Epstein-Barr virus molecular pathogenesis in the nasopharynx and the role of LMP1 in lytic infection

eb病毒（EBV）在鼻咽部的感染可以是潜伏性或溶解性的，但eb病毒相关的鼻咽癌（NPC）是潜伏性感染。由于以下几个原因，溶性感染和复制失败对eb病毒在鼻咽部的发病机制很重要。EBV裂解基因促进癌症机制，包括基因组不稳定和炎症细胞因子的产生。此外，裂解性感染的病毒脱落补充了当地的病毒库。有趣的是，EBV溶解蛋白的IgA抗体在鼻咽癌患者中升高，有时在鼻咽癌发病前几年升高。鼻咽上皮由多种细胞类型组成，大致分为层状上皮和假层状呼吸上皮。这些上皮组织中细胞类型的分类不能在二维培养中进行研究。这一差距使得明确地阐明EBV在鼻咽上皮中的分子发病机制具有挑战性。一种变革性的方法是使用三维细胞培养模型。在本研究中，我们利用不同类型的鼻咽气液界面（ALI）三维细胞培养模型，通过诱导编码Zebra的即早基因（BZLF1）的表达，证明了EBV蛋白潜伏膜蛋白1 （LMP1）在裂解诱导中是必需的。在此应用中，我们将使用在ALI培养中重新激活的HK1 EBV感染细胞系（称为HK1-EBV ALI），以及第二个[从头EBV感染]模型，其中原代鼻咽细胞播种在器官型筏（称为鼻筏）中。这两种三维培养模型在分析潜力方面都是高度允许的。我们专注于将LMP1重新定义为裂解诱导所需的毒力因子，这修改了目前承认LMP1是EBV癌蛋白的范式。在HK1-EBV ALI或鼻筏中去除或抑制LMP1会减弱斑马诱导。相反，在HK1-EBV细胞中引入LMP1的表达会产生分化依赖的超溶表型。引人注目的是，LMP1是EBV编码基因中最多样化的基因之一，但在鼻咽癌肿瘤中高度保守，但LMP1序列变异与溶性感染的功能意义尚不完全清楚。我们证明LMP1相互作用组在溶解性感染期间完全重新连接。我们假设LMP1序列变异会影响斑马诱导，而这种反应可能依赖于分化依赖转录因子（TF）的差异激活。在本文中，我们试图通过激活或诱导三种目的的tf表达来定义LMP1参与诱导Zebra的机制。在一个目标中，我们将研究LMP1序列变异如何影响与斑马诱导相关的LMP1功能基因组学。在另外两个目标中，我们将使用蛋白质阵列和单细胞rna测序方法的组合来阐明激活斑马表达的分化依赖性和lmp1诱导的tf。这一建议有望阐明LMP1序列进化在裂解潜能方面的重要性，并定义在裂解感染过程中LMP1的重新布线机制如何重新定义LMP1作为毒力因子。

Epstein-Barr virus (EBV) infection in the nasopharynx can be latent or lytic but EBV-associated nasopharyngeal carcinomas (NPC) are latently-infected. Lytic infection, and the failure to replicate, in response to epithelial differentiation is important to EBV pathogenesis in the nasopharynx for several reasons. EBV lytic genes promote cancer mechanisms including genome instability and the production of inflammatory cytokines. Furthermore, virus shed from lytic infection replenishes the local reservoir. Intriguingly, IgA antibodies to EBV lytic proteins are elevated in NPC patients sometimes rising years prior to the onset of NPC. The nasopharyngeal epithelium is composed of many cell types which are broadly divided into stratified epithelia and pseudostratified respiratory epithelia. The assortment of cell types in these epithelial tissues cannot be studied in 2-D culture. This gap has made it challenging to elucidate EBV molecular pathogenesis in the nasopharyngeal epithelium with any clarity. One transformative approach is the use of 3-D cell culture models. In this proposal, we demonstrate with different types of nasopharyngeal air-liquid interface (ALI) 3-D cell culture models that the EBV protein, latent membrane protein 1 (LMP1), is required for lytic induction through induced expression of the immediate-early gene (BZLF1) encoding Zebra. In this application, we will use a HK1 EBV-infected cell line reactivated in ALI culture (known as HK1-EBV ALI), and a second [de novo EBV infection] model in which primary nasopharyngeal cells are seeded in organotypic rafts (referred as nasal-rafts). Both these 3-D culture models are highly permissive in terms of lytic potential. We focus on redefining LMP1 as a virulence factor required for lytic induction, which revises the current paradigm acknowledging LMP1 as an EBV oncoprotein. The removal or inhibition of LMP1 in HK1-EBV ALI or in nasal-rafts, blunts Zebra induction. Conversely, the introduced expression of LMP1 in HK1-EBV cells produces a differentiation-dependent superlytic phenotype. Strikingly, LMP1 is one of the most divergent EBV- encoded genes but is highly conserved in NPC tumors, yet the functional significance of LMP1 sequence variation in relation to lytic infection is completely unknown. We show evidence that the LMP1 interactome is completely rewired during lytic infection. We hypothesize that LMP1 sequence variation impacts Zebra induction and this response would be dependent on the differential activation of differentiation-dependent transcription factors (TF). In this proposal, we seek to define LMP1’s mechanisms involved in the induction of Zebra through activation or induced expression of TFs in three Aims. In one Aim, we will study how LMP1 sequence variation impacts LMP1 functional genomics in relation to Zebra induction. In the other two Aims, we will use a combination of protein array and single cell RNA-sequencing methods to elucidate differentiation-dependent and LMP1-induced TFs that activate Zebra expression. This proposal is expected to shed light on the significance of LMP1 sequence evolution in regard to lytic potential and to define how rewiring of LMP1 mechanisms during lytic infection redefines LMP1 as a virulence factor.