Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

Abstract

The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells. The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major oncoprotein, LMP1, into vesicles for secretion. We have recently described a role of the host cell protein CD63 in regulating intracellular signaling of the viral oncoprotein by shuttling LMP1 into exosomes. Here, we provide strong evidence of the utility of CD63-dependent EVs in regulating global intracellular signaling, including mTOR activation by LMP1. We also demonstrate a key role of CD63 in coordinating endosomal and autophagic processes to regulate LMP1 levels within the cell. Overall, this study offers new insights into the complex intersection of cellular secretory and degradative mechanisms and the implications of these processes in viral replication.

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

“…Since LMP1 has previously been shown to increase EV production, we wondered whether TM5-6 similarly enhances EV levels. Nanoparticle tracking analysis of cells transfected with LMP1 confirmed previous findings of elevated EV production (P Ͻ 0.05) (28,57); however, expression of TM5-6 produced levels of particles comparable to those of control GFP-transfected cells (P ϭ 0.16) ( Fig. 7D).…”

Epstein‐Barr virus‐encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan‐2 and synaptotagmin‐like‐4 in nasopharyngeal carcinoma cells

“…35,36 Previous studies indicated that CD63 is a critical player in shuttling LMP1 into EVs or exosomes. [17][18][19] However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood.…”

“…PI3K/Akt and MAPK/ERK signaling pathways to promote malignant cell growth, invasion, and metastasis. 15,16 CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of EV production, [17][18][19] and it has been found that C-terminal farnesylation of UCH-L1 is also one of the mechanisms by which LMP1 is sorted to EVs. 20 Therefore, LMP1 cannot only be loaded in EVs, but might also be involved in the regulation of EV secretion.…”

A cell-based assay for CD63-containing extracellular vesicles

“…Based on the origin of EVs a number of proteins are well established as markers. Among these are the tetraspanins characterized by four membrane spanning helices, including most notably CD63 [27][28][29][30][31]. CD63 is predominantly localized to the intraluminal vesicles (ILVs) of late endosomes and MVBs and is thus enriched on exosomes [31][32][33].…”

“…Among these are the tetraspanins characterized by four membrane spanning helices, including most notably CD63 [27][28][29][30][31]. CD63 is predominantly localized to the intraluminal vesicles (ILVs) of late endosomes and MVBs and is thus enriched on exosomes [31][32][33]. Cytoplasmic proteins including members of the endosomal sorting complex required for transport (ESCRT) machinery, syntenin, and certain chaperones are also enriched in EVs [32,34].…”