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. 2008 Jan;6(1):e8.
doi: 10.1371/journal.pbio.0060008.

The viral oncoprotein LMP1 exploits TRADD for signaling by masking its apoptotic activity

Frank Schneider  1 , Julia Neugebauer, Janine Griese, Nicola Liefold, Helmut Kutz, Cinthia Briseño, Arnd Kieser
Affiliations

The viral oncoprotein LMP1 exploits TRADD for signaling by masking its apoptotic activity

Frank Schneider et al. PLoS Biol. 2008 Jan.

Abstract

The tumor necrosis factor (TNF)-receptor 1-associated death domain protein (TRADD) mediates induction of apoptosis as well as activation of NF-kappaB by cellular TNF-receptor 1 (TNFR1). TRADD is also recruited by the latent membrane protein 1 (LMP1) oncoprotein of Epstein-Barr virus, but its role in LMP1 signaling has remained enigmatic. In human B lymphocytes, we have generated, to our knowledge, the first genetic knockout of TRADD to investigate TRADD's role in LMP1 signal transduction. Our data from TRADD-deficient cells demonstrate that TRADD is a critical signaling mediator of LMP1 that is required for LMP1 to recruit and activate I-kappaB kinase beta (IKKbeta). However, in contrast to TNFR1, LMP1-induced TRADD signaling does not induce apoptosis. Searching for the molecular basis for this observation, we characterized the 16 C-terminal amino acids of LMP1 as an autonomous and unique virus-derived TRADD-binding domain. Replacing the death domain of TNFR1 by LMP1's TRADD-binding domain converts TNFR1 into a nonapoptotic receptor that activates NF-kappaB through a TRAF6-dependent pathway, like LMP1 but unlike wild-type TNFR1. Thus, the unique interaction of LMP1 with TRADD encodes the transforming phenotype of viral TRADD signaling and masks TRADD's pro-apoptotic function.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TRADD Gene Targeting in Human DG75 B Lymphocytes
(A) Knockout strategy. Given base positions relate to the transcriptional start site at position +1. The complete TRADD open reading frame (black boxes, exons 2–5 with ATG at position +3218) was deleted by homologous recombination with pTRADDko.1 (first allele) and pTRADDko.2 (second allele). C, CMV promoter; P, promoter; SV, SV40 promoter; TK, thymidine kinase. (B) Southern blot analysis of the resulting DG75 clones with EcoRI-digested chromosomal DNA and the external probe 1. The genealogy of clones is illustrated by arrows. WT, TRADD wild-type allele; KO, targeted and Cre-deleted alleles 1 or 2, respectively. (C) TRADD protein expression. Immunoblot analysis using the mouse anti-TRADD antibody.
Figure 2
Figure 2. TRADD Is Essential for IKKβ Activation by LMP1, but It Is Dispensable for JNK Induction
(A) The knockout of TRADD in DG75 B lymphocytes abolishes TNFR1 activation of the NF-κB pathway. DG75 TRADD+/+ or TRADD–/– cells were stimulated with 200 ng ml−1 soluble human TNFα (Roche) for the indicated times. Levels of phospho-I-κB and I-κB were analyzed on immunoblots. αTubulin served as the loading control. (B) LMP1 activation of IKKβ requires TRADD. DG75 TRADD+/+ or TRADD–/– cells were electroporated with the indicated LMP1 constructs and Flag-IKKβ. Flag-IKKβ activity was monitored in immunocomplex kinase assays. GST-I-κBα phosphorylation was quantified by a phosphoimager and is given as x-fold induction. Immunoprecipitated Flag-IKKβ and the expressed HA-LMP1 constructs were detected using the anti-Flag (M2) and anti-HA (12CA5) antibodies, respectively. LMP1-CD40 was visualized by the anti-CD40 antibody. IB, immunoblot; IP, immunoprecipitation. (C) Ectopic TRADD expression rescues LMP1 activation of IKKβ in TRADD–/– cells. IKKβ assays were performed in DG75 TRADD–/– cells as in (B). Where indicated, TRADD was expressed at physiological levels by co-transfecting 1 μg of pACYC184-1012.4, which carries the complete human TRADD gene including the TRADD promoter. (D) JNK1 activation by LMP1 is independent of TRADD. HA-JNK1 immunocomplex kinase assays in DG75 TRADD+/+ and TRADD–/– cells are shown. Immunoprecipitated HA-JNK1 was dectected with the anti-JNK1 (C17) antibody.
Figure 3
Figure 3. TRADD Mediates the Interaction of IKKβ with CTAR2
(A) DG75 TRADD+/+ or TRADD–/– cells were electroporated with the indicated HA-LMP1 constructs and Flag-IKKβ. Twenty-four h post transfection, the LMP1 signaling complex was immunoprecipitated using the anti-HA (12CA5) antibody, which was covalently coupled to protein A sepharose beads. Precipitated HA-LMP1 proteins were detected by the anti-HA (12CA5) antibody, and IKKβ and TRADD were detected by the anti-IKKβ and mouse anti-TRADD antibodies, respectively. (B) IKKβ interacts with the LMP1 signaling complex in lymphoblastoid cells. LCL 3 cells were generated by transformation of primary human B cells with a recombinant maxi-EBV, in which the wild-type LMP1 gene had been replaced by HA-tagged LMP1. HA-LMP1 was immunoprecipitated from LCL 3 lysates using anti-HA (12CA5) beads. Parallel immunoprecipitations from LCL 721 cells expressing untagged LMP1 served as a negative control. Precipitated proteins were detected by the mouse anti-LMP1 and the rabbit anti-IKKβ antibodies.
Figure 5
Figure 5. LMP1′s TRADD-Binding Domain (LTB) Induces LMP1-Type Signaling but Does Not Activate Apoptosis in the Context of the TNFR1 Signaling Domain
(A) The LTB is functional in the context of the TNFR1 signaling domain. NF-κB reporter assays in HEK293 cells. Data are mean values of three independent experiments ± standard deviation. (B) The transferred LTB determines TRAF6-dependent NF-κB activation. NF-κB reporter assays in TRAF6–/– MEFs are shown. Where indicated (black bars), TRAF6 was co-transfected. Data are mean values of four independent experiments ± standard deviation; statistics: two-tailed Student's t-test. (C) Lack of apoptosis induction by LTB in the context of the TNFR1 signaling domain. Transient cell death assays in human BJAB B lymphocytes are shown. The NF-κB pathway was blocked by co-expression of domaint-negative I-κBα(S32/36A). Where indicated, the cells were incubated in the presence of 2 μM zVAD-fmk (zVAD), a pan-caspase inhibitor. PI, propidium iodide. Data are mean values of three independent experiments ± standard deviation. (D) Apoptosis induction by LMP1-TNFR1 is dependent on TRADD. Cell death assays in DG75 TRADD+/+ and DG75 TRADD–/– cells are shown. Data are mean values of three independent experiments ± standard deviation; statistics: two-tailed Student's t-Test.
Figure 4
Figure 4. Amino Acids 371–386 of LMP1 Encompass the Functional TRADD-Binding Domain
(A) Domain swapping constructs. HA-LMP1-TNFR1 is a chimera of the LMP1 transmembrane domain and the signaling domain of TNFR1. HA-LMP1-TNFR1ΔDD lacks the TNFR1 death domain (DD). HA-LMP1-TNFR1-LTB carries aa 371–386 of LMP1 instead of the TNFR1 death domain. EC, extracellular domain; LTB, LMP1 TRADD-binding domain; TM, transmembrane domain; wt, wild type. (B) Expression in HEK293 cells. Cells were lysed in the presence of 0.1% NP40, and proteins were detected on immunoblots of total cell lysates by the anti-HA (12CA5) antibody. (C) TRADD recruitment into lipid rafts. HEK293 cells were transfected with the indicated constructs together with expression vectors for TRADD and p35. Twenty-four h post transfection, lipid rafts were isolated. Fraction 2 contains lipid rafts (R), as detected on dot blots by the raft marker GM1. The anti-TRADD (H278) and anti-HA (12CA5) antibodies were used to visualize TRADD and HA-tagged constructs on immunoblots, respectively. (D) Replacing the TNFR1 death domain, aa 371–386 of LMP1 are sufficient to recruit TRADD to the TNFR1 signaling domain. HEK293 cells were transfected with the indicated constructs together with expression vectors for TRADD wild type and p35. The HA-tagged constructs (asterisks) were immunoprecipitated via HA and detected by the anti-HA (12CA5) antibody. The mouse anti-TRADD antibody was used to stain TRADD. IP, immunoprecipitation; wt, wild type. (E) Interaction of LTB with TRADD is independent of a functional TRADD death domain. TRADD(296–299A) was co-transfected together with the indicated contructs. TRADD(296–299A) was detected by the anti-TRADD (H278) antibody. n.s., non-specific band.
Figure 6
Figure 6. TNFR1 Is Converted into a Nonapoptotic Receptor upon Replacement of Its Death Domain by the TRADD-Binding Site of LMP1
(A) Schematic depiction of the chimeras. (B) Ectopic expression of the indicated constructs in TNFR1/TNFR2 double-negative MEFs, detected by the anti-TNFR1 (H5) antibody. (C) Both TNFR1 wild type and TNFR1-LTB induce NF-κB. NF-κB reporter assays in TNFR1/TNFR2 double-negative MEFs are shown. Data have been corrected for protein expression levels of TNFR1 constructs and are mean values of three independent experiments ± standard deviation. (D) TNFR1, but not TNFR1-LTB, induces apoptosis. TNFR1/TNFR2 double-negative MEFs were electroporated with the indicated amounts of constructs together with GFP. Five h post transfection, the cells were stained with Annexin V-Cy5 (AnV-Cy5) and analyzed by flow cytometry. AnV-Cy5+/GFP+ staining indicated apoptosis induced by the transfected constructs. In (C) and (D), the cells were incubated with 2 μM zVAD-fmk (zVAD) throughout the experiment where indicated.
Figure 7
Figure 7. Schematic Model of TRADD's Role in Nonapoptotic Signaling Induced by the TRADD-Binding Domain of LMP1
The unique interaction of the 16 C-terminal amino acids of LMP1 (LTB) with TRADD prevents apoptosis induction through TRADD, which is an intrinsic and transferable function of LTB. Additional LMP1 sequences are not required for masking TRADD's apoptotic activity. TRADD mediates the recruitment of IKKβ to the LMP1 signaling complex through a yet unidentified factor. Activation of IKKβ requires a parallel pathway that is dependent on TRAF6. TRAF6 activation of IKKβ most likely involves TAK1 and TAB1 (not depicted). The JNK pathway is TRADD-independent and works through TRAF6 and, probably, BS69. To keep this model concise, only key molecules of CTAR2 signaling are shown. Refer to the text for more details.
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