Note: *, co-first authors; **, co-correspondence authors.
- Abstract BibTeX EndNote URLWang, L. W., Wang, Z., Ersing, I., Nobre, L., Guo, R., Jiang, S., Trudeau, S., Zhao, B., Weekes, M. P. & Gewurz, B. E. Epstein-Barr virus subverts mevalonate and fatty acid pathways to promote infected B-cell proliferation and survival. PLOS Pathogens 15, 1-35 (2019).Author summary EBV, the first human tumor virus identified, persistently infects >95% of adults worldwide. Upon infection of small, resting B-lymphocytes, EBV establishes a state of viral latency, where viral oncoproteins and non-coding RNAs activate host pathways to promote rapid B-cell proliferation. EBV’s growth-transforming properties are closely linked to the pathogenesis of multiple immunoblastic lymphomas, particularly in immunosuppressed hosts. While EBV oncogenes important for B-cell transformation have been identified, knowledge remains incomplete of how these EBV factors remodel cellular metabolism, a hallmark of human cancers. Using a recently established proteomic map of EBV-mediated B-cell growth transformation, we found that EBV induces biosynthetic pathways that convert acetyl-coenzyme A (acetyl-CoA) into isoprenoids, steroids, terpenoids, cholesterol, and long-chain fatty acids. Viral nuclear antigens cooperated with EBV-activated host transcription factors to upregulate rate-limiting enzymes of these biosynthetic pathways. The isoprenoid geranylgeranyl pyrophosphate was identified as a key product of the EBV-induced mevalonate pathway. Our studies highlighted GGPP roles in Rab protein activation, and Rab13 was identified as a highly EBV-upregulated GTPase critical for LMP1 and LMP2A trafficking and signaling. These studies identify multiple EBV-induced metabolic enzymes important for B-cell transformation, including potential therapeutic targets.
- BibTeX EndNote URLWang, L. W., Shen, H., Nobre, L., Ersing, I., Paulo, J. A., Trudeau, S., Wang, Z., Smith, N. A., Ma, Y., Reinstadler, B., Nomburg, J., Sommermann, T., Cahir-McFarland, E., Gygi, S. P., Mootha, V. K., Weekes, M. P. & Gewurz, B. E. Epstein-Barr-Virus-Induced One-Carbon Metabolism Drives B Cell Transformation. Cell Metabolism (2019). doi:10.1016/j.cmet.2019.06.003
- BibTeX EndNote URLJiang, C., Trudeau, S. J., Cheong, T. -C., Guo, R., Teng, M., Wang, L. W., Wang, Z., Pighi, C., Gautier-Courteille, C., Ma, Y., Jiang, S., Wang, C., Zhao, B., Paillard, L., Doench, J. G., Chiarle, R. & Gewurz, B. E. CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation. Cell Reports 28, 1307--1322.e8 (2019).
- Abstract BibTeX EndNote URLGebre, M., Nomburg, J. L. & Gewurz, B. E. CRISPR–Cas9 Genetic Analysis of Virus–Host Interactions. Viruses 10, (2018).Clustered regularly interspaced short palindromic repeats (CRISPR) has greatly expanded the ability to genetically probe virus–host interactions. CRISPR systems enable focused or systematic, genomewide studies of nearly all aspects of a virus lifecycle. Combined with its relative ease of use and high reproducibility, CRISPR is becoming an essential tool in studies of the host factors important for viral pathogenesis. Here, we review the use of CRISPR–Cas9 for the loss-of-function analysis of host dependency factors. We focus on the use of CRISPR-pooled screens for the systematic identification of host dependency factors, particularly in Epstein–Barr virus-transformed B cells. We also discuss the use of CRISPR interference (CRISPRi) and gain-of-function CRISPR activation (CRISPRa) approaches to probe virus–host interactions. Finally, we comment on the future directions enabled by combinatorial CRISPR screens.
- Abstract BibTeX EndNote URLZhang, H., Li, Y., Wang, H. -B., Zhang, A., Chen, M. -L., Fang, Z. -X., Dong, X. -D., Li, S. -B., Du, Y., Xiong, D., He, J. -Y., Li, M. -Z., Liu, Y. -M., Zhou, A. -J., Zhong, Q., Zeng, Y. -X., Kieff, E., Zhang, Z., Gewurz, B. E., Zhao, B. & Zeng, M. -S. Ephrin receptor A2 is an epithelial cell receptor for Epstein–Barr virus entry. Nature Microbiology 3, 164--171 (2018).Epstein–Barr virus (EBV) is causally associated with nasopharyngeal carcinoma, 10% of gastric carcinoma and various B cell lymphomas 1 . EBV infects both B cells and epithelial cells 2 . Recently, we reported that epidermal growth factor and Neuropilin 1 markedly enhanced EBV entry into nasopharyngeal epithelial cells 3 . However, knowledge of how EBV infects epithelial cells remains incomplete. To understand the mechanisms through which EBV infects epithelial cells, we integrated microarray and RNA interference screen analyses and found that Ephrin receptor A2 (EphA2) is important for EBV entry into the epithelial cells. EphA2 short interfering RNA knockdown or CRISPR–Cas9 knockout markedly reduced EBV epithelial cell infection, which was mostly restored by EphA2 complementary DNA rescue. EphA2 overexpression increased epithelial cell EBV infection. Soluble EphA2 protein, antibodies against EphA2, soluble EphA2 ligand EphrinA1, or the EphA2 inhibitor 2,5-dimethylpyrrolyl benzoic acid efficiently blocked EBV epithelial cell infection. Mechanistically, EphA2 interacted with EBV entry proteins gH/gL and gB to facilitate EBV internalization and fusion. The EphA2 Ephrin-binding domain and fibronectin type III repeats domain were essential for EphA2-mediated EBV infection, while the intracellular domain was dispensable. This is distinct from Kaposi’s sarcoma-associated herpesvirus infection through EphA2 4 . Taken together, our results identify EphA2 as a critical player for EBV epithelial cell entry.
- Abstract BibTeX EndNote URLWang, L. W., Jiang, S. & Gewurz, B. E. Epstein-Barr Virus LMP1-Mediated Oncogenicity. Journal of Virology 91, (2017).Epstein-Barr virus latent membrane protein 1 (LMP1) is expressed in multiple human malignancies, including nasopharyngeal carcinoma and Hodgkin and immunosuppression-associated lymphomas. LMP1 mimics CD40 signaling to activate multiple growth and survival pathways, in particular, NF-κB. LMP1 has critical roles in Epstein-Barr virus (EBV)-driven B-cell transformation, and its expression causes fatal lymphoproliferative disease in immunosuppressed mice. Here, we review recent developments in studies of LMP1 signaling, LMP1-induced host dependency factors, mouse models of LMP1 lymphomagenesis, and anti-LMP1 immunotherapy approaches.
- Abstract BibTeX EndNote Download URLErsing, I. *, Nobre, L. *, Wang, L. W., Soday, L., Ma, Y., Paulo, J. A., Narita, Y., Ashbaugh, C. W., Jiang, C., Grayson, N. E., Kieff, E., Gygi, S. P., Weekes, M. P. ** & Gewurz, B. E. **. A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells. Cell Reports 19, 1479--1493 (2017).Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.
- Abstract BibTeX EndNote URLMa, Y. *, Walsh, M. J. *, Bernhardt, K., Ashbaugh, C. W., Trudeau, S. J., Ashbaugh, I. Y., Jiang, S., Jiang, C., Zhao, B., Root, D. E., Doench, J. G. & Gewurz, B. E. CRISPR/Cas9 Screens Reveal Epstein-Barr Virus-Transformed B Cell Host Dependency Factors. Cell Host & Microbe 21, 580--591.e7 (2017).Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma (BL) and immunosuppression-related lymphomas. These B cell malignancies arise by distinct transformation pathways and have divergent viral and host expression programs. To identify host dependency factors resulting from these EBV+, B cell-transformed cell states, we performed parallel genome-wide CRISPR/Cas9 loss-of-function screens in BL and lymphoblastoid cell lines (LCLs). These highlighted 57 BL and 87 LCL genes uniquely important for their growth and survival. LCL hits were enriched for EBV-induced genes, including viral super-enhancer targets. Our systematic approach uncovered key mechanisms by which EBV oncoproteins activate the PI3K/AKT pathway and evade tumor suppressor responses. LMP1-induced cFLIP was found to be critical for LCL defense against TNFα-mediated programmed cell death, whereas EBV-induced BATF/IRF4 were critical for BIM suppression and MYC induction in LCLs. Finally, EBV super-enhancer-targeted IRF2 protected LCLs against Blimp1-mediated tumor suppression. Our results identify viral transformation-driven synthetic lethal targets for therapeutic intervention.
- Abstract BibTeX EndNote Download URLMinamitani, T. *, Ma, Y. *, Zhou, H., Kida, H., Tsai, C. -Y., Obana, M., Okuzaki, D., Fujio, Y., Kumanogoh, A., Zhao, B., Kikutani, H., Kieff, E. **, Gewurz, B. E. ** & Yasui, T. **. Mouse model of Epstein–Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease. Proceedings of the National Academy of Sciences 114, 4751-4756 (2017).Epstein–Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.
- Abstract BibTeX EndNote Download URLHunter, J. E., Butterworth, J. A., Zhao, B., Sellier, H., Campbell, K. J., Thomas, H. D., Bacon, C. M., Cockell, S. J., Gewurz, B. E. & Perkins, N. D. The NF-kappaB subunit c-Rel regulates Bach2 tumour suppressor expression in B-cell lymphoma. Oncogene 35, 3476-3484 (2016).The REL gene, encoding the NF-kappaB subunit c-Rel, is frequently amplified in B-cell lymphoma and functions as a tumour-promoting transcription factor. Here we report the surprising result that c-rel-/- mice display significantly earlier lymphomagenesis in the c-Myc driven, Emu-Myc model of B-cell lymphoma. c-Rel loss also led to earlier onset of disease in a separate TCL1-Tg-driven lymphoma model. Tumour reimplantation experiments indicated that this is an effect intrinsic to the Emu-Myc lymphoma cells but, counterintuitively, c-rel-/- Emu-Myc lymphoma cells were more sensitive to apoptotic stimuli. To learn more about why loss of c-Rel led to earlier onset of disease, microarray gene expression analysis was performed on B cells from 4-week-old, wild-type and c-rel-/- Emu-Myc mice. Extensive changes in gene expression were not seen at this age, but among those transcripts significantly downregulated by the loss of c-Rel was the B-cell tumour suppressor BTB and CNC homology 2 (Bach2). Quantitative PCR and western blot analysis confirmed loss of Bach2 in c-Rel mutant Emu-Myc tumours at both 4 weeks and the terminal stages of disease. Moreover, Bach2 expression was also downregulated in c-rel-/- TCL1-Tg mice and RelA Thr505Ala mutant Emu-Myc mice. Analysis of wild-type Emu-Myc mice demonstrated that the population expressing low levels of Bach2 exhibited the earlier onset of lymphoma seen in c-rel-/- mice. Confirming the relevance of these findings to human disease, analysis of chromatin immunoprecipitation sequencing data revealed that Bach2 is a c-Rel and NF-kappaB target gene in transformed human B cells, whereas treatment of Burkitt's lymphoma cells with inhibitors of the NF-kappaB/IkappaB kinase pathway or deletion of c-Rel or RelA resulted in loss of Bach2 expression. These data reveal a surprising tumour suppressor role for c-Rel in lymphoma development explained by regulation of Bach2 expression, underlining the context-dependent complexity of NF-kappaB signalling in cancer.
- Abstract BibTeX EndNote Download URLBlondel, C. J., Park, J. S., Hubbard, T. P., Pacheco, A. R., Kuehl, C. J., Walsh, M. J., Davis, B. M., Gewurz, B. E., Doench, J. G. & Waldor, M. K. CRISPR/Cas9 Screens Reveal Requirements for Host Cell Sulfation and Fucosylation in Bacterial Type \III\ Secretion System-Mediated Cytotoxicity. Cell Host & Microbe 20, 226 - 237 (2016).Summary Type \III\ secretion systems (T3SSs) inject bacterial effector proteins into host cells and underlie the virulence of many gram-negative pathogens. Studies have illuminated bacterial factors required for T3SS function, but the required host processes remain largely undefined. We coupled CRISPR/Cas9 genome editing technology with the cytotoxicity of two Vibrio parahaemolyticus \T3SSs\ (T3SS1 and T3SS2) to identify human genome disruptions conferring resistance to T3SS-dependent cytotoxicity. We identity non-overlapping genes required for T3SS1- and T3SS2-mediated cytotoxicity. Genetic ablation of cell surface sulfation reduces bacterial adhesion and thereby alters the kinetics of T3SS1-mediated cytotoxicity. Cell surface fucosylation is required for T3SS2-dependent killing, and genetic inhibition of fucosylation prevents membrane insertion of the T3SS2 translocon complex. These findings reveal the importance of ubiquitous surface modifications for \T3SS\ function, potentially explaining the broad tropism of V. parahaemolyticus, and highlight the utility of genome-wide CRISPR/Cas9 screens to discover processes underlying host-pathogen interactions.
- Abstract BibTeX EndNote Download URLMinamitani, T., Yasui, T., Ma, Y., Zhou, H., Okuzaki, D., Tsai, C. -Y., Sakakibara, S., Gewurz, B. E., Kieff, E. & Kikutani, H. Evasion of affinity-based selection in germinal centers by Epstein–Barr virus LMP2A. Proceedings of the National Academy of Sciences 112, 11612-11617 (2015).Epstein–Barr virus (EBV) infects germinal center (GC) B cells and establishes persistent infection in memory B cells. EBV-infected B cells can cause B-cell malignancies in humans with T- or natural killer-cell deficiency. We now find that EBV-encoded latent membrane protein 2A (LMP2A) mimics B-cell antigen receptor (BCR) signaling in murine GC B cells, causing altered humoral immune responses and autoimmune diseases. Investigation of the impact of LMP2A on B-cell differentiation in mice that conditionally express LMP2A in GC B cells or all B-lineage cells found LMP2A expression enhanced not only BCR signals but also plasma cell differentiation in vitro and in vivo. Conditional LMP2A expression in GC B cells resulted in preferential selection of low-affinity antibody-producing B cells despite apparently normal GC formation. GC B-cell–specific LMP2A expression led to systemic lupus erythematosus-like autoimmune phenotypes in an age-dependent manner. Epigenetic profiling of LMP2A B cells found increased H3K27ac and H3K4me1 signals at the zinc finger and bric-a-brac, tramtrack domain-containing protein 20 locus. We conclude that LMP2A reduces the stringency of GC B-cell selection and may contribute to persistent EBV infection and pathogenesis by providing GC B cells with excessive prosurvival effects.
- Abstract BibTeX EndNote Download URLZhou, H., Schmidt, S. C. S., Jiang, S., Willox, B., Bernhardt, K., Liang, J., Johannsen, E. C., Kharchenko, P., Gewurz, B. E., Kieff, E. & Zhao, B. Epstein-Barr Virus Oncoprotein Super-enhancers Control B Cell Growth. Cell Host & Microbe 17, 205 - 216 (2015).Summary Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), we identified four \EBV\ oncoproteins and five EBV-activated NF-κB subunits co-occupying ∼1,800 enhancer sites. Of these, 187 had markedly higher and broader histone \H3K27ac\ signals, characteristic of super-enhancers, and were designated “EBV super-enhancers.” \EBV\ super-enhancer-associated genes included the \MYC\ and \BCL2\ oncogenes, which enable ŁCL\ proliferation and survival. \EBV\ super-enhancers were enriched for B cell transcription factor motifs and had high co-occupancy of \STAT5\ and \NFAT\ transcription factors (TFs). \EBV\ super-enhancer-associated genes were more highly expressed than other ŁCL\ genes. Disrupting \EBV\ super-enhancers by the bromodomain inhibitor \JQ1\ or conditionally inactivating an \EBV\ oncoprotein or NF-κB decreased \MYC\ or \BCL2\ expression and arrested ŁCL\ growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions.
- Abstract BibTeX EndNote Download URLGreenfeld, H., Takasaki, K., Walsh, M. J., Ersing, I., Bernhardt, K., Ma, Y., Fu, B., Ashbaugh, C. W., Cabo, J., Mollo, S. B., Zhou, H., Li, S. & Gewurz, B. E. TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation. PLOS Pathogens 11, 1-35 (2015).Author Summary The linear ubiquitin assembly complex (LUBAC) plays crucial roles in immune receptor-mediated NF-kB and MAP kinase pathway activation. Comparatively little is known about the extent to which microbial pathogens use LUBAC to activate downstream pathways. We demonstrate that TRAF1 enhances EBV oncoprotein LMP1 TES1/CTAR1 domain mediated MAP kinase and canonical NF-kB activation. LMP1 TES1 signaling induces association between TRAF1 and LUBAC, and triggers M1-polyubiquitin chain attachment to TRAF1 complexes. TRAF1 and LMP1 complexes are decorated by M1-polyubiquitin chains in LCL extracts. TRAF2 plays a key role in LMP1-induced LUBAC recruitment and M1-chain attachment to TRAF1 complexes. TRAF1 and LMP1 complexes are modified by lysine 63-linked polyubiquitin chains in LCL extracts, and TRAF2 is a target of LMP1-induced K63-ubiquitin chain attachment. Thus, the TRAF1:TRAF2 heterotrimer may coordinate ubiquitin signaling downstream of TES1. Depletion of TRAF1 or the LUBAC subunit HOIP impairs LCL growth and survival. Thus, although TRAF1 is the only TRAF without a RING finger ubiquitin ligase domain, TRAF1 nonetheless has important roles in ubiqutin-mediated signal transduction downstream of LMP1. Our work suggests that LUBAC is important for EBV-driven B-cell proliferation, and suggests that LUBAC may be a novel therapeutic target in EBV-associated lymphoproliferative disorders.
- Abstract BibTeX EndNote Download URLZhao, B., Barrera, L. A., Ersing, I., Willox, B., Schmidt, S. C. S., Greenfeld, H., Zhou, H., Mollo, S. B., Shi, T. T., Takasaki, K., Jiang, S., Cahir-McFarland, E., Kellis, M., Bulyk, M. L., Kieff, E. & Gewurz, B. E. The NF-κB Genomic Landscape in Lymphoblastoid B Cells. Cell Reports 8, 1595 - 1606 (2014).Summary 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.
- Abstract BibTeX EndNote Download URLZhou, X., Massol, R. H., Nakamura, F., Chen, X., Gewurz, B. E., Davis, B. M., Lencer, W. I. & Waldor, M. K. Remodeling of the Intestinal Brush Border Underlies Adhesion and Virulence of an Enteric Pathogen. mBio 5, (2014).Intestinal colonization by Vibrio parahaemolyticus—the most common cause of seafood-borne bacterial enteritis worldwide—induces extensive disruption of intestinal microvilli. In orogastrically infected infant rabbits, reorganization of the apical brush border membrane includes effacement of some microvilli and marked elongation of others. All diarrhea, inflammation, and intestinal pathology associated with V. parahaemolyticus infection are dependent upon one of its type 3 secretion systems (T3SS2); however, translocated effectors that directly mediate brush border restructuring and bacterial adhesion are not known. Here, we demonstrate that the effector VopV is essential for V. parahaemolyticus intestinal colonization and therefore its pathogenicity, that it induces effacement of brush border microvilli, and that this effacement is required for adhesion of V. parahaemolyticus to enterocytes. VopV contains multiple functionally independent and mechanistically distinct domains through which it disrupts microvilli. We show that interaction between VopV and filamin, as well as VopV’s previously noted interaction with actin, mediates enterocyte cytoskeletal reorganization. VopV’s multipronged approach to epithelial restructuring, coupled with its impact on colonization, suggests that remodeling of the epithelial brush border is a critical step in pathogenesis.IMPORTANCE Colonization of the small bowel by Vibrio parahaemolyticus, the most common bacterial agent of seafood-borne enteric disease, induces extensive structural changes in the intestinal epithelium. Here, we show that this diarrheal pathogen’s colonization and virulence depend upon VopV, a bacterial protein that is transferred into host epithelial cells. VopV induces marked rearrangement of the apical epithelial cell membrane, including elimination of microvilli, by two means: through interaction with actin and through a previously unrecognized interaction with the actin-cross-linking protein filamin. VopV-mediated “effacement” of microvilli enables V. parahaemolyticus to adhere to host cells, although VopV may not directly mediate adhesion. VopV’s effects on microvillus structure and bacterial adhesion likely account for its essential role in V. parahaemolyticus intestinal pathogenesis. Our findings suggest a new role for filamin in brush border maintenance and raise the possibility that microvillus effacement is a common strategy among enteric pathogens for enhancing adhesion to host cells.
- Abstract BibTeX EndNote Download URLIannetti, A., Ledoux, A. C., Tudhope, S. J., Sellier, H., Zhao, B., Mowla, S., Moore, A., Hummerich, H., Gewurz, B. E., Cockell, S. J., Jat, P. S., Willmore, E. & Perkins, N. D. Regulation of p53 and Rb Links the Alternative NF-κB Pathway to EZH2 Expression and Cell Senescence. PLOS Genetics 10, 1-20 (2014).Author Summary Although the classical NF-κB pathway is frequently associated with the induction of cellular senescence and the senescence associated secretory phenotype (SASP), the role of the alternative NF-κB pathway, which is frequently activated in hematological malignancies as well as some solid tumors, has not been defined. We therefore investigated the role of the alternative NF-κB pathway in this process. Here we report that NF-κB2 and RelB, the effectors of the alternative NF-κB pathway, suppress senescence through inhibition of p53 activity. Using primary human fibroblasts, we demonstrate that this is accomplished through NF-κB2/RelB dependent control of a previously unknown pathway, incorporating regulation of CDK4 and 6 expression as well as regulators of p21WAF1 and p53 protein stability. Loss of NF-κB2/RelB results in suppression of retinoblastoma (Rb) tumour suppressor phosphorylation, which in turn leads to inhibition of EZH2 expression and de-repression of p53 activity. Interestingly, we find that CD40 ligand stimulation of cells from Chronic Lymphocytic Leukemia patients, which strongly induces the alternative NF-κB pathway, also induces EZH2 expression. We propose that the alternative NF-κB pathway can promote tumorigenesis through suppression of p53 dependent senescence, a process that may have relevance to cancer cells retaining wild type p53.
- Abstract BibTeX EndNote Download URLErsing, I., Bernhardt, K. & Gewurz, B. E. NF-κB and IRF7 Pathway Activation by Epstein-Barr Virus Latent Membrane Protein 1. Viruses 5, 1587 (2013).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.
- Abstract BibTeX EndNote Download URLZhou*, X., Gewurz*, B. E., Ritchie, J. M., Takasaki, K., Greenfeld, H., Kieff, E., Davis, B. M. & Waldor, M. K. A Vibrio parahaemolyticus \T3SS\ Effector Mediates Pathogenesis by Independently Enabling Intestinal Colonization and Inhibiting \TAK1\ Activation. Cell Reports 3, 1690 - 1702 (2013).Summary Vibrio parahaemolyticus type \III\ secretion system 2 (T3SS2) is essential for the organism’s virulence, but the effectors required for intestinal colonization and induction of diarrhea by this pathogen have not been identified. Here, we identify a type \III\ secretion system (T3SS2)-secreted effector, VopZ, that is essential for V. parahaemolyticus pathogenicity. VopZ plays distinct, genetically separable roles in enabling intestinal colonization and diarrheagenesis. Truncation of VopZ prevents V. parahaemolyticus colonization, whereas deletion of VopZ amino acids 38–62 abrogates V. parahaemolyticus-induced diarrhea and intestinal pathology but does not impair colonization. VopZ inhibits activation of the kinase \TAK1\ and thereby prevents the activation of \MAPK\ and NF-κB signaling pathways, which lie downstream. In contrast, the VopZ internal deletion mutant cannot counter the activation of pathways regulated by TAK1. Collectively, our findings suggest that VopZ’s inhibition of \TAK1\ is critical for V. parahaemolyticus to induce diarrhea and intestinal pathology.
- Abstract BibTeX EndNote Download URLGewurz, B. E., Towfic, F., Mar, J. C., Shinners, N. P., Takasaki, K., Zhao, B., Cahir-McFarland, E. D., Quackenbush, J., Xavier, R. J. & Kieff, E. Genome-wide siRNA screen for mediators of NF-κB activation. Proceedings of the National Academy of Sciences 109, 2467-2472 (2012).Although canonical NFκB is frequently critical for cell proliferation, survival, or differentiation, NFκB hyperactivation can cause malignant, inflammatory, or autoimmune disorders. Despite intensive study, mammalian NFκB pathway loss-of-function RNAi analyses have been limited to specific protein classes. We therefore undertook a human genome-wide siRNA screen for novel NFκB activation pathway components. Using an Epstein Barr virus latent membrane protein (LMP1) mutant, the transcriptional effects of which are canonical NFκB-dependent, we identified 155 proteins significantly and substantially important for NFκB activation in HEK293 cells. These proteins included many kinases, phosphatases, ubiquitin ligases, and deubiquinating enzymes not previously known to be important for NFκB activation. Relevance to other canonical NFκB pathways was extended by finding that 118 of the 155 LMP1 NF-κB activation pathway components were similarly important for IL-1β–, and 79 for TNFα–mediated NFκB activation in the same cells. MAP3K8, PIM3, and six other enzymes were uniquely relevant to LMP1-mediated NFκB activation. Most novel pathway components functioned upstream of IκB kinase complex (IKK) activation. Robust siRNA knockdown effects were confirmed for all mRNAs or proteins tested. Although multiple ZC3H-family proteins negatively regulate NFκB, ZC3H13 and ZC3H18 were activation pathway components. ZC3H13 was critical for LMP1, TNFα, and IL-1β NFκB-dependent transcription, but not for IKK activation, whereas ZC3H18 was critical for IKK activation. Down-modulators of LMP1 mediated NFκB activation were also identified. These experiments identify multiple targets to inhibit or stimulate LMP1-, IL-1β–, or TNFα–mediated canonical NFκB activation.
- Abstract BibTeX EndNote URLZhao, B., Mar, J. C., Maruo, S., Lee, S., Gewurz, B. E., Johannsen, E., Holton, K., Rubio, R., Takada, K., Quackenbush, J. & Kieff, E. Epstein–Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines. Proceedings of the National Academy of Sciences 108, 337-342 (2011).EBV nuclear antigen 3C (EBNA3C) is an essential transcription factor for EBV transformed lymphoblast cell line (LCL) growth. To identify EBNA3C-regulated genes in LCLs, microarrays were used to measure RNA abundances in each of three different LCLs that conditionally express EBNA3C fused to a 4-OH-Tamoxifen–dependent estrogen receptor hormone binding domain (EBNA3CHT). At least three RNAs were assayed for each EBNA3CHT LCL under nonpermissive conditions, permissive conditions, and nonpermissive conditions with wild-type EBNA3C transcomplementation. Using a two-way ANOVA model of EBNA3C levels, we identified 550 regulated genes that were at least 1.5-fold up- or down-regulated with false discovery rates < 0.01. EBNA3C-regulated genes overlapped significantly with genes regulated by EBNA2 and EBNA3A consistent with coordinated effects on cell gene transcription. Of the 550 EBNA3C-regulated genes, 106 could be placed in protein networks. A seeded Bayesian network analysis of the 80 most significant EBNA3C-regulated genes suggests that RAC1, LYN, and TNF are upstream of other EBNA3C-regulated genes. Gene set enrichment analysis found enrichment for MAP kinase signaling, cytokine–cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecules, implicating these pathways in EBNA3C effects on LCL growth or survival. EBNA3C significantly up-regulated the CXCL12 ligand and its CXCR4 receptor and increased LCL migration. CXCL12 up-regulation depended on EBNA3C's interaction with the cell transcription factor, RBPJ, which is essential for LCL growth. EBNA3C also up-regulated MYC 1.3-fold and down-regulated CDKN2A exons 2 and 3, shared by p16 and p14, 1.4-fold, with false discovery rates < 5 × 10−4.
- Abstract BibTeX EndNote Download URLGewurz, B. E., Mar, J. C., Padi, M., Zhao, B., Shinners, N. P., Takasaki, K., Bedoya, E., Zou, J. Y., Cahir-Mcfarland, E., Quackenbush, J. & Kieff, E. Canonical NF-κB Activation Is Essential for Epstein-Barr Virus Latent Membrane Protein 1 TES2/CTAR2 Gene Regulation. Journal of Virology 85, 6764-6773 (2011).Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) transforms rodent fibroblasts and is expressed in most EBV-associated malignancies. LMP1 (transformation effector site 2 TES2/C-terminal activation region 2 CTAR2) activates NF-κB, p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and interferon regulatory factor 7 (IRF7) pathways. We have investigated LMP1 TES2 genome-wide RNA effects at 4 time points after LMP1 TES2 expression in HEK-293 cells. By using a false discovery rate (FDR) of <0.001 after correction for multiple hypotheses, LMP1 TES2 caused >2-fold changes in 1,916 mRNAs; 1,479 RNAs were upregulated and 437 were downregulated. In contrast to tumor necrosis factor alpha (TNF-α) stimulation, which transiently upregulates many target genes, LMP1 TES2 maintained most RNA effects through the time course, despite robust and sustained induction of negative feedback regulators, such as IκBα and A20. LMP1 TES2-regulated RNAs encode many NF-κB signaling proteins and secondary interacting proteins. Consequently, many LMP1 TES2-regulated RNAs encode proteins that form an extensive interactome. Gene set enrichment analyses found LMP1 TES2-upregulated genes to be significantly enriched for pathways in cancer, B- and T-cell receptor signaling, and Toll-like receptor signaling. Surprisingly, LMP1 TES2 and IκBα superrepressor coexpression decreased LMP1 TES2 RNA effects to only 5 RNAs, with FDRs of <0.001-fold and >2-fold changes. Thus, canonical NF-κB activation is critical for almost all LMP1 TES2 RNA effects in HEK-293 cells and a more significant therapeutic target than previously appreciated.
- Abstract BibTeX EndNote Download URLBoehm, D., Gewurz, B. E., Kieff, E. & Cahir-McFarland, E. Epstein-Barr latent membrane protein 1 transformation site 2 activates NF-κB in the absence of NF-κB essential modifier residues 133–224 or 373–419. Proceedings of the National Academy of Sciences 107, 18103-18108 (2010).Epstein Barr virus latent membrane protein 1 (LMP1) induces NF-κB activation through transformation effector sites (TES) 1 and 2, both of which are critical for B-lymphocyte transformation. TES2 principally activates canonical NF-κB, which we confirm is NF-κB essential modifier (NEMO)-dependent and requires an intact ubiquitin binding in A20 binding inhibitor of NF-κB and NEMO (UBAN) domain. LMP1 TES2 activated NF-κB in Jurkat cell lines harboring NEMO truncated at 372 (A45) or NEMO with an in-frame deletion of 133–224 (2C), whereas TNFα, 12-O-Tetradecanoylphorbol-13-acetate, human T-cell leukemia virus 1 Tax, and CD40 did not. In both A45 and 2C Jurkat cell lines, LMP1 TES2-mediated NF-κB activation was blocked by siRNAs to TNFα receptor-associated factor 6 and NEMO, by IκB kinase inhibitors, and by the IκBα superrepressor, indicating that the NEMO mutants function to support canonical NF-κB activation. Expression of A45 or 2C mutants in NEMO-deficient murine embryonic fibroblasts reproduced the Jurkat phenotypes: LMP1 TES2 activated NF-κB in fibroblasts lacking NEMO amino acids 133–224 or 373–419, but TNFα and Tax did not. Further analysis indicated that TES2 did not activate NF-κB in cells expressing the double deletion mutant Δ133–224/Δ372–419. These data provide further evidence of the essential role for NEMO in LMP1 TES2 NF-κB activation and highlight the importance of unique domains within NEMO for sensing distinct NF-κB stimuli.
- Abstract BibTeX EndNote Download URLSong, Y. -J., Izumi, K. M., Shinners, N. P., Gewurz, B. E. & Kieff, E. IRF7 activation by Epstein–Barr virus latent membrane protein 1 requires localization at activation sites and TRAF6, but not TRAF2 or TRAF3. Proceedings of the National Academy of Sciences 105, 18448--18453 (2008).Epstein–Barr virus (EBV) latent infection membrane protein 1 (LMP1), a constitutively aggregated and activated pseudoreceptor, activates IFN regulatory factor 7 (IRF7) through RIP1. We now report that the LMP1 cytoplasmic carboxyl terminal amino acids 379–386 bound IRF7 and activated IRF7. IRF7 activation required TRAF6 and RIP1, but not TRAF2 or TRAF3. LMP1 Y384YD386, which are required for TRADD and RIP1 binding and for NF-κB activation, were not required for IRF7 binding, but were required for IRF7 activation, implicating signaling through TRADD and RIP1 in IRF7 activation. Association with active LMP1 signaling complexes was also critical for IRF7 activation because (i) a dominant-negative IRF7 bound to LMP1, blocked IRF7 association and activation, but did not inhibit LMP1 induced NF-κB or TBK1 or Sendai virus-mediated IFN stimulated response element activation; and (ii) two different LMP1 transmembrane domain mutants, which fail to aggregate, each bound IRF7 and prevented LMP1 from binding and activating IRF7 in the same cell, but did not prevent NF-κB activation. Thus, efficient IRF7 activation required association with LMP1 CTAR2 in proximity to LMP1 CTAR2 mediated kinase activation sites.
- Abstract BibTeX EndNote Download URLGewurz, B. E., Ploegh, H. L. & Tortorella, D. US2, a Human Cytomegalovirus-encoded Type I Membrane Protein, Contains a Non-cleavable Amino-terminal Signal Peptide. Journal of Biological Chemistry 277, 11306-11313 (2002).The human cytomegalovirus US2 gene product targets major histocompatibility class I molecules for degradation in a proteasome-dependent fashion. Degradation requires interaction between the endoplasmic reticulum (ER) lumenal domains of US2 and class I. While ER insertion of US2 is essential for US2 function, US2 lacks a cleavable signal peptide. Radiosequence analysis of glycosylated US2 confirms the presence of the NH2 terminus predicted on the basis of the amino acid sequence, with no evidence for processing by signal peptidase. Despite the absence of cleavage, the US2 NH2-terminal segment constitutes its signal peptide and is sufficient to drive ER translocation of chimeric reporter proteins, again without further cleavage. The putative US2 signal peptide c-region is responsible for the absence of cleavage, despite the presence of a suitable −3,−1 amino acid motif for signal peptidase recognition. In addition, the US2 signal peptide affects the early processing events of the nascent polypeptide, altering the efficiency of ER insertion and subsequentN-linked glycosylation. To our knowledge, US2 is the first example of a membrane protein that does not contain a cleavable signal peptide, yet otherwise behaves like a type I membrane glycoprotein.
- Abstract BibTeX EndNote Download URLGewurz, B. E., Wang, E. W., Tortorella, D., Schust, D. J. & Ploegh, H. L. Human Cytomegalovirus US2 Endoplasmic Reticulum-Lumenal Domain Dictates Association with Major Histocompatibility Complex Class I in a Locus-Specific Manner. Journal of Virology 75, 5197-5204 (2001).The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome. We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus. Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2's ability to broadly downregulate class I molecules. The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry. These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules. Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2. The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E. We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM. The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules.
- Abstract BibTeX EndNote Download URLGewurz, B. E., Gaudet, R., Tortorella, D., Wang, E. W. & Ploegh, H. L. Virus subversion of immunity: a structural perspective. Current Opinion in Immunology 13, 442 - 450 (2001).Over the past year, we have witnessed the discovery of further virus immuno-evasins — proteins that alter the host immune response. Although many of these factors have been described over the past decade, the structural basis underlying their biology has lagged behind. Structural data have now been obtained for several such proteins. Major advances of the past year include the structures of a viral chemokine-binding protein, of an intact viral regulator of complement activation and of an immuno-evasin with its cellular target.
- Abstract BibTeX EndNote Download URLGewurz, B. E., Gaudet, R., Tortorella, D., Wang, E. W., Ploegh, H. L. & Wiley, D. C. Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2. Proceedings of the National Academy of Sciences 98, 6794-6799 (2001).Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cell-surface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the α3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.