title: TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation.
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.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Hannah
authors.initials: H
authors.lastname: Greenfeld
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Takasaki
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Walsh
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Ina
authors.initials: I
authors.lastname: Ersing
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Katharina
authors.initials: K
authors.lastname: Bernhardt
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Yijie
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Bishi
authors.initials: B
authors.lastname: Fu
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Camille W
authors.initials: CW
authors.lastname: Ashbaugh
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Jackson
authors.initials: J
authors.lastname: Cabo
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Mollo
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Hufeng
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Shitao
authors.initials: S
authors.lastname: Li
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Gewurz
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000821:virology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D002472:Cell Transformation, Viral; D020031:Epstein-Barr Virus Infections / Q000276:immunology* / Q000378:metabolism / Q000821:virology; D057809:HEK293 Cells; D004854:Herpesvirus 4, Human / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D047990:TNF Receptor-Associated Factor 1 / Q000235:genetics / Q000378:metabolism*; D047992:TNF Receptor-Associated Factor 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C037662:EBV-associated membrane antigen, Epstein-Barr virus; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D047990:TNF Receptor-Associated Factor 1; D047992:TNF Receptor-Associated Factor 2; D014763:Viral Matrix Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D008239:Lysine