LMP1, a viral relative of the TNF receptor family, signals principally from intracellular compartments

Abstract

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded, ligand-independent receptor that mimics CD40. We report here that LMP1 signals principally from intracellular compartments. LMP1 associates simultaneously with lipid rafts and with its signaling molecules, tumor necrosis factor-receptor (TNF-R)-associated factors (TRAFs) and TNF-R1-associated death domain protein (TRADD) intracellularly, although it can be detected at low levels at the plasma membrane, indicating that most of LMP1's signaling complex resides in intracellular compartments. LMP1's signaling is independent of its accumulation at the plasma membrane in different cells, and as demonstrated by a mutant of LMP1 which has significantly reduced localization at the plasma membrane yet signals as efficiently as does wild-type LMP1. The fusion of the transmembrane domain of LMP1 to signaling domains of CD40, TNF-R1 and Fas activates their signaling; we demonstrate that a fusion of LMP1 with CD40 recruits TRAF2 intracellularly. Our results imply that members of the TNF-R family can signal from intracellular compartments containing lipid rafts and may do so when they act in autocrine loops.

Fig. 1. LMP1 co-localizes with internalized cholera toxin. 721 cells, 293 or CNE cells transiently expressing LMP1 were incubated with 2.5 µg/ml FITC-conjugated cholera toxin (green) at 4°C for 30 min. Cells were washed twice with cold medium and either fixed, or shifted to 37°C for 30 min then fixed. The fixed cells were then stained for LMP1 (red). Yellow indicates co-localization of LMP1 and CTxB. At 4°C, most of LMP1 did not co-localize with CTxB in 721 cells (A, the first and second rows). In ∼16% of 721 cells, LMP localized to the cap-like structures which co-localized with surface-bound CTxB (A, third and fourth rows, see text). LMP1 also co-localized with internalized CTxB in 721 cells (B). White arrowheads in (B) indicate co-localization of LMP1 and CTxB at the perinuclear structures after CTxB had moved intracellularly on incubating live cells at 37°C for 30 min. In 293 and CNE cells, co-localization of LMP1 and surface-bound CTxB was rarely detected (C and E). LMP1 at the perinuclear and vesicular-like structures did co-localize with internalized CTxB (D and F).

Fig. 2. The fraction of LMP1 accessible to cleavage in live cells by chymotrypsin differs among cell types. (A) The first extracellular loop of LMP1 can be cleaved by chymotrypsin when LMP1 is at the cell surface. The cleaved product can be separated from intact LMP1 by SDS–PAGE and identified by western blotting using an antibody against the C-terminal cytoplasmic domain of LMP1 (Liebowitz et al., 1986). (B) Live 721 cells were treated with different concentrations of chymotrypsin for different periods of time as indicated and as described in Materials and methods. The chymotrypsin-treated cells were lysed in 1× RIPA. Cell lysates from 5 × 10⁴ cells for each sample were separated by SDS–PAGE and transferred to a nitrocellulose membrane. The LMP1 was detected with a polyclonal rabbit anti-LMP1 antibody followed by ³⁵S-labeled goat anti-rabbit antibodies. The cleaved LMP1 and the uncleaved LMP1 were quantified. The percentage of cleavage was calculated as [cleaved LMP1/(cleaved LMP1 + uncleaved LMP1)] and plotted. Shown is a representative of three independent experiments. (C) Live 721 cells, and 293, CNE and BJAB cells transiently expressing LMP1 were treated with 1000 U/ml chymotrypsin for 10 min at room temperature. The fractions of LMP1 being cleaved were calculated as described in (B).

Fig. 3. Electron microscopy confirms that the majority of LMP1 does not localize at the plasma membrane. LMP1’s subcellular localization was examined in 721 (A) and 293 (B) cells by immunogold electron microscopy as described in Materials and methods. Arrowheads in (A) indicate examples of staining of LMP1 at the plasma membrane and at intracellular compartments. N, nuclear; PM, plasma membrane; scale bar = 1 µm. The fraction of LMP1 at the cell surface was quantified by counting the number of particles from randomly chosen cells (C). The difference between 721 and 293 cells was significant (Wilcoxon one-sided test, P < 0.05). ND, not determined. The silver enhancement of the fine gold particles leads to their dimensions varying. Each deposition was counted once independently of its size.

Fig. 4. LMP1 associates with its signaling molecules intracellularly. 721 cells were stained for LMP1 (red) and TRAF2 (green) (A) and for LMP1 (red) and TRADD (green) (B). Yellow indicates co-localization of red and green. Arrowheads in (A) and (B) indicate co-localization of LMP1 with TRAF2 and TRADD at the perinuclear structures. 293 cells transiently expressing TRAF2–GFP (green) (C), TRAF2–GFP and LMP1 (D), TRAF2–GFP and CATLMP1 (E), and TRAF2–GFP and Sub2CΔ55LMP1 (F) were stained for LMP1 (red) and TRADD (blue). White indicates co-localization of the three colors. Arrowheads in (D) indicate the perinuclear structures where LMP1 recruited TRAF2–GFP and co-localized with TRADD. In cells expressing lower levels of TRAF2–GFP, LMP1 recruited all TRAF2–GFP to its own sites (compare the upper cell in D with those in C). Co-localization of the three proteins was only observed at these perinuclear structures. CATLMP1, which can bind TRAF2–GFP and TRADD but lacks its membrane-spanning domains (E), localizes homogeneously in the cytoplasm but did not affect the distribution of TRAF2–GFP. Sub2CΔ55LMP1 (F), which has a mutated TRAF-binding site and lacks a TRADD-binding site, localized similarly to wild-type LMP1 but did not recruit TRAF2–GFP to the perinuclear structures (arrowhead) and vesicular-like structures. Both CATLMP1 and Sub2CΔ55LMP1 are defective in signaling. (G) The fraction of LMP1 co-precipitated with TRAFs was measured in 721 and 293 cells. Cell lysates were immunoprecipitated with antibodies against TRAF1, 2 or 3 as described in Materials and methods. The immunoprecipitates were separated by SDS–PAGE, transferred to nitrocellulose and detected with antibodies to LMP1. Different amounts of the cell lysate used for each immunoprecipitation were loaded as standard for calculation of the fraction of LMP1 being co-precipitated with TRAFs.

Fig. 5. 3LLMP1 which is not accessible detectably to cleavage by chymotrypsin signals as efficiently as does wild-type LMP1. (A) A diagram shows the leucine to alanine substitutions in 3LLMP1. (B) 293 cells transiently expressing LMP1 or 3LLMP1 were treated with 1000 U/ml chymotrypsin for 10 min at room temperature as described in Materials and methods. LMP1 was analyzed by SDS–PAGE and western blotting. The blot was overdeveloped in order to detect as little as 0.1% of LMP1 being cleaved. The signals therefore are not linear. Activation of NF-κB-mediated transcription (C) and JNK (D) by both wild-ype LMP1 and 3LLMP1 was assayed in 293 cells. For both assays, the basal activities in the absence of LMP1 were set as 1. The fold of induction of activities by LMP1 is shown on the y-axis. (E) 3LLMP1 (red) recruited TRAF2–GFP (green) and co-localized with TRADD (blue) at the perinuclear structures (white arrowhead) in 293 cells. (F) The fraction of 3LLMP1 co-precipitated with TRAFs was measured as described in Figure 4G and was found to be similar to that of wild-type LMP1.

Fig. 6. An LMP1–CD40 chimera recruits TRAF2 to intracellular compartments. 293 cells expressing TRAF2–GFP (green) and LMP1–CD40 (red), which has the N-terminus and the transmembrane domain of LMP1 fused to the cytoplasmic tail of CD40, were fixed and stained with anti-CD40 antibodies. Arrowheads indicate co-localization of TRAF2–GFP and LMP1–CD40 at the perinuclear structures (yellow). TRAF2–GFP was recruited to sites where LMP1–CD40 localized (compare TRAF2-GFP’s localization in this figure with that in Figure 4C).