Transmembrane Domains Mediate Intra- and Extracellular Trafficking of Epstein-Barr Virus Latent Membrane Protein 1.

EBV latent membrane protein 1 (LMP1) is released from latently infected tumor cells in small membrane-enclosed extracellular vesicles (EVs). Accumulating evidence suggests that LMP1 is a major driver of EV content and functions. LMP1-modified EVs have been shown to influence recipient cell growth, migration, differentiation, and regulation of immune cell function. Despite the significance of LMP1-modified exosomes, very little is known about how this viral protein enters or manipulates the host EV pathway. In this study, LMP1 deletion mutants were generated to assess protein regions required for EV trafficking. Following transfection of LMP1 or mutant plasmids, EVs were collected by differential centrifugation, and the levels of specific cargo were evaluated by immunoblot analysis. The results demonstrate that, together, the N terminus and transmembrane region 1 of LMP1 are sufficient for efficient sorting into EVs. Consistent with these findings, a mutant lacking the N terminus and transmembrane domains 1 through 4 (TM5-6) failed to be packaged into EVs, and exhibited higher colocalization with endoplasmic reticulum and early endosome markers than the wild-type protein. Surprisingly, TM5-6 maintained the ability to colocalize and form a complex with CD63, an abundant exosome protein that is important for the incorporation of LMP1 into EVs. Other mutations within LMP1 resulted in enhanced levels of secretion, pointing to potential positive and negative regulatory mechanisms for extracellular vesicle sorting of LMP1. These data suggest new functions of the N terminus and transmembrane domains in LMP1 intra- and extracellular trafficking that are likely downstream of an interaction with CD63.