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.IMPORTANCE 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.

Keywords: Epstein-Barr virus; exosomes; extracellular vesicles; herpesvirus; microvesicles; protein trafficking; tetraspanin; trafficking.

FIG 1

The C-terminal cytoplasmic tail of LMP1 is not required for EV packaging. (A) Schematic depiction of WT GFP-tagged LMP1 and deletion mutants. LMP1 is a six-pass transmembrane protein with N- and C-terminal cytoplasmic tails. Various GFP-tagged mutants of the full-length wild-type LMP1 protein were constructed by PCR mutagenesis and used throughout the study. (B) The levels of nontagged LMP1 and GFP-LMP1 were compared in cell lysates and EVs. (C) Lysate immunoblot analysis of cells transiently transfected with LMP1 or mutants containing deletions in the C terminus (1-233, 1-203, and 1-187) (equal protein masses were loaded). (D) EVs (equal volumes were loaded) were harvested by differential centrifugation and analyzed by immunoblot analysis for GFP-tagged proteins and common EV markers (Alix, HSC70, Flotillin-2, and CD81). (E) Semiquantitative immunoblot analysis of the results of 3 independent experiments. The levels of LMP1 mutant secretion are relative to wild-type LMP1 secretion and normalized to cellular expression and EV production [(LMP1_EV/HSC70_EV)/(LMP1_cell/HSC70_cell)]. The error bars indicate standard deviations. (F) Cell lysates and (G) EVs harvested from GFP, GFP-LMP1, or GFP-tagged N (1-27) and C (187-386) termini of LMP1. Cells and EVs were harvested by differential centrifugation and analyzed by immunoblotting for expression with anti-GFP antibody and common EV markers (Alix, HSC70, and Flotillin-2). TM, transmembrane domains.

FIG 2

Transmembrane domains control the vesicular release of LMP1 from cells. HEK293 cells were transfected with GFP, GFP-LMP1, or transmembrane deletion mutants (TMΔ5-6, TMΔ3-4, TM3-6, TM5-6, and TM3-4). (A) Whole-cell and (B) EV lysates were separated by SDS-PAGE and analyzed by immunoblot analysis for GFP and common EV markers (Alix, HSC70, Flotillin-2, and CD81). (C) Semiquantitative immunoblot analysis of the results of four to six independent experiments examining relative vesicular LMP1 secretion of the transmembrane deletion mutants. Immunoblot analysis of (D) cell and (E) vesicle lysates for deletion mutants (TM1-2ΔC and TM1-4ΔC). (F) Semiquantitative immunoblot analysis from three independent experiments showing the relative EV LMP1 secretion for TM1-2ΔC and TM1-4ΔC deletion mutants. Immunoblot analysis of (G) cell and (H) vesicle lysates for mutants (TM1ΔC, TMΔ1-2, TMΔ1-4, and Δ1-25). (I) Semiquantitative immunoblot analysis from three independent experiments showing the relative EV LMP1 secretion for TM1ΔC, TMΔ1-2, TMΔ1-4, and Δ1-25 deletion mutants. The error bars indicate standard deviations.

FIG 3

Mutation of the lipid-raft anchoring domain FWLY increases LMP1 EV release. Immunoblot analysis of the levels of GFP-tagged wild-type or mutant LMP1 in (A and D) cell lysates or (B and E) purified membranes. (C and F) CD63 was detected by immunoblotting in all isolated membranes. (G) Whole-cell or (H) EV lysates were analyzed by immunoblot analysis for SNAP, HSC70, Alix, and Flotillin-2 following transfection with SNAP, SNAP-LMP1, or the LMP1-FWLY mutant. (I) Semiquantitative immunoblot analysis of the results of 3 independent experiments. The levels of LMP1 mutant secretion are relative to wild-type LMP1 secretion and normalized to cellular expression and EV production. The error bars indicate standard deviations.

FIG 4

LMP1 mutants that fail to traffic to EVs exhibit altered subcellular localization. HEK293 cells were transfected with the indicated LMP1 constructs and imaged 24 h posttransfection by live-cell confocal microscopy. Scale bars, 10 μm.

FIG 5

TM5-6 accumulates in ER and early endosome membranes. HEK293 cells were cotransfected with GFP-tagged LMP1 constructs and RFP-tagged (A) Sec61B, (B) Rab5, or (C) eNos. Live-cell confocal images were acquired 24 h posttransfection. The images were analyzed using Imaris software. (D) Colocalization was quantified using Pearson's correlation coefficient and graphed with standard errors of the mean (n ≥ 12 cells). Representative maximum-projection images are shown. **, P < 0.001. Scale bars, 10 μm.

FIG 6

TM5-6 exhibits altered colocalization with endolysosomal markers. HEK293 cells were cotransfected with GFP-tagged LMP1 constructs and RFP-tagged (A) Rab7, (B) Rab11, or (C) LAMP1. Live-cell confocal images were acquired 24 h posttransfection. The images were analyzed using Imaris software. (D) Colocalization was quantified using Pearson's correlation coefficient and graphed with standard errors of the mean (n ≥ 12 cells). Representative maximum-projection images are shown. *, P < 0.05; **, P < 0.001. Scale bars, 10 μm.

FIG 7

TM5-6 colocalizes and associates with CD63. (A) HEK293 cells were cotransfected with GFP-tagged LMP1 constructs and CD63-RFP. Live-cell confocal images were acquired 24 h posttransfection. The images were analyzed using Imaris software. Representative maximum-projection images are shown. Scale bar, 10 μm. (B) Colocalization was quantified using Pearson's correlation coefficient and graphed with standard errors of the mean (n ≥ 12 cells). (C) CD63 knockout cells were cotransfected with CD63-BirA* plus WT LMP1 or CD63-BirA* plus TM5-6 or transfected with CD63-BirA* alone. Biotinylated protein complexes were isolated and separated by SDS-PAGE and analyzed by immunoblotting with LMP1 and CD63-specific antibodies. (D) EVs were harvested from HEK293 cells transiently transfected with GFP, GFP-LMP1, or TM5-6 and quantified by nanoparticle tracking. The data are represented as total numbers of particles harvested per cell. *, P < 0.05. (E and F) HEK293 cells were transfected with GFP-TM5-6, SNAP-LMP1 or cotransfected with GFP-TM5-6 and SNAP-LMP1. (E) Whole-cell and (F) EV lysates were separated by SDS-PAGE and analyzed by immunoblot analysis for GFP, SNAP, and common EV markers (Alix and HSC70).

FIG 8

Summary of LMP1 mutant data. P, packaged; EP, enhanced packaging; MP, moderate packaging; RP, reduced packaging; PP, punctate perinuclear; SPS, scattered punctate structures; DP, diffuse perinuclear; DC, diffuse in cytoplasm; NA, not analyzed.