Extracellular vesicles (EVs) are membrane enclosed vesicles released from cells that act as mediators of intercellular communication and are involved in normal physiological processes as well as pathological conditions. EVs can transfer functional vesicles which can promote cell growth, migration, differentiation, and regulate immune cell function in the recipient cells. Latent membrane protein 1 (LMP1), an Epstein-Barr virus (EBV) major oncoprotein has been shown to enhance the secretion EVs from infected cells. LMP1 is an important viral protein that contributes to tumor growth, metastasis and microenvironment remodeling. Transfer of the LMP1-modified EVs can activate MAPK/ERK and PI3K/Akt signaling functions within neighboring uninfected cells. Currently there is limited understanding of the mechanisms responsible for orchestrating trafficking of LMP1 into EVs and tumor microenvironment remodeling. The following work describe the cellular machineries utilized by LMP1 to secreted in EVs and the effects of the EVs on recipient cells. Our studies in Chapter 2 focused on determining the LMP1 domains important for EV trafficking. Using various LMP1 constructs with deletion mutations in the C-terminal and transmembrane domains, we showed that the N-terminus and transmembrane region one (TM1) is sufficient to guide efficient sorting into the EV pathway. Additionally, a mutant lacking the N-terminus and transmembrane domains 1 through 4 (TM5-6) that fails to be packaged into EVs displayed an altered endo-lysosomal trafficking being retained in the endoplasmic reticulum and early endosomes. We further showed existence of potential positive and negative regulatory mechanisms of LMP1 secretion. Overall, this study provides a detailed analysis of the roles different LMP1 structural domains play in endosomal sorting and EV packaging. In chapter 3, we investigated the role of the ESCRT dependent pathway and associated proteins in mediating LMP1 cargo sorting and EV secretion. Our results demonstrated that LMP1 utilizes Hrs, Syntenin-1 and specific components of the ESCRT-III complex (CHMP4B, 5 and 6) for release from the cell and enhancement of EV production. Additionally, our data revealed that efficient secretion of LMP1 modified EVs promotes cell attachment, proliferation, migration and tumor growth. These data suggest that LMP1 interacts with ESCRT machinery components to enter the endocytic route for efficient EV sorting and release to extracellular space. Finally, to understand the molecular mechanisms underlying LMP1 remodeling of the tumor microenvironment, we hypothesized that LMP1 alters the content and functions of the EVs and the LMP1 modified EVs induce transformation process in the recipient cells. Our findings reveal that LMP1 expression alters the EV protein and microRNA content packaged into EV. The LMP1-modified EVs also enhance recipient cell adhesion, proliferation, migration, invasion concomitant with the activation of ERK, AKT, and NF-κb signaling pathways. The LMP1 modified vesicles induced transcriptome reprogramming by upregulating gene expression in recipient cells. Collectively, our findings begin to demonstrate the mechanisms in which LMP1 modified EVs are secreted from infected cells and establish a tumor-permissive microenvironment by increasing gene expression of ECM interaction proteins. Our findings from these studies contribute to advance the current knowledge on understanding protein trafficking, EV cargo sorting, EV secretion and mechanisms of tumor microenvironment remodeling
