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Integration site distribution of expressed proviruses
Miklík, Dalibor ; Hejnar, Jiří (advisor) ; Kejnovský, Eduard (referee) ; Indik, Stanislav (referee)
To establish efficient expression of their genes, retroviruses integrate proviral copies into the genomes of the cells they have infected. Epigenetic events, however, silence expression of the integrated proviruses. This silencing protects host cells from harmful viral spread, but also creates a reservoir of latent proviruses that subsequently hinders the cure of retroviral (e.g., HIV-1) infections. Furthermore, the silencing of retrovirus-derived integrative vectors complicates their application in transgenesis and gene therapy. The goal of this thesis is to describe the interaction between retroviral expression and host (epi)genomic environment at the site of proviral integration. To pursue the goal, we sought to define the (epi)genomic environment of the proviruses, which expression is not affected by the epigenetic silencing. Diverse retroviral vectors derived from avian sarcoma and leukosis virus (ASLV), murine leukemia virus (MLV), and human immunodeficiency virus type 1 (HIV-1) were used as model retroviral systems, and expression stability of the vectors in human cell lines was examined. In order to identify the features unique to integration sites of the active proviruses, we sorted the cells positive for the proviral expression, identified their proviral integration sites, and compared them to...
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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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Host factors involved in Rous Sarcoma Virus replication
Štafl, Kryštof ; Svoboda, Jan (advisor) ; Horníková, Lenka (referee)
Rous sarcoma virus (RSV) takes a place of honor among retroviruses. Research of RSV allows us to uncover the secret of the origin and evolution of life, the mechanisms of tumorigenesis and the interaction between viruses and their hosts. Viruses are not able to replicate themselves without host cells. They exploit a number of cellular pathways and factors and they can reprogram the cells to produce great amounts of viral progeny. They exert pressure on their host that leads to developement of new types of cellular proteins, which then results in resistance of the cells. This thesis focuses on the host factors involved in the RSV replication cycle. It summarizes the current knowledge about the replication cycle of retroviruses and the host factors that are necessary for productive infection: cellular receptors, endocytic and secretory pathways, nuclear transport, proteosynthesis, replication of proviruses and its stimulation. The restriction mechanisms of cells are also taken into account. The current knowledge about RSV is compared with facts on mammalian retroviruses and gaps in research are highlighted. The influence of the host cell factor absence, host specificity and cellular permissiveness are correlated and discussed.
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