The Nanoarchitecture of the KSHV LANA Tether and Approaches to Its Disruption
Grant, Margaret, Microbiology - School of Medicine, University of Virginia
Kedes, Dean, Department of Microbiology, University of Virginia
Smith, M, Department of Microbiology, University of Virginia
Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies and presents a special concern to immunocompromised individuals. Its latency-associated nuclear antigen protein (LANA) tethers latent viral genomes to host chromatin, thereby maintaining viral infection, and as such, is an attractive target for therapeutic intervention. In an effort to better understand this protein and its tethering function, we have used super-resolution microscopy to examine LANA tethers, obtaining information that remained obscured in earlier studies using standard epifluorescence microscopy. We have determined several characteristics of these tethers, including the folding properties of the underlying viral DNA and occupancy of LANA on its available viral binding sites. Quantitative data support the prediction of a coiled-coil feature in LANA dimers, and computer modeling of a minimal LANA tether illustrates the importance of viral DNA bending and nucleosome positioning on tether structure. Preliminary data examining LANA tethers during cellular mitosis suggest a potential role for mitotic machinery in manipulating tether positioning and condensation. This work also begins to address the relative timing of host chromosome condensation and LANA tether formation. These promising early results compel further study of the mitotic LANA tether and its dynamics. Finally, proof-of-concept experiments disrupting LANA expression via CRISPR-Cas9 gene editing pave the way for future manipulation of the KSHV genome and present a potential avenue for downstream therapeutic intervention.
PHD (Doctor of Philosophy)
KSHV, Superresolution Microscopy, dSTORM, CRISPR-Cas9
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