Antibody concentrations of three EBV proteins in Guangzhou and Beijing.

<p>(A) Average concentrations of VCA-IgG were measured in all the samples. (B) Average concentrations of EBNA-IgG were measured in all the samples. (C) Average concentrations of EA-IgG were measured in all the samples.</p

EBV-specific antibody seropositivity status by ELISA.

<p>EBV-specific antibody seropositivity status by ELISA.</p

EBV seropositivity rates in Guangzhou and Beijing.

<p>(A) Four EBV antibodies were screened in 805 serum samples from Guangzhou. (B) Four EBV antibodies were screened in 973 serum samples from Beijing.</p

Demographic information for the samples in the study.

<p>Demographic information for the samples in the study.</p

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

<div><p>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.</p></div

Analysis of LMP1 and TRAF1 domains important for M1-pUb attachment.

<p>A) 293 cells were co-transfected with FLAG-TRAF1, and with either wildtype LMP1 (WT), an LMP1 mutant that signal only from the TES2 domain (TES2), an LMP1 mutant that signals only from the TES1 domain (TES1), or an LMP1 double mutant (DM) that does not signal from either TES1 or TES2. Immuno-purified FLAG-TRAF1 complexes and whole cell lysates were blotted, as indicated. B) 293 cells were transfected with FLAG-tagged WT LMP1, LMP1 1–231, or LMP1 DM and untagged TRAF1. Purified FLAG complexes or lysates were blotted as indicated. C) 293 cells were co-transfected with untagged LMP1 and FLAG-tagged GFP, TRAF1 1–416, TRAF1 183–416, or TRAF1 264–416. FLAG complexes or lysates were blotted, as indicated. A-C are representative of triplicate experiments.</p

TRAF2, HOIP, HOIL-1L, and SHARPIN, but not cIAP1/2, are important for LMP1 1-231-induced M1-pUb chain attachment to TRAF1 complexes.

<p>A) 72 hours after siRNA transfection of 293 TRAF1 cells, LMP1 1–231 expression was induced for 16 hours. FLAG-TRAF1 complexes and lysates were WB, as indicated. B) 293 TRAF1 cells were treated with a SMAC mimetic peptide to deplete cells of cIAP ligases, and were then induced for LMP1 1–231 expression in the presence of the SMAC mimetic, as indicated. FLAG-TRAF1 IPs and lysates were blotted as indicated. A-B are representative of three independent experiments.</p

LMP1 1–231 expression induces K63-pUb chain attachment to TRAF2.

<p>293 cells were co-transfected with FLAG-tagged GFP, TRAF1 (T1), TRAF2 (T2), TRAF3 (T3), or LMP1 constructs, HA-LMP1, and untagged TRAF1 for 24 hours, as indicated. 1% SDS was added to whole cell lysates, and samples were boiled for 5 minutes to denature complexes. SDS was diluted to 0.1%, and anti-FLAG IP was performed. Western blots were performed, as indicated. A-D are representative of three independent experiments.</p

Depletion of HOIP or TRAF1 impairs LCL growth and survival.

<p>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.</p

LMP1 is a target of M1-pUb chain attachment.

<p>A) M1-pUb chains, immunopurified from control EBV-negative Burkitt lymphoma BL2 cells or from GM12878 LCLs under denaturing conditions, and whole cell lysate were blotted for LMP1. B) M1-pUb chains were immune-purified under denaturing conditions from 293 TRAF1 cells, uninduced or induced for LMP1 1–231 expression for 16 hours. LMP1 1–231 is untagged and has no lysine residues. M1-pUb IPs or whole cell lysate was WB for LMP1 or M1-pUb, as indicated. C) 293 TRAF1 cells were transfected with control or anti-HOIP siRNAs, and 72 hours later, LMP1 1–231 expression was induced for 16 hours. M1-pUb IPs were blotted for LMP1 or total poly-Ub, and lysates were blotted as indicated. A-C are representative of three independent experiments.</p