RESUMO
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.
Assuntos
Linfócitos B/metabolismo , Transformação Celular Viral , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/metabolismo , Fator 1 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Proteínas da Matriz Viral/metabolismo , Linfócitos B/imunologia , Linfócitos B/virologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Células HEK293 , Herpesvirus Humano 4/imunologia , Humanos , Lisina/metabolismo , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fator 1 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/antagonistas & inibidores , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
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.
Assuntos
Linfócitos B/virologia , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Fator B do Complemento/metabolismo , Herpesvirus Humano 4/fisiologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linfoma de Burkitt/virologia , Linhagem Celular , Transformação Celular Viral , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Fator Regulador 2 de Interferon/metabolismo , Fatores Reguladores de Interferon/metabolismo , Mutagênese , NF-kappa B/metabolismo , Proteínas Oncogênicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
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.