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-induc...