The role of polycomb repressive complex proteins in Marek's Disease Virus (MDV) latency and lymphomagenesis
Date
2018
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Publisher
University of Delaware
Abstract
Marek’s Disease Virus (MDV) is the causative agent of Marek’s disease (MD), a pathology associated with a rapid formation of T-cell lymphomas in chickens. MDV-induced T-cell lymphomagenesis is associated with the latent infection of CD4+ T-cells causing tumor formation within a few weeks of infection. A specific viral oncoprotein (Meq) and splice variants of this protein (Meq/vIL8 and MeqvIL8Δexon3) are expressed as MDV establishes latency, in primary lymphomas and in lymphoma-derived cell lines. Furthermore, we have discovered that these latently-associated splice-variant proteins interact with a host polycomb repressive complex (PRC-1) protein, Bmi-1 (B-cell Moloney leukemia virus integration site 1). PRCs regulate cell differentiation and development through targeted modification of chromatin, resulting in the silencing of gene expression. Specifically, Bmi-1 is associated with gene silencing through chromatin remodeling via ubiquitin ligase activity of lysine 119 on histone 2A (H2AK119). Yet, this complex does not act alone. PRC-2 protein, EZH2 (Enhancer of zeste homolog 2), will initially tri-methylate lysine 27 of histone 3 (H3K27me3), forming a docking site for PRC-1. ☐ The focus of this project was to characterize the interactions between the viral oncoproteins of MDV (Meq and two of its splice-variants, Meq/vIL8 and Meq/vIL8Δexon3) and host PRC proteins, as they related to cellular transformation and the maintenance of viral latency. This was done through inhibition of Bmi-1 and EZH2 through chemical inhibitors (PTC-209 and GSK-126, respectively) and with siRNA knock-downs examining viral reactivation in MDV-transformed T-lymphoma cell lines (UA22 and UD35 cells). UA22 cells have been transformed by RB1B-pp38-smGFP (with a GFP tag downstream of pp38, a major early lytic phase antigen). To this end, viral reactivation has been examined through flow cytometry analysis of GFP expression. Viral reactivation has also been examined through RT-qPCR, and viral genome copy numbers via qPCR. Further interaction between Bmi-1 and Meq proteins was also characterized through bioimaging methods (co-localization, FRET, and FRAP analysis). Our data suggest that EZH2-mediated histone trimethylation is involved in MDV genome silencing during latency, and that Bmi-1-mediated repression is longer term and is primarily involved in cellular transformation. Moreover, we found that Meq-splice-variant proteins have increased affinity for Bmi-1, causing its translocation from the nucleoplasm into the nucleolus, and decreasing its mobility. Our findings extend our previous research and further associate MDV latency with cellular transformation pathways involving PRCs, pathways identified in numerous human malignancies, including Hodgkin’s lymphoma.
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Biological sciences