Reprogramming of energy metabolism by oncogenic Marek's disease virus (MDV) in chicken embryo fibroblasts (CEFs)
Date
2014
Authors
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Publisher
University of Delaware
Abstract
Marek's disease (MD), the most prevalent clinically-diagnosed cancer in the animal kingdom, is a herpesvirus infection that rapidly induces aggressive T-cell lymphomas in chickens. This disease, caused by Marek's disease virus (MDV), a double-stranded DNA alphaherpesvirus, is prevalent worldwide. While this oncogenic herpesvirus and its cell-associated, non-sterilizing vaccinations remain good models for herpesvirus oncology and immunotherapy, respectively, the complete mechanisms of lymphoma development and progression remain unclear. The elucidation of these mechanisms remains an important topic of research.
Tumorigenesis is a multistep process in which transformed cells tend to acquire a number of biological "hallmarks of cancer" including: sustained proliferation, loss of tumor suppression, resistance to apoptosis, immortalization, angiogenesis, invasion, and metastasis. After accumulating these hallmarks of cancer, oncogenically-transformed cells shift from being benign to being fully malignant. This transition requires increased energy metabolism to support cell proliferation and growth during tumorigenesis. The increased metabolic need of these cells, in part, helped the discovery of two hallmarks of cancer: reprogramming of energy metabolism and immune evasion. German physiologist Otto Warburg had documented this switch from oxidative phosphorylation to anaerobic glycolysis and fermentation in tumorigenic cells, a process termed the Warburg effect. Viruses have developed multiple processes that aid in their efficient replication within cells. The processes of genomic and structural protein replication and assembly require substantial energy demands, and therefore it is likely that many viruses affect cellular metabolism in similar ways as cellular transformation. Specifically, MDV represents an important model of the Warburg effect due to its ability to not only replicate in multiple cell types (CEF, B-cells, T-cells, etc.), but also due to its ability to transform CD4 + T-cells. This work was aimed at identifying the MDV-mediated effects on cellular metabolism during infection. In this study, we hypothesized that oncogenic MDVs induce metabolic changes during replication similar to previously documented cancers, contributing to tumorigenesis in affected birds. To test this hypothesis, we performed qRT-PCR and Western blot analyses of MDV-infected chicken embryo fibroblasts (CEF) to determine if infection with MDV reprograms glucose and glutamine metabolism during lytic replication in cell culture. To address whether this reprogramming is MDV common, specific to oncogenic strains, or to specific gene products of MDV-1, we examined targeted gene expression during infections of CEF with: vaccine strains HVT, SB-1, and CVI-988, pathogenic MDV-1 strains: CU-2, RB-1B, rMd5, and TK (TKING), as well as two rMd5-based recombinant strains: rMd5-delta-Meq and rMd5-delta-pp38. We found significant up-regulation in the transcription of glycolytic genes (HIF-1alpha, SLC2A1, HK2, LDHA, and SLC16A3 ) and glutaminolysis genes (SLC7A5 and GLS ) in cells infected with CU-2, RB-1B, rMd5 and TKING strains. Both TKING-infected CEF and TKING-transformed spleen tumors also showed biologically-significant up-regulation of HIF-1alpha, SLC16A3, and SLC7A5 genes. Furthermore, oncogenic MDV infection of CEFs showed statistically significant increases in expression of several glycolytic and glutaminolytic genes when compared to both vaccine and rMd5-based recombinant strains. These changes in expression were not observed by Western blot analysis, however. This discrepancy in RNA and protein levels may have been due to decreased solubility of many of the membrane-associated transport proteins. At the transcript but not the protein levels, these data support the hypothesis that virulent MDV-1 strains may affect glycolysis and glutaminolysis during lytic infection and in TKING-induced tumors. These data await further characterization via definitive assays for metabolic activity and additional assessments of protein expression (immunofluorescence or mass spectrometry).