The effect of mutations in the Meq oncoprotein of Marek's disease virus (MDV) on lymphomas composition
Date
2014
Authors
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Publisher
University of Delaware
Abstract
Marek's Disease (MD) is a highly transmissible immunosuppressive alpha-herpes-virus infection of chickens caused by Marek's disease virus (MDV). MDV infects chickens and can cause immunosuppression, paralysis, and rapid induction of T-cell lymphomas. The exact mechanism of tumor formation is still unknown, but involves the expression of Meq, a basic-leucine zipper (bZIP) protein encoded by the virus. Losses due to MD are currently controlled through the near ubiquitous use of vaccines. Despite the success of these vaccines, field strains of MDV have increased in virulence since the early 1970s. A main focus of our laboratory is the molecular basis of the increase in virulence of MDV field strains. We have found that mutations in the proline-rich repeat (PRR) domains of Meq correlate with increased virulence. To determine the effect of these mutations on virulence, we examined the composition (in terms of cell populations) of lymphomas induced by strains RB-1B, rMd5, and rMDV-1137 (a recombinant having the meq gene of RB-1B in the background strain rMd5). We found that RB-1B and rMDV-1137-induced lymphomas had greater numbers of CD30+ CD4+ T-cells, compared to rMd5. As the meq genes of RB-1B and rMd5 have only 3 amino acid changes in the PRR region, our data suggest that mutations in Meq may affect tumor development and progression. To extend these results, we used flow cytometry to identify the cellular composition of archived tumor suspension samples and cell lines induced by MDVs of different virulence levels (virulent, very virulent and very virulent plus MDVs). We first validated this technique using defined cell lines that were fixed prior to staining, thawed from frozen storage and stained prior to fixation, or were actively growing at the time of staining. Next, we determined the cellular composition and surface antigen expression of selected MDV induced lymphomas and MDV-transformed cell lines of different pathotypes (vMDV, vvMDV, and vv+MDV) respectively. Our results suggested that MDV induced lymphomas are heterogeneous and changes in tumor composition appear unrelated to virus pathotype. Since we did not see pathotype-associated effects on tumor composition, we performed two additional sets of experiments to test the effect of Meq mutations on: (1) the T-cell receptor (TCR) spectratype of defined MDV-transformed cell lines and tumors induced by different pathotypes MDVs via PCR analysis of the rearranged TCR to determine whether lymphomas induced by vv+MDVs were comprised of polyclonal or monoclonal expansion events; and (2) the interaction of Meq proteins with specific and common cellular proteins detected by proteomic analysis of Meq-co-immunoprecipitations from MDV transformed cell lines MSB-1 (transformed by vMDV strain,BC-1), UD35 (transformed by vvMDV strain, RB-1B) and UA53 (transformed by a vv+MDV strain, TK).