiveness against a model retrovirus - Rous sarcoma virus. The research is conducted on duck cells which are semipermis- sive to the subgroup C of Rous sarcoma virus. The virus can enter those cells, but it is not able to produce enough infectious viral progeny. Two blocks of the viral replication cycle in the duck cells are described in the thesis. The first one is the probably not optimal cellular receptor recognition. The second one is in the late phase of the replication cycle when the viral proteins are synthesized. The amount of the envelope glyco- protein coding mRNA is reduced due to the altered splicing ratios, and the virions produced from the duck cells are less infectious. This block is recessive and can be partially omitted by cell fusions with permissive chicken cells; therefore, the block is not caused by specific restriction fac- tors in sensu stricto. Additionally, the influence of mutations in duck adapted Rous...
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Experimental system for the mouse polyomavirus life cycle study
Pergner, Jiří ; Mašek, Tomáš (referee) ; Španielová, Hana (advisor)
Experimental system for the mouse polyomavirus life cycle study Abstract: Murine polyomavirus (MPyV) is the prototype of the Polyomaviridae family. This family includes also some important human pathogens (BKV, JCV, Merkel cell polyomavirus). Due to their specific properties viruses within this family may serve as versatile vectors for gene therapy or recombinant vaccine production. New methodological approaches may help to understand some yet unknown facts about MPyV life cycle. Clarification of some processes during murine polyomavirus life cycle may be also important to fully exploit polyomaviruses for therapeutic purposes. The aim of this diploma thesis was to preparare two innovative experimental systems that extend possibilities of studying the life cycle of MPyV. The first part of the diploma thesis focusses on construction of recombinant MPyV which expresses yellow fluorescent protein (EYFP) in the early stages of infection. Such virus can be very useful for studying the infection spreading by live- cell imaging and Fluorescence-Activated Cell Sorting (FACS) and can be employed for co- localization studies of YFP-tagged LT antigen with certain cellular proteins. Second part of the diploma thesis describes preparation of a hybrid cell line prepared by fusion of mouse and monkey cells. This new cell...
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