Epstein-Barr virus is a ?-herpesvirus that infects nearly 95% of adults worldwide. A potent immune response prevents disease in the majority of those infected. However, immune suppression can lead to latent EBV-driven B-cell lymphomas. It is our ultimate goal to define the mechanisms by which EBV establishes latency and how these processes go awry leading to disease. In this proposal, we aim to define the cellular metabolic restriction to EBV transformation of primary human B cells. It is our central hypothesis that EBV-induced hyper-proliferation initially upon infection leads to an imbalance in energy metabolism resulting in a depletion of purine nucleotides and telomere-specific DNA damage causing permanent cell cycle arrest. We have formulated our hypothesis based on preliminary data characterizing the metabolic state of EBV-infected cells that begin to proliferate, then either arrest or continue to long-term outgrowth. In cells that arrest, we detected a failure to up-regulate oxidative phosphorylation as well as genes controlled by nuclear respiratory factor 1. Arrested cells display activated p53 and AMP kinase, which suppresses mTOR leading to elevated basal autophagy. Arrested cells also have depleted purine nucleotides and providing exogenous nucleosides rescues transformation of primary B cells by EBV. Finally, expression of the viral latent membrane proteins is delayed until late infection and their activation of NF-kappaB and Akt mitigate autophagy in B cells through inducing surface expression of the glucose transporter, Glut1. Based on these data, we propose that early EBV infected cells display elevated autophagy as a mechanism to compensate for nutrient deprivation during hyperproliferation. These starved cells are depleted fur purine nucleotides triggering a DNA damage response that causes permanent growth arrest. At later times during infection, EBV suppresses autophagy through increased glucose import. The rationale for this proposed research is that understanding the control of B-cell metabolism by EBV during latent infection will provide important clues to how this virus mimics B-cell maturation and may provide new therapeutic targets for EBV-associated lymphomas. We plan to test our central hypothesis and complete the objectives outlined in this proposal through the following specific aims: 1) Determine the mechanism by which EBV overcomes the early metabolic barrier to B-cell transformation, 2) Determine the consequences of nucleotide pool imbalance on the growth-suppressive DDR at telomeres following EBV-mediated B-cell infection, and 3) Define the role of NF-kappaB/Akt-mediated glucose import in suppressing nutrient deprivation-induced autophagy.
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