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的或溶解性的，但EBV相关的鼻咽癌（NPC）是潜伏性感染。由于几个原因，响应于上皮分化的裂解性感染和复制失败对于鼻咽中的EBV发病机制是重要的。EBV裂解基因促进癌症机制，包括基因组不稳定性和炎性细胞因子的产生。此外，溶菌性感染所释放的病毒会使当地的宿主死亡。有趣的是，NPC患者中EBV裂解蛋白的伊加抗体升高，有时在NPC发病前几年升高。鼻咽上皮由多种细胞组成，大致可分为复层上皮和假复层呼吸上皮。这些上皮组织中细胞类型的分类不能在二维培养中研究。这一差距使得它具有挑战性，以任何清晰阐明鼻咽上皮细胞中的EBV分子发病机制。一种变革性的方法是使用3-D细胞培养模型。在这个建议中，我们证明了与不同类型的鼻咽气液界面（ALI）的3-D细胞培养模型的EBV蛋白，潜伏膜蛋白1（LMP 1），是需要通过诱导表达的立即早期基因（BZLF 1）编码斑马裂解诱导。在本申请中，我们将使用在ALI培养物中再活化的HK 1 EBV感染的细胞系（称为HK 1-EBV ALI）和第二种[从头EBV感染]模型，其中原代鼻咽细胞接种在器官型筏（称为鼻筏）中。这两种3-D培养模型在裂解潜力方面都是高度允许的。我们专注于将LMP 1重新定义为裂解诱导所需的毒力因子，这修改了目前承认LMP 1为EBV癌蛋白的范式。在HK 1-EBV ALI或鼻筏中去除或抑制LMP 1会减弱Zebra诱导。相反，在HK 1-EBV细胞中引入LMP 1的表达产生分化依赖性超裂解表型。引人注目的是，LMP 1是最不同的EBV编码基因之一，但在NPC肿瘤中高度保守，然而LMP 1序列变异与裂解性感染相关的功能意义完全未知。我们的证据表明，LMP 1相互作用组是完全重新布线溶解感染。我们假设LMP 1序列变异影响Zebra诱导，并且这种反应将依赖于分化依赖性转录因子（TF）的差异激活。在这个提议中，我们试图通过激活或诱导表达三个目标中的TF来定义LMP 1参与诱导Zebra的机制。在一个目标中，我们将研究LMP 1序列变异如何影响LMP 1功能基因组学与斑马诱导。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Zebra表达的分化依赖性和LMP 1诱导的TF。这一提议有望阐明LMP 1序列进化在裂解潜力方面的意义，并确定裂解感染期间LMP 1机制的重新布线如何将LMP 1重新定义为毒力因子。

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.