EBV gp350 is packaged into exosomes, confers B-cell tropism, and reactivates specific T cells EBV has a profound B-cell tropism that is mainly conveyed by gp350, which is the major EBV glycoprotein in the viral envelope and the ligand for cellular CD21 (CR2) on B cells. We knew from previous work that exosomes can transport ectopically expressed proteins such as green fluorescent protein (GFP), which is presumably present as a cargo in the exosomal lumen. In addition, several groups provided evidence that surface proteins are incorporated in exosome membranes [5] . We therefore asked whether gp350 could also become an integral part of exosomes and confer B-cell tropism to these vesicles. To answer this question, we cotransfected 293 cells with expression plasmids encoding BLLF1, the gene of gp350, and gfp. Three days later, we isolated vesicles from the supernatants of transfected HEK293 cells as described in Material and Methods and analyzed them by immunoblots for the presence of gp350 and exosome markers. Gp350 was detected in vesicles that floated at a density between 1.03 and 1.08 into an OptiPrep TM gradient, corresponding to a density between 1.13 to 1.18 in a sucrose gradient and thus in the density described for exosomes. The gradient also revealed the co-sedimentation of gp350 with the exosome markers hps70, tsg101 and CD63, indicating the nature of the gp350+ vesicles as exosomes ( Figure 1A ). Flow cytometry of exosomes coupled to latex beads revealed that gp350 is presumably located within the exosome membrane because it could be targeted with a specific antibody ( Figure 1B ). To demonstrate that gp350 confers B-cell tropism also to exosomes, we incubated gp350+/gfp+ exosomes with PBMCs from a healthy donor for one day and then quantified exosome binding by measuring GFP fluorescence by flow cytometry. This assay revealed that gp350+/gfp+ exosomes had an EBV-like tropism because they bound to CD19+ B cells but not to CD19negative cells ( Figure 1C ).