Metabolic stress is a barrier to Epstein-Barr virus mediated B-cell immortalization

Early after infection, Epstein-Barr Virus (EBV) induces a transient period of hyper-proliferation that is suppressed by the activation of the DNA damage response and a G1/S phase growth arrest. This growth arrest prevents long-term outgrowth of the majority of infected cells. We developed a method to isolate and characterize infected cells that arrest after this early burst of proliferation. We used microarray analysis to uncover changes in gene expression that could give us a better understanding of the pathways that attenuate immortalization. Human PBMCs were labeled with CellTrace Violet and infected with the B95-8 strain of EBV. At 4 days post-infection, the cells were labeled with a second proliferation dye, CFSE. Cells that proliferated are called PP and those that initially proliferated and then arrested are called PA. Total mRNA was isolated from sorted PA and PP cells using an RNeasy kit . The RNA was processed using an Ambion MessageAmp Premier Package and hybridized to a Human Genome U133 Plus. 2.0 Chip by the Duke Center for Genomic and Computational Biology Microarray Core. The resultant CEL files were RMA normalized (Partek) and the data was analyzed with GenePattern and GSEA v2.