Exosomes are small vesicles secreted from cells that contain specific profiles of functional proteins and RNAs. Exosomes participate in a recently discovered mechanism of cellular communication, and therefore have been implicated in many biological processes. There is accumulating evidence that alterations in normal exosome content or function can contribute to disease. In the case of human cancers, exosomes have been shown to function in immune cell modulation, angiogenesis, cell proliferation, migration, and tumor cell invasion. Based on data from our studies and others, it is also becoming clear that viruses utilize and modify the host cell exosome pathway. For example, cancer cells infected with the Epstein-Barr virus (EBV) release exosomes that are enriched in viral products like the major viral oncogene latent membrane protein 1 (LMP1) and virally- encoded miRNAs. We have previously shown that LMP1 alters the cargo of exosomes released from infected cells and that these LMP1-modifed exosomes can exert oncogenic signaling functions on neighboring uninfected cells. Despite the importance of inter-cellular transmission of LMP1-modifeid exosomes, very little is known about how this viral protein actually enters and manipulates the exosome pathway. The overall goal of these studies is to determine the mechanism of LMP1 trafficking to the site of exosome formation and its release from the cell. We hypothesize that LMP1 exosomal trafficking modulates the components and functions of exosomes by altering endocytic routes and membrane microdomains. To test this, we aim to: 1.) determine the vesicular trafficking pathway traveled by LMP1 for exosome targeting using a combination of dominant negative and shRNA constructs directed against specific endocytic components and pathways identified in our preliminary studies, and 2.) investigate the LMP1-CD63 protein interaction network critical for LMP1 exosome secretion using affinity purification and quantitative mass spectrometry of interaction networks and sub-cellular compartments.
The Epstein-Barr virus is considered the cause of multiple human cancers, and has been estimated to account for approximately 200,000 new cancers worldwide each year. The viral protein, latent membrane protein 1, is consistently detected in many of the EBV-associated cancers, and is released from cells in exosomes where it can exhort its functions on the tumor microenvironment. Thus, exosomal LMP1 likely contributes to pathogenesis within the host. In this project, we will determine the molecular mechanism of LMP1 exosome targeting and release from the cell that will aid in the design of novel therapeutics to combat EBV-associated cancers.
