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Article

Visualization of Marek’s Disease Virus Genomes in Living Cells during Lytic Replication and Latency

1
Institut für Virologie, Freie Universität Berlin, Robert von Ostertag-Straße 7-13, 14163 Berlin, Germany
2
Department of Biology, Molecular Biophysics, Humboldt-Universität zu Berlin, Invalidenstraße 42, 10115 Berlin, Germany
3
Institut für Chemie und Biochemie, Freie Universität Berlin, Altensteinstr. 23a, 14195 Berlin, Germany
4
Veterinary Centre for Resistance Research (TZR), Freie Universität Berlin, 14163 Berlin, Germany
*
Author to whom correspondence should be addressed.
Academic Editors: Charles Grose, Ravi Mahalingam and Joel Rovnak
Viruses 2022, 14(2), 287; https://doi.org/10.3390/v14020287
Received: 30 November 2021 / Revised: 24 January 2022 / Accepted: 26 January 2022 / Published: 29 January 2022
Visualization of the herpesvirus genomes during lytic replication and latency is mainly achieved by fluorescence in situ hybridization (FISH). Unfortunately, this technique cannot be used for the real-time detection of viral genome in living cells. To facilitate the visualization of the Marek’s disease virus (MDV) genome during all stages of the virus lifecycle, we took advantage of the well-established tetracycline operator/repressor (TetO/TetR) system. This system consists of a fluorescently labeled TetR (TetR-GFP) that specifically binds to an array of tetO sequences. This tetO repeat array was first inserted into the MDV genome (vTetO). Subsequently, we fused TetR-GFP via a P2a self-cleaving peptide to the C-terminus of the viral interleukin 8 (vIL8), which is expressed during lytic replication and latency. Upon reconstitution of this vTetO-TetR virus, fluorescently labeled replication compartments were detected in the nucleus during lytic replication. After validating the specificity of the observed signal, we used the system to visualize the genesis and mobility of the viral replication compartments. In addition, we assessed the infection of nuclei in syncytia as well as lytic replication and latency in T cells. Taken together, we established a system allowing us to track the MDV genome in living cells that can be applied to many other DNA viruses. View Full-Text
Keywords: Marek’s disease virus; live-cell genome visualization; lytic replication; T cells; latency; genome integration; TetO/TetR system Marek’s disease virus; live-cell genome visualization; lytic replication; T cells; latency; genome integration; TetO/TetR system
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MDPI and ACS Style

Vychodil, T.; Wight, D.J.; Nascimento, M.; Jolmes, F.; Korte, T.; Herrmann, A.; Kaufer, B.B. Visualization of Marek’s Disease Virus Genomes in Living Cells during Lytic Replication and Latency. Viruses 2022, 14, 287. https://doi.org/10.3390/v14020287

AMA Style

Vychodil T, Wight DJ, Nascimento M, Jolmes F, Korte T, Herrmann A, Kaufer BB. Visualization of Marek’s Disease Virus Genomes in Living Cells during Lytic Replication and Latency. Viruses. 2022; 14(2):287. https://doi.org/10.3390/v14020287

Chicago/Turabian Style

Vychodil, Tereza, Darren J. Wight, Mariana Nascimento, Fabian Jolmes, Thomas Korte, Andreas Herrmann, and Benedikt B. Kaufer. 2022. "Visualization of Marek’s Disease Virus Genomes in Living Cells during Lytic Replication and Latency" Viruses 14, no. 2: 287. https://doi.org/10.3390/v14020287

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