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

Abstract

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. EBV infection contributes to the development of cancers, such as nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin's disease, and posttransplant lymphomas, in immunocompromised or genetically susceptible individuals. LMP1 is an important viral protein expressed by EBV in these cancers. LMP1 is secreted in extracellular vesicles (EVs), and the transfer of LMP1-modified EVs to uninfected cells can alter their physiology. Understanding the cellular machinery responsible for sorting LMP1 into EVs is limited, despite the importance of LMP1-modified EVs. Here, we illustrate the roles of different regions of LMP1 in EV packaging. Our results show that the N terminus and TM1 are sufficient to drive LMP1 EV trafficking. We further show the existence of potential positive and negative regulatory mechanisms for LMP1 vesicle sorting. These findings provide a better basis for future investigations to identify the mechanisms of LMP1 targeting to EVs, which could have broad implications in understanding EV cargo sorting.

Epstein-Barr virus subverts mevalonate and fatty acid pathways to promote infected B-cell proliferation and survival

“…Rab13 has pleiotropic roles, including the regulation of glucose transporter 4 trafficking [101], macrophage phagocytosis [102, 103], autophagy [104], lymphocyte trafficking [105], anoikis and metastasis [106–108]. Notably, LMP1 traffics to intracellular membranes to initiate signaling or to be secreted in exosomes [75, 94, 95, 109–112], though the chaperone protein(s) required for intracellular sorting are incompletely defined. Our results suggest that EBV co-opts Rab13 for yet another function: to chaperone oncogenic LMP1 and LMP2A to appropriate sub-cellular compartments where they can then activate downstream pathways.…”

Extracellular Vesicles in Herpes Viral Spread and Immune Evasion

“…On the other hand, EVs may also transfer virus-encoded proteins and nucleic acids independently of the viral particles. For instance, both simian immunodeficiency virus (SIV) ( McNamara et al, 2018 ) and HIV-1 ( Lenassi et al, 2010 ; Pereira and daSilva, 2016 ; Puzar Dominkus et al, 2017 ) induce the release of Nef protein in exosomes; EBV LMP1 is also secreted in EVs ( Meckes et al, 2010 ; Nkosi et al, 2018 ) and functional exosomes containing miRNAs have been detected in human clinical samples and mouse models of KSHV-associated malignancies ( Chugh et al, 2013 ).…”

Epstein-Barr virus LMP1 manipulates the content and functions of extracellular vesicles to enhance metastatic potential of recipient cells

“…An ideal purification method would retain EV functional properties [ 60 , 61 ]. In the case of LMP1 modified EVs, we have previously shown that expression of this viral oncoprotein enhances the release of smaller EVs in different cell lines with an enrichment of different exosomal markers [ 49 , 50 ]. In this study, the EVs used were isolated by a combination of tangential flow filtration (TFF, 100kDa cutoff) followed by precipitation using PEG-6000 then ultracentrifugation to remove PEG and contaminating protein complexes ( Fig 1A ).…”

“…Numerous studies have reported the significant role of EVs play in cell growth, invasion, and metastasis of diverse cancers [ 46 , 47 ]. EBV infected cells release EVs that can contain the viral proteins, LMP1 and LMP2, and virally encoded miRNAs [ 42 , 48 – 50 ]. EBV has been demonstrated to modify protein content and cargo of EVs released from latently infected B-cells with most of the significant changes correlating to LMP1 expression [ 51 ].…”