The Deubiquitinating Enzym A20 Negatively Regulates LMP1 Activation of IRF7

The deubiquitinating enzyme (DUB) A20 is an anti-apoptotic protein which is induced by the EBV oncoprotein LMP1, but its role in EBV oncogenesis has not been investigated. A20 is a member of the ovarian tumor (OTU) DUB family, and its DUB activity is required for termination of TLR signaling leading to NF-κB activation, and for blockage of TNF-induced cytotoxicity and apoptosis. IRF7 has oncogenic properties, and we have recently shown that LMP1 activates IRF7 through K63-linked ubiquitination which requires RIP and TRAF6 (Huye/Ning et al. Mol. Cell. Biol, 2007; Ning et al. Mol. Cell. Biol., 2008). 

In this study, we show that A20 negatively regulates IRF7 transcriptional activity induced by LMP1. Deletion or mutation of A20 C-terminal Zinc finger motifs had no effect on the inhibition of IRF7 activity, whereas deletion of the N-terminal OTU domain ablated the ability of A20 to inhibit IRF7. Correspondingly, A20 N-terminus but not C-terminus interacts physically with IRF7. Moreover, A20 interacts with IRF7 endogenously in EBV latently infected type 3 cells in which expression of both A20 and IRF7 are constitutively induced by the high level of endogenous LMP1. Knockdown of endogenous A20 in Raji cells by expression of A20 shRNA vectors increases endogenous IRF7 activity and ubiquitination. In vitro deubiquitination assay results show that IRF7 is a substrate for the DUB A20 which removes K63-linked polyubiquitin chains from IRF7. Thus, A20 acts as a DUB to negatively regulate LMP1-stimulated IRF7 activity, and may participate in regulation of LMP1 oncogenic mechanisms at least through regulation of IRF7 activity.