Host pathways regulating Epstein-Barr virus-mediated B cell growth transformation

爱泼斯坦-巴尔病毒是一种γ-疱疹病毒，感染了全世界近95%的成年人。在大多数感染者中，强有力的免疫反应可以预防疾病。然而，免疫抑制可导致潜伏的eb病毒驱动的b细胞淋巴瘤。我们的最终目标是确定EBV建立潜伏期的机制以及这些过程如何出错导致疾病。在这个提议中，我们的目的是确定细胞代谢限制EBV转化的原代人B细胞。我们的中心假设是，ebv在感染后最初诱导的超增殖导致能量代谢失衡，导致嘌呤核苷酸耗竭和端粒特异性DNA损伤，导致永久性细胞周期停滞。我们根据初步数据制定了我们的假设，这些数据描述了ebv感染细胞的代谢状态，这些细胞开始增殖，然后停止或继续长期生长。在停滞的细胞中，我们检测到氧化磷酸化和核呼吸因子1控制基因的上调失败。阻滞细胞显示活化的p53和AMP激酶，抑制mTOR导致基底自噬升高。被抑制的细胞嘌呤核苷酸也被耗尽，提供外源性核苷可以挽救原代B细胞被EBV转化。最后，病毒潜伏膜蛋白的表达延迟到感染后期，NF-kappaB和Akt的激活通过诱导葡萄糖转运体Glut1的表面表达来减轻B细胞的自噬。基于这些数据，我们提出早期EBV感染的细胞表现出升高的自噬作为一种机制来补偿过度增殖期间的营养剥夺。这些饥饿的细胞嘌呤核苷酸耗尽，引发DNA损伤反应，导致永久性生长停滞。在感染后期，EBV通过增加葡萄糖进口抑制自噬。这项研究的基本原理是了解eb病毒在潜伏感染期间对b细胞代谢的控制，将为这种病毒如何模拟b细胞成熟提供重要线索，并可能为eb病毒相关淋巴瘤提供新的治疗靶点。我们计划通过以下具体目标来验证我们的中心假设并完成本提案中概述的目标：1)确定EBV克服b细胞转化早期代谢屏障的机制；2)确定EBV介导的b细胞感染后核苷酸池失衡对端粒生长抑制DDR的影响；3)确定NF-kappaB/ akt介导的葡萄糖进口在抑制营养剥夺诱导的自噬中的作用。

Epstein-Barr virus is a γ-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.