Publikationsdatenbank

Marek’s disease virus genome replication, telomere integration, and live-cell visualization (2022)

Art

Hochschulschrift

Autor

Vychodil, Tereza (WE 5)

Quelle

Berlin: Mensch und Buch Verlag, 2022 — XII, 108 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/36984

Kontakt

Institut für Virologie

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51833
virologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Herpesviruses maintain their genome lifelong in its host by establishing a latent phase of infection. Marek’s disease virus (MDV) is a chicken herpesvirus causing fatal lymphomas and the vaccination against MD is considered to be the first example for vaccination against an agent causing cancer. MDV, as well as some other herpesviruses, achieves latency by incorporating its genome into host telomeres. However, many steps and factors involved in genome integration and reactivation process remain unclear. Our current knowledge about herpesvirus integration is mainly based on results from animal experiments and fluorescence in situ hybridization (FISH). However, FISH cannot be used for a real-time detection of integrated MDV. In this thesis, I established a visualization system utilizing the tetracycline operator/repressor (tetO/TetR) system to optically detect and track MDV genomes without any other additional staining in living cells during different stages of infection. MDV genome consists of two unique regions both flanked by two inverted repeat regions. Genes found in repeat regions are present twice in the genome, several of which are involved in MDV pathogenesis, tumorigenesis, and latency. Therefore, I generated a platform virus lacking the majority of internal repeats (ΔIRLS-HR) to facilitate a straightforward mutation of the virus genome in the repeat region. I demonstrated that such a virus can rapidly restore the deleted repeat region when short terminal sequences are left for homologous recombination. Conversely, viruses unable to restore the internal repeats (ΔIRLS) showed impaired replication and pathogenesis in infected chickens corroborating previous findings in related herpesviruses where deletion of inverted repeat region affected virus pathogenicity. To generate MDV mutants whose genome can be directly detected by microscopy, the tetO sequence was inserted into the ΔIRLS-HR backbone. Fluorescently labeled TetR proteins that specifically bind to tetO sequence were expressed either from cells or from the virus itself. Using this visualization technique, replicating virus genomes, genesis and mobility of viral replication compartments and latent virus were detected. Moreover, these viruses also express another fluorescent protein with a different color for unambiguous identification of infected cells during the lytic stage of virus infection. The combination of both fluorescent proteins revealed not only that MDV can induce syncytia in primary chicken embryo cells, but also that the virus does not replicate in all nuclei of a syncytium. Taken together, ΔIRLS-HR serves as an excellent platform to generate recombinant viruses and in combination with tetO/TetR system we established a powerful tool to visualize virus genomes and to trace the impact of different mutations or gene deletions on the behavior of MDV during lytic replication, latency, and reactivation.