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Genomic preferences in integration and expression of retroviruses and retrovirus-derived vectors
Miklík, Dalibor ; Hejnar, Jiří (advisor) ; Žíla, Vojtěch (referee)
Integration and provirus establishment are the key steps of retroviral life cycle. Genome-wide studies show that the integration is not a random process and that groups of related retroviruses display distinguishable patterns of integration preferences. The most rated genomic features forming the integration preferences of retroviruses are transcription units, transcription start sites and CpG islands. Whereas extending knowledge of genome structure, new, mainly epigenetic marks, which have a relationship to retroviral preferences are being defined. The integration into a specific genome region has a straight influence on the provirus expression and therefore on the production of virus progeny. While integration into some regions results in provirus silencing which is managed and maintained by variety of factors, some other genomic regions are vice-versa capable of stable provirus expression maintenance. These findings have implications for construction of safe and efficient retroviral vectors as well as for use of modified retroviruses as markers for an epigenetic and expression profile determination of genome regions.
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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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Functional genome analysis using the retroviral integration sites permissive for provirus expression in human cells
Miklík, Dalibor ; Hejnar, Jiří (advisor) ; Španielová, Hana (referee)
The expression of retroviral genes depends on the establishment of the provirus - the DNA copy of retroviral genome integrated into the host genome. The transcriptional state of provirus is then influenced by the environment at the site of integration. The phenomenon of proviral silencing is an obstacle to the usage of retroviral vectors and a barrier to the eradication of human immunodeficiency virus type 1 (HIV-1) from infected individuals. Taking advantage of single cell clones bearing one provirus, this diploma thesis investigates the distribution of (epi)genomic features at the sites occupied by stably expressed proviruses. In total, long-term expression profiles of 245 and 255 clones carrying avian sarcoma-leucosis virus (ASLV) and HIV-1, respectively, were obtained. The database-based analysis of 42 integration sites of ASLV and three integration sites of HIV-1 proviruses shows that proviral stable expression highly correlates with the transcriptional start sites (TSS) at the sites of integration. Histone marks characteristic for the proximity of active TSSs and regulatory elements at the sites of integration of stably expressed proviruses confirm this finding. The results presented in this thesis could inspire other analyses investigating the relationship between the integration site and the...
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Genomic preferences in integration and expression of retroviruses and retrovirus-derived vectors
Miklík, Dalibor ; Hejnar, Jiří (advisor) ; Žíla, Vojtěch (referee)
Integration and provirus establishment are the key steps of retroviral life cycle. Genome-wide studies show that the integration is not a random process and that groups of related retroviruses display distinguishable patterns of integration preferences. The most rated genomic features forming the integration preferences of retroviruses are transcription units, transcription start sites and CpG islands. Whereas extending knowledge of genome structure, new, mainly epigenetic marks, which have a relationship to retroviral preferences are being defined. The integration into a specific genome region has a straight influence on the provirus expression and therefore on the production of virus progeny. While integration into some regions results in provirus silencing which is managed and maintained by variety of factors, some other genomic regions are vice-versa capable of stable provirus expression maintenance. These findings have implications for construction of safe and efficient retroviral vectors as well as for use of modified retroviruses as markers for an epigenetic and expression profile determination of genome regions.
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