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的，也可以是裂解的，但EBV相关的鼻咽癌(NPC)是潜伏感染的。由于上皮性分化，裂解性感染和复制失败是鼻咽部EBV致病的重要原因。EBV裂解基因促进癌症机制，包括基因组不稳定和炎性细胞因子的产生。此外，从裂解感染中排出的病毒补充了当地的水库。有趣的是，EB病毒裂解蛋白的IgA抗体在鼻咽癌患者中升高，有时在鼻咽癌发病前几年上升。鼻咽上皮由多种细胞类型组成，大致分为复层上皮和假复层呼吸道上皮。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。这一差距使得鼻咽上皮中EBV分子发病机制的阐明变得具有挑战性。一种变革性的方法是使用3D细胞培养模型。在这项建议中，我们用不同类型的鼻咽气-液界面(ALI)三维细胞培养模型证明，EBV蛋白，潜伏膜蛋白1(LMP1)，是通过诱导编码Zebra的即刻早期基因(BZLF1)的表达来进行裂解诱导的。在这项应用中，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)，以及第二种[从头开始EBV感染]模型，在该模型中，原代鼻咽细胞被种植在器官型筏(称为鼻筏)中。这两种3D培养模型在裂解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，这修订了目前承认LMP1为EBV癌蛋白的范式。在HK1-EBV ALI或鼻筏中LMP1的去除或抑制会钝化斑马的诱导。相反，在HK1-EBV细胞中引入LMP1的表达产生了依赖于分化的超裂解表型。值得注意的是，LMP1是EBV编码的最具差异性的基因之一，但在鼻咽癌中高度保守，然而LMP1序列变异与溶血性感染有关的功能意义尚不完全清楚。我们的证据表明，LMP1相互作用体在裂解感染过程中完全重新连接。我们假设LMP1序列变异影响斑马的诱导，这种反应将依赖于分化依赖的转录因子(TF)的差异激活。在这项研究中，我们试图从三个方面定义LMP1的S机制，该机制通过激活或诱导转录因子的表达来诱导斑马。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测序相结合的方法来阐明分化依赖和LMP1诱导的激活Zebra表达的转录因子。这一建议有望阐明LMP1序列进化在裂解潜力方面的意义，并定义LMP1在裂解感染过程中机制的重新连接如何重新定义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.