NF-κB and IRF7 Pathway Activation by Epstein-Barr Virus Latent Membrane Protein 1

The principal Epstein-Barr virus (EBV) oncoprotein, Latent Membrane Protein 1 (LMP1), is expressed in most EBV-associated human malignancies. LMP1 mimics CD40 receptor signaling to provide infected cells with constitutive NF-κB, MAP kinase, IRF7, and PI3 kinase pathway stimulation. EBV-transformed B-cells are particularly dependent on constitutive NF-κB activity, and rapidly undergo apoptosis upon NF-κB blockade. Here, we review LMP1 function, with special attention to current understanding of the molecular mechanisms of LMP1-mediated NF-κB and IRF7 pathway activation. Recent advances include the elucidation of transmembrane motifs important for LMP1 trafficking and ligand-independent signaling, analysis of genome-wide LMP1 gene targets, and the identification of novel cell proteins that mediate LMP1 NF-κB and IRF7 pathway activation

Epstein-Barr virus subverts mevalonate and fatty acid pathways to promote infected B-cell proliferation and survival.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with multiple human malignancies. EBV drives B-cell proliferation, which contributes to the pathogenesis of multiple lymphomas. Yet, knowledge of how EBV subverts host biosynthetic pathways to transform resting lymphocytes into activated lymphoblasts remains incomplete. Using a temporal proteomic dataset of EBV primary human B-cell infection, we identified that cholesterol and fatty acid biosynthetic pathways were amongst the most highly EBV induced. Epstein-Barr nuclear antigen 2 (EBNA2), sterol response element binding protein (SREBP) and MYC each had important roles in cholesterol and fatty acid pathway induction. Unexpectedly, HMG-CoA reductase inhibitor chemical epistasis experiments revealed that mevalonate pathway production of geranylgeranyl pyrophosphate (GGPP), rather than cholesterol, was necessary for EBV-driven B-cell outgrowth, perhaps because EBV upregulated the low-density lipoprotein receptor in newly infected cells for cholesterol uptake. Chemical and CRISPR genetic analyses highlighted downstream GGPP roles in EBV-infected cell small G protein Rab activation. Rab13 was highly EBV-induced in an EBNA3-dependent manner and served as a chaperone critical for latent membrane protein (LMP) 1 and 2A trafficking and target gene activation in newly infected and in lymphoblastoid B-cells. Collectively, these studies identify highlight multiple potential therapeutic targets for prevention of EBV-transformed B-cell growth and survival

Epstein-Barr-Virus-Induced One-Carbon Metabolism Drives B Cell Transformation.

Epstein-Barr virus (EBV) causes Burkitt, Hodgkin, and post-transplant B cell lymphomas. How EBV remodels metabolic pathways to support rapid B cell outgrowth remains largely unknown. To gain insights, primary human B cells were profiled by tandem-mass-tag-based proteomics at rest and at nine time points after infection; >8,000 host and 29 viral proteins were quantified, revealing mitochondrial remodeling and induction of one-carbon (1C) metabolism. EBV-encoded EBNA2 and its target MYC were required for upregulation of the central mitochondrial 1C enzyme MTHFD2, which played key roles in EBV-driven B cell growth and survival. MTHFD2 was critical for maintaining elevated NADPH levels in infected cells, and oxidation of mitochondrial NADPH diminished B cell proliferation. Tracing studies underscored contributions of 1C to nucleotide synthesis, NADPH production, and redox defense. EBV upregulated import and synthesis of serine to augment 1C flux. Our results highlight EBV-induced 1C as a potential therapeutic target and provide a new paradigm for viral onco-metabolism

Differentiation between Polymorphisms and Resistance-Associated Mutations in Human Cytomegalovirus DNA Polymerase▿ †

Specific mutations in the human cytomegalovirus (HCMV) DNA polymerase (pUL54) are known to confer resistance against all currently licensed drugs for treatment of HCMV infection and disease. Following the widespread use of antivirals, the occurrence of HCMV drug resistance is constantly increasing. Recently, diagnostic laboratories have started to replace phenotypic drug resistance testing with genotypic resistance testing. However, the reliability and success of genotypic testing highly depend on the availability of high-quality phenotypic resistance data for each individual mutation and for combinations of mutations, with the latter being increasingly found in patients' HCMV isolates. We performed clonal marker transfer experiments to investigate the impacts of 7 different UL54 point mutations and also of combinations of these mutations on drug susceptibility and viral replicative fitness. We show that several mutations—S695T, A972V, K415R, S291P, and A692V—of suspected but uncertain drug susceptibility phenotype, either alone or in combination, were not relevant to antiviral drug resistance. In contrast, the combination of two mutations individually characterized previously—E756K and D413E—conferred high-grade loss of susceptibility to all three antivirals. Our results have been added to the newly available database of all published HCMV resistance mutations (http://www.informatik.uni-ulm.de/ni/mitarbeiter/HKestler/hcmv/index.html). These data will allow better interpretation of genotypic data and further improve the basis for drug resistance testing

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

The NF-κB Genomic Landscape in Lymphoblastoid B Cells

The nuclear factor κB (NF-κΒ) subunits RelA, RelB, cRel, p50, and p52 are each critical for B cell development and function. To systematically characterize their responses to canonical and noncanonical NF-κB pathway activity, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) analysis in lymphoblastoid B cell lines (LCLs). We found a complex NF-κB-binding landscape, which did not readily reflect the two NF-κB pathway paradigms. Instead, 10 subunit-binding patterns were observed at promoters and 11 at enhancers. Nearly one-third of NF-κB-binding sites lacked κB motifs and were instead enriched for alternative motifs. The oncogenic forkhead box protein FOXM1 co-occupied nearly half of NF-κB-binding sites and was identified in protein complexes with NF-κB on DNA. FOXM1 knockdown decreased NF-κB target gene expression and ultimately induced apoptosis, highlighting FOXM1 as a synthetic lethal target in B cell malignancy. These studies provide a resource for understanding mechanisms that underlie NF-κB nuclear activity and highlight opportunities for selective NF-κB blockade

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

LMP1 and TRAF1 complexes are modified by M1-pUb chains.

<p>A) FLAG-LMP1 complexes were immuno-purified from LCLs that express FLAG-LMP1 from the EBV genome at physiologic levels [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004890#ppat.1004890.ref018" target="_blank">18</a>]. GM12878 LCLs were used as a negative control. M1-linked polyubiquitin chain (M1-pUb) content was analyzed by WB, using a highly chain specific antibody. * indicates antibody heavy chain background. FLAG-LMP1 migrates at a slightly higher than untagged LMP1. B) FLAG complexes were immuno-purified from GM12878 LCLs that stably express either FLAG- GFP or FLAG-TRAF1, and were immuno-blotted, as indicated. C) 293 cells were co-transfected with FLAG- GFP, FLAG-TRAF1, or FLAG-TRAF2, and either empty PSG5-vector control or untagged LMP1 1–386 for 24 hours. Purified FLAG complexes or whole cell lysates were blotted for FLAG or LMP1, as indicated. D) FLAG-LMP1 LCLs were transduced with lentiviruses that express control shGFP or an independent shRNA against HOIP. Following puromycin selection, FLAG LMP1-immuno-purified complexes and lysates were blotted, as indicated. E) Whole cell extracts from 293 TRAF1 cells, uninduced or induced for LMP1 1–231 expression for 16 hours, were blotted for M1-pUb, LMP1, or tubulin. A-E are representative of three independent experiments.</p