Defining the Mechanisms of Epstein-Barr Virus Persistence and Recurrence

摘要 我们的最终目标是确定口腔中EBV潜伏期建立和重新激活的分子机制 空洞。在这项提案中，我们的目标是描述EBV如何篡夺B细胞成熟程序以促进细胞存活和 平衡潜伏期驱动的增殖、分化和裂解的细胞状态连续体的建立 重新激活。我们的中心假设是EBV通过模仿B细胞建立潜伏感染 生发中心(GC)反应，随后促进激活和分化的连续体，即 平衡以使得能够访问支持裂解再激活的细胞状态。我们已经阐明了我们的中心假设 基于包括单细胞基因表达在内的初步数据，扁桃体B细胞和 EBV永生化细胞以及EBV新的自发裂解株的特征。我们发现 EBV潜伏感染通过时间调控抗凋亡分子MCL-1和BFL-1促进GC拟态。vbl.使用 我们发现了一系列基因表达的连续体，其中NFB信号/激活(如： NFKB2、MYC、IRF8)与浆母细胞分化(如CD38、PRDM1、XBP1)呈显著负相关。 此外，EBV裂解基因在与高分化状态最相似的细胞中表达，表明 EBV感染细胞不断采样这种分化状态以切换到裂解状态的模型 重新激活。最后，使用新的EBV毒株，我们描述了一种持续的、自发的转换到 短暂性和可逆性的生产性感染。因此，这项拟议研究的理由是 了解EBV如何建立潜伏期并重新激活以应对生产性感染，有助于深入了解 从口腔中清除EBV感染细胞的治疗方法。潜在的分子电路 控制这些细胞命运的决定也可能提供关于B细胞可塑性的重要新信息 成熟状态。我们计划测试我们的中心假设，并通过以下方式完成本提案中的目标 以下三个具体目标：i)确定EBV促进B细胞的分子机制 模拟扁桃体B细胞成熟的存活，II)定义支持一种新的 在单个EBV永生化的B细胞中的激活/分化连续体，以及III)定义生化 以及一种新描述的EBV复发表型的细胞生物学特征，在该表型中潜伏感染的细胞 产生病毒粒子并返回到它们的基本潜伏状态。

ABSTRACT Our ultimate goal to define the molecular mechanisms for EBV latency establishment and reactivation in the oral cavity. In this proposal, we aim to characterize how EBV usurps B-cell maturation programs for cell survival and the establishment of a continuum of cell states balancing latency-driven proliferation, differentiation, and lytic reactivation. It is our central hypothesis that EBV establishes B-cell latent infection through mimicry of the germinal center (GC) reaction and subsequently promotes a continuum of activation and differentiation that is balanced to enable access to a cell state supporting lytic reactivation. We have formulated our central hypothesis based on preliminary data including single-cell gene expression, chromatin conformation of tonsillar B cells and EBV-immortalized cells as well as characterization of new spontaneously lytic strains of EBV. We found that EBV latent infection promotes GC mimicry through temporal regulation of anti-apoptotic MCL-1 and BFL-1. Using scRNA-seq of LCLs, we discovered a continuum of gene expression where NFB signaling/activation (e.g. NFKB2, MYC, IRF8) is strongly anti-correlated with plasmablast differentiation (e.g. CD38, PRDM1, XBP1). Furthermore, EBV lytic genes were expressed in cells most similar to the high differentiation state suggesting a model whereby EBV-infected cells constantly sample this differentiated state to enable a switch to lytic reactivation. Finally, using new strains of EBV we describe a paradigm of a persistent, spontaneous switch to productive infection that is transient and reversible. Therefore, the rationale for this proposed research is that understanding how EBV establishes latency and reactivates to productive infection provides insight into therapeutic modalities to eliminate EBV-infected cells from the oral cavity. The underlying molecular circuitry controlling these cell fate decisions may also provide important new information regarding the plasticity of B-cell maturation states. We plan to test our central hypothesis and complete the objectives in this proposal through the following three specific aims: i) to determine the molecular mechanisms by which EBV promotes B-cell survival mimicking tonsillar B-cell maturation, ii) to define the underlying molecular circuitry supporting a novel activation/differentiation continuum within individual EBV-immortalized B cells, and iii) to define the biochemical and cell biological features of a newly described EBV recurrence phenotype in which latently infected cells produce virions and return to their basal latent state.