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

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.

SUBMITTER: Schneider F

PROVIDER: S-EPMC2174972 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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

Schneider Frank F Neugebauer Julia J Griese Janine J Liefold Nicola N Kutz Helmut H Briseño Cinthia C Kieser Arnd A

PLoS biology 20080101 1

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 da ...[more]

PMID: 18198944

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Project description:The Epstein-Barr virus (EBV)-encoded oncoprotein latent membrane protein 1 (LMP1) constitutively activates nuclear factor κB (NFκB) from intracellular membranes to promote cell growth and survival. LMP1 associates with CD63 in intracellular membranes and is released via exosomes. Whether tumour necrosis factor (TNF) receptor-associated factors (TRAFs) mediate LMP1 NFκB signalling from endosomes and modulate exosomal sorting is unknown. In this article, we show that LMP1-TRAF2 signalling complexes accumulate at endosomes in a palmitoylation-dependent manner, thereby driving LMP1-dependent oncogenicity. Palmitoylation is a reversible post-translational modification and is considered to function as a membrane anchor for proteins. Mutagenesis studies showed that LMP1-TRAF2 trafficking to endosomes is dependent on one single cysteine residue (C78), a known palmitoylation site of LMP1. Notably, growth assays in soft agar revealed that oncogenic properties of the palmitoylation-deficient LMP1 mutant C78A were diminished compared to wild-type LMP1. Since LMP1 recruitment of TRAF2 and downstream NFκB signalling were not affected by a disturbance in palmitoylation, the specific localization of LMP1 at endosomal membranes appears crucial for its transforming potential. The importance of palmitoylation for trafficking to and signalling from endosomal membranes was not restricted to LMP1, as similar observations were made for the cellular oncoproteins Src and Fyn. Despite abundant LMP1-TRAF2 association at endosomal membranes TRAF2 could not be detected in exosomes by Western blotting or proteomics. Interestingly, point mutations that prevented TRAF binding strongly promoted the sorting and release of LMP1 via exosomes. These observations reveal that LMP1-TRAF2 complexes at endosomes support oncogenic NFκB activation and suggest that LMP1 dissociates from the activated signalling complexes upon sorting into intraluminal vesicles. We propose that "signalling endosomes" in EBV-infected tumour cells can fuse with the plasma membrane, explaining LMP1 release via exosomes.

Project description:EBV infects normal oral keratinocytes (NOKs) and plays an essential role in undifferentiated nasopharyngeal carcinoma (NPC). We previously showed that the EBV oncogene, LMP1, promotes proliferation and inhibits spontaneous differentiation in telomerase-immortalized NOKs grown in growth factor-restricted conditions. Here we have further examined the phenotypes of NOKs infected with wild-type EBV (WT EBV) versus an LMP1-deleted EBV mutant (ΔLMP1 EBV) in growth factor-restricted conditions. RNA-seq results show that WT EBV-infected NOKs not only have reduced differentiation, but also decreased expression of genes activated by the integrated stress response (ISR) pathway, in comparison to the ΔLMP1 EBV-infected cells. The ISR pathway is mediated by increased phosphorylation of the eIF2α translation initiation factor, leading to decreased translation of most cellular proteins but increased expression of some proteins, including ATF4 and CHOP. Immunoblot analyses confirmed that WT EBV-infected NOKs have decreased phosphorylation of eIF2α in comparison to uninfected and ΔLMP1 EBV-infected cells and showed that expression of LMP1 alone is sufficient to inhibit eIF2α phosphorylation. We found that LMP1 decreases the activity of two different eIF2α kinases, PERK and GCN2, in WT EBV-infected NOKs, resulting in decreased expression of the ISR-induced transcription factors, ATF4 and CHOP, in WT EBV-infected versus uninfected and ΔLMP1 EBV-infected NOKs. Furthermore, we found that both GCN2 and PERK activity are required for efficient TPA-induced lytic EBV reactivation and TPA-mediated epithelial cell differentiation. In addition, we demonstrate that over-expression of CHOP is sufficient to induce both lytic EBV reactivation and epithelial cell differentiation in WT EBV-infected NOKs and NPC cells and show that this effect is mediated by CHOP activation of the differentiation-inducing transcription factors, KLF4 and BLIMP1. Our results suggest that inhibition of the ISR pathway by the EBV oncoprotein, LMP1, may promote early NPC development by preventing epithelial cell differentiation and lytic EBV reactivation.

Project description:UnlabelledThe latent infection of Epstein-Barr virus (EBV) is associated with 1% of human cancer incidence. Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) that mediate EBV replication during latency. In this study, we detail the mechanisms that drive cellular PARylation during latent EBV infection and the effects of PARylation on host gene expression and cellular function. EBV-infected B cells had higher PAR levels than EBV-negative B cells. Moreover, cellular PAR levels were up to 2-fold greater in type III than type I latently infected EBV B cells. We identified a positive association between expression of the EBV genome-encoded latency membrane protein 1 (LMP1) and PAR levels that was dependent upon PARP1. PARP1 regulates gene expression by numerous mechanisms, including modifying chromatin structure and altering the function of chromatin-modifying enzymes. Since LMP1 is essential in establishing EBV latency and promoting tumorigenesis, we explored the model that disruption in cellular PARylation, driven by LMP1 expression, subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP1 inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes. Inhibition of PARP1, or abrogation of PARP1 expression, also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP1 activity in LMP1-induced gene expression and cellular transformation associated with LMP1. In summary, we identified a novel mechanism by which LMP1 drives expression of host tumor-promoting genes by blocking generation of the inhibitory histone modification H3K27me3 through PARP1 activation.ImportanceEBV is causally linked to several malignancies and is responsible for 1% of cancer incidence worldwide. The EBV-encoded protein LMP1 is essential for promoting viral tumorigenesis by aberrant activation of several well-known intracellular signaling pathways. We have identified and defined an additional novel molecular mechanism by which LMP1 regulates the expression of tumor-promoting host genes. We found that LMP1 activates the cellular protein PARP1, leading to a decrease in a repressive histone modification, accompanied by induction in expression of multiple cancer-related genes. PARP1 inhibition or depletion led to a decrease in LMP1-induced cellular transformation. Therefore, targeting PARP1 activity may be an effective treatment for EBV-associated malignancies.

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

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

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