EBV latent membrane protein 1 effects on plakoglobin, cell growth, and migration

EBV latent membrane protein 1 effects on plakoglobin, cell growth, and migration. packaging of LMP1. Nanoparticle tracking TUG-891 analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this Rabbit polyclonal to ZNF658 vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-B and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-B activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling. IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitt’s lymphoma, and Hodgkin’s lymphoma. In the context of cancer, EBV hijacks the exosomal pathway to modulate cell-to-cell signaling by secreting viral components such as an oncoprotein, LMP1, into host cell membrane-bound EVs. Trafficking of LMP1 into exosomes is associated with increased oncogenicity of these secreted vesicles. However, we have only a limited understanding of the mechanisms surrounding exosomal cargo packaging, including viral proteins. Here, we describe a role of LMP1 in EV production that requires CD63 and provide an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Finally, we present further evidence of the role of CD63 in limiting LMP1-induced noncanonical NF-B and ERK activation. Our findings have implications for future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in viral-associated tumorigenesis. through the activation of intracellular signaling pathways (7,C9). When present in the host cell, LMP1 acts as a mimic of CD40, a tumor necrosis factor receptor (TNFR) (8, 10), activating NF-B, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), phosphatidylinositol 3-kinase (PI3K)/Akt, and c-Jun N-terminal kinase (JNK) pathways. The activation of these pathways results in upregulation of multiple genes involved with regulation of apoptosis, cell cycle progression, cell proliferation, migration, and invasion (8, 11,C16). Notably, LMP1 can signal in the absence of a ligand (17) through recruitment of TNFR-associated factors (TRAFs) to interaction sites TUG-891 at C-terminal activation region (CTAR) domains (18, 19). Localization of LMP1 to perinuclear regions of the cell is believed to be necessary to mediate these signaling functions, independent of the transmembrane protein aggregation on the plasma membrane (20). LMP1 has also been demonstrated to localize to internal Golgi and multivesicular body (MVB) compartments and is packaged into exosomes for release from the cell (21). Exosomes are a population of small (40 to 150 nm) endocytically derived extracellular vesicles. Extracellular vesicles (EVs) broadly encompass a variety of vesicle populations, including exosomes, microvesicles, apoptotic bodies, and viral particles (22,C25). These vesicle populations reflect a diversity of sizes, densities, and intracellular origins of EVs. While microvesicles are considered larger EVs shed directly from the plasma membrane into the extracellular milieu, exosomes are produced from inward budding events on the limiting membrane of late endosomal organelles, forming intraluminal vesicles in MVBs. Similar to mechanisms of egress used by viral particles, MVBs can fuse with the plasma membrane to release exosomes into the extracellular space (22, 24). Functionally, exosomes have been revealed to play a role in cell-to-cell communication and modulation of immune responses (26,C29). Thus, it is likely that packaging of LMP1 into these vesicles mediates a TUG-891 number of functions, including facilitation of viral replication, immunosuppression, the establishment of latency, and promotion of cell growth. Exosomal trafficking of LMP1 has been linked to both the intra- and intercellular signaling capabilities of the viral protein. For instance, blockage of exosomal LMP1 secretion has been demonstrated to lead to TUG-891 downstream intracellular NF-B overstimulation within the cell, as measured by luciferase reporter assay (30). Additionally, transfer of LMP1-containing.