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.

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.