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TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation.

Greenfeld H, Takasaki K, Walsh MJ, Ersing I, Bernhardt K, Ma Y, Fu B, Ashbaugh CW, Cabo J, Mollo SB, Zhou H, Li S, Gewurz BE - PLoS Pathog. (2015)

Bottom Line: TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders.We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity.Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
The Epstein-Barr virus (EBV) encoded oncoprotein Latent Membrane Protein 1 (LMP1) signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC), and stimulated linear (M1)-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs) were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63)-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63-linked polyubiquitin chains on LMP1 complexes may facilitate downstream canonical NF-kB pathway activation. Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.

No MeSH data available.


Related in: MedlinePlus

Depletion of HOIP or TRAF1 impairs LCL growth and survival.A) GM12878 LCLs were transduced with lentiviruses that express control shGFP or one of five independent anti-HOIP shRNAs on day 0. Transduced cells were selected with puromcyin on day 2 post-transduction, and then analyzed by quantitative CellTiter-Glo luminescent cell viability assays on the indicated days post transduction. Average and standard deviations from triplicate experiments are shown. WB whole cell lysates obtained four days after transduction are shown below the growth curves. B) GM12878 stable Cas9+ cells were transduced with lentiviruses that express a control anti-GFP sgRNA or an anti-HOIP sgRNA on day 0. Transduced cells were selected by puromcyin on day 2 post-transduction, and then analyzed by CellTiter-glo at the indicated timepoints post-transduction. Western blot of whole cell lysates from Day 6 post-transduction demonstrated HOIP depletion from the cell population. C) GM12878 LCLs were transduced with lentiviruses that express shGFP or one of five independent TRAF1 shRNAs. Transduced cells were selected with puromcyin on day 2, and then analyzed by CellTiter-Glo on the indicated days post-transduction. Average and standard deviations from triplicate experiments are shown. WB of day 4 whole cell lysates are shown below. Student’s one-tailed T-test *P < 0.05, ** P < 0.01, *** P<0.001.
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ppat.1004890.g008: Depletion of HOIP or TRAF1 impairs LCL growth and survival.A) GM12878 LCLs were transduced with lentiviruses that express control shGFP or one of five independent anti-HOIP shRNAs on day 0. Transduced cells were selected with puromcyin on day 2 post-transduction, and then analyzed by quantitative CellTiter-Glo luminescent cell viability assays on the indicated days post transduction. Average and standard deviations from triplicate experiments are shown. WB whole cell lysates obtained four days after transduction are shown below the growth curves. B) GM12878 stable Cas9+ cells were transduced with lentiviruses that express a control anti-GFP sgRNA or an anti-HOIP sgRNA on day 0. Transduced cells were selected by puromcyin on day 2 post-transduction, and then analyzed by CellTiter-glo at the indicated timepoints post-transduction. Western blot of whole cell lysates from Day 6 post-transduction demonstrated HOIP depletion from the cell population. C) GM12878 LCLs were transduced with lentiviruses that express shGFP or one of five independent TRAF1 shRNAs. Transduced cells were selected with puromcyin on day 2, and then analyzed by CellTiter-Glo on the indicated days post-transduction. Average and standard deviations from triplicate experiments are shown. WB of day 4 whole cell lysates are shown below. Student’s one-tailed T-test *P < 0.05, ** P < 0.01, *** P<0.001.

Mentions: Aberrant HOIP activity has recently been implicated in the pathogenesis of the activated B cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) [87,88]. LUBAC inhibition was synthetically lethal to ABC DLBCL, but not the germinal center lymphoma subtype, which have lower NF-kB activity [88]. Given these and our results, we tested the effect of HOIP knockdown on GM12878 LCL growth and survival. Interestingly, by comparison with a non-targeting shGFP control, HOIP depletion by five independent shRNAs significantly impaired LCL growth in biological triplicate assays (Fig 8A). While four anti-HOIP shRNAs yielded very similar effects, a fifth anti-HOIP shRNA (shRNA #3 on the growth curve) had a statistically significant effect in the same direction, but a more modest growth phenotype. This attenuated phenotype may reflect partial rescue by an off-target shRNA effect, or partial rescue by an alternatively spliced HOIP transcript that lacks the shRNA targeting sequence, and which results in a truncated protein not recognized by our anti-HOIP antibody. To further validate the overall shRNA result, we used CRISPR/Cas9 mutagenesis in GM12878 cells that stably express Cas9 to deplete HOIP (Methods). An anti-HOIP exon 1 small guide RNA (sgRNA) knocked down HOIP expression and caused a statistically significant decrease in LCL growth, by comparison with a control anti-GFP sgRNA (Fig 8B). CRISPR/Cas9 edited cells with residual HOIP expression, for example as a result of mono-allelic HOIP disruption, may account for residual LCL growth observed in this experiment. Anti-HOIP sgRNA expression triggered marked induction of caspase 3 and 7 activity, and to a lesser extent, caspase 8 activity (S11 Fig). Western blot analysis of GM12878 whole cell lysates obtained 6 days after transduction with sgRNA-expressing lentiviruses also demonstrated cleaved caspases 3, 7, 9 and cleaved PARP. Overall, these results suggest that HOIP depletion predominantly triggers the intrinsic apoptosis pathway. While TRAF1-independent HOIP roles may also have contributed to this phenotype, it nonetheless suggests an important role for M1-pUb chains in LCL growth and survival, and highlights LUBAC as a potential therapeutic target in EBV-associated lymphoproliferative disorders. We next tested the effect of TRAF1 knockdown in GM12878 LCLs, and found that five independent TRAF1 shRNAs each significantly impaired LCL growth relative to the shRNA control (Fig 8C). We note that all five TRAF1 shRNAs had similar effects on LCL growth, despite variation in the extent of TRAF1 knockdown evident on whole cell extract western blot four days after lentivirus transduction. This result raises the possibility that GM12878 are quite sensitive to TRAF1 depletion, and that even partial TRAF1 depletion impaired cell proliferation. Alternatively, off-target shRNA effects may also have contributed to shRNA effects on LCL growth, in particular for shRNA #3, which depleted TRAF1 to the least extent. TRAF1 levels may also have become more similar across shRNA conditions at subsequent timepoints. Nonetheless, the ability of all five TRAF1 shRNAs to impair LCL proliferation argues against off-target effects being solely responsible for the observed phenotypes. Collectively, our results support an important role for TES1 and TRAF1-dependent M1-pUb chains in the LCL immortalized growth phenotype.


TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation.

Greenfeld H, Takasaki K, Walsh MJ, Ersing I, Bernhardt K, Ma Y, Fu B, Ashbaugh CW, Cabo J, Mollo SB, Zhou H, Li S, Gewurz BE - PLoS Pathog. (2015)

Depletion of HOIP or TRAF1 impairs LCL growth and survival.A) GM12878 LCLs were transduced with lentiviruses that express control shGFP or one of five independent anti-HOIP shRNAs on day 0. Transduced cells were selected with puromcyin on day 2 post-transduction, and then analyzed by quantitative CellTiter-Glo luminescent cell viability assays on the indicated days post transduction. Average and standard deviations from triplicate experiments are shown. WB whole cell lysates obtained four days after transduction are shown below the growth curves. B) GM12878 stable Cas9+ cells were transduced with lentiviruses that express a control anti-GFP sgRNA or an anti-HOIP sgRNA on day 0. Transduced cells were selected by puromcyin on day 2 post-transduction, and then analyzed by CellTiter-glo at the indicated timepoints post-transduction. Western blot of whole cell lysates from Day 6 post-transduction demonstrated HOIP depletion from the cell population. C) GM12878 LCLs were transduced with lentiviruses that express shGFP or one of five independent TRAF1 shRNAs. Transduced cells were selected with puromcyin on day 2, and then analyzed by CellTiter-Glo on the indicated days post-transduction. Average and standard deviations from triplicate experiments are shown. WB of day 4 whole cell lysates are shown below. Student’s one-tailed T-test *P < 0.05, ** P < 0.01, *** P<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440769&req=5

ppat.1004890.g008: Depletion of HOIP or TRAF1 impairs LCL growth and survival.A) GM12878 LCLs were transduced with lentiviruses that express control shGFP or one of five independent anti-HOIP shRNAs on day 0. Transduced cells were selected with puromcyin on day 2 post-transduction, and then analyzed by quantitative CellTiter-Glo luminescent cell viability assays on the indicated days post transduction. Average and standard deviations from triplicate experiments are shown. WB whole cell lysates obtained four days after transduction are shown below the growth curves. B) GM12878 stable Cas9+ cells were transduced with lentiviruses that express a control anti-GFP sgRNA or an anti-HOIP sgRNA on day 0. Transduced cells were selected by puromcyin on day 2 post-transduction, and then analyzed by CellTiter-glo at the indicated timepoints post-transduction. Western blot of whole cell lysates from Day 6 post-transduction demonstrated HOIP depletion from the cell population. C) GM12878 LCLs were transduced with lentiviruses that express shGFP or one of five independent TRAF1 shRNAs. Transduced cells were selected with puromcyin on day 2, and then analyzed by CellTiter-Glo on the indicated days post-transduction. Average and standard deviations from triplicate experiments are shown. WB of day 4 whole cell lysates are shown below. Student’s one-tailed T-test *P < 0.05, ** P < 0.01, *** P<0.001.
Mentions: Aberrant HOIP activity has recently been implicated in the pathogenesis of the activated B cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) [87,88]. LUBAC inhibition was synthetically lethal to ABC DLBCL, but not the germinal center lymphoma subtype, which have lower NF-kB activity [88]. Given these and our results, we tested the effect of HOIP knockdown on GM12878 LCL growth and survival. Interestingly, by comparison with a non-targeting shGFP control, HOIP depletion by five independent shRNAs significantly impaired LCL growth in biological triplicate assays (Fig 8A). While four anti-HOIP shRNAs yielded very similar effects, a fifth anti-HOIP shRNA (shRNA #3 on the growth curve) had a statistically significant effect in the same direction, but a more modest growth phenotype. This attenuated phenotype may reflect partial rescue by an off-target shRNA effect, or partial rescue by an alternatively spliced HOIP transcript that lacks the shRNA targeting sequence, and which results in a truncated protein not recognized by our anti-HOIP antibody. To further validate the overall shRNA result, we used CRISPR/Cas9 mutagenesis in GM12878 cells that stably express Cas9 to deplete HOIP (Methods). An anti-HOIP exon 1 small guide RNA (sgRNA) knocked down HOIP expression and caused a statistically significant decrease in LCL growth, by comparison with a control anti-GFP sgRNA (Fig 8B). CRISPR/Cas9 edited cells with residual HOIP expression, for example as a result of mono-allelic HOIP disruption, may account for residual LCL growth observed in this experiment. Anti-HOIP sgRNA expression triggered marked induction of caspase 3 and 7 activity, and to a lesser extent, caspase 8 activity (S11 Fig). Western blot analysis of GM12878 whole cell lysates obtained 6 days after transduction with sgRNA-expressing lentiviruses also demonstrated cleaved caspases 3, 7, 9 and cleaved PARP. Overall, these results suggest that HOIP depletion predominantly triggers the intrinsic apoptosis pathway. While TRAF1-independent HOIP roles may also have contributed to this phenotype, it nonetheless suggests an important role for M1-pUb chains in LCL growth and survival, and highlights LUBAC as a potential therapeutic target in EBV-associated lymphoproliferative disorders. We next tested the effect of TRAF1 knockdown in GM12878 LCLs, and found that five independent TRAF1 shRNAs each significantly impaired LCL growth relative to the shRNA control (Fig 8C). We note that all five TRAF1 shRNAs had similar effects on LCL growth, despite variation in the extent of TRAF1 knockdown evident on whole cell extract western blot four days after lentivirus transduction. This result raises the possibility that GM12878 are quite sensitive to TRAF1 depletion, and that even partial TRAF1 depletion impaired cell proliferation. Alternatively, off-target shRNA effects may also have contributed to shRNA effects on LCL growth, in particular for shRNA #3, which depleted TRAF1 to the least extent. TRAF1 levels may also have become more similar across shRNA conditions at subsequent timepoints. Nonetheless, the ability of all five TRAF1 shRNAs to impair LCL proliferation argues against off-target effects being solely responsible for the observed phenotypes. Collectively, our results support an important role for TES1 and TRAF1-dependent M1-pUb chains in the LCL immortalized growth phenotype.

Bottom Line: TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders.We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity.Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
The Epstein-Barr virus (EBV) encoded oncoprotein Latent Membrane Protein 1 (LMP1) signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC), and stimulated linear (M1)-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs) were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63)-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63-linked polyubiquitin chains on LMP1 complexes may facilitate downstream canonical NF-kB pathway activation. Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.

No MeSH data available.


Related in: MedlinePlus