The A20 deubiquitinase activity negatively regulates LMP1 activation of IRF7

Abstract

A20 possesses both deubiquitinase (DUB) and ubiquitin E3 ligase activities that are required for termination of Toll-like receptor (TLR) signaling leading to NF-kappaB activation and for blockage of tumor necrosis factor (TNF)-induced cytotoxicity and apoptosis. A20 is induced by the Epstein-Barr virus (EBV) oncoprotein LMP1. However, its dual ubiquitin-editing activities have not been investigated in the context of either EBV infection or IRF7 responses. Both A20 and IRF7 have oncogenic properties. We have recently shown that LMP1 activates IRF7 through K63-linked ubiquitination which requires RIP1 and TRAF6, but how this ubiquitination event is regulated has not been studied. Here, 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 DUB-deficient truncation or point mutation ablated the ability of A20 to inhibit IRF7. Correspondingly, the A20 N-terminal DUB domain, but not the C-terminal E3 ligase domain, interacts physically with IRF7. Transient expression of A20 reduced K63-linked ubiquitination of IRF7 in vivo, but an in vitro deubiquitination assay with purified constituents shows that IRF7 did not act as a substrate for A20 DUB activity. Moreover, A20 interacts with IRF7 endogenously in latently EBV-infected type 3 Raji cells, in which expression of both A20 and IRF7 is constitutively induced by the considerable level of endogenous LMP1. Knockdown of endogenous A20 in Raji cells by expression of A20 short hairpin RNA (shRNA) vectors increases endogenous IRF7 activity and ubiquitination, as well as the protein level of LMP1, a target of IRF7. Thus, A20 negatively regulates LMP1-stimulated IRF7 ubiquitination and activity in EBV latency, and its DUB activity is indispensable for this function. Finally, we discussed the regulation and function of IRFs in EBV latency.

FIG. 1.

A20 inhibits IRF7 transcriptional activity. 293 cells in 24-well plates were transfected with 0.1 μg IRF7, 0.1 μg A20, 0.02 μg LMP1, 50 ng IFN-α4-Luc, and 20 ng Renilla luciferase. Cells were treated with TLR ligands at concentrations indicated in Materials and Methods for 6 h before harvest for measurement of luciferase activity. Results are representative of at least three independent experiments. Results are the averages ± SE of duplicates for each sample. Western blots show the expression levels of transfected IRF7 and A20 in the tested samples. (A) A20 inhibits LMP1-promoted IRF7 activity. (B) A20 inhibits IRF7 activity stimulated by poly(I-C)/TLR3. (C) A20 inhibits IRF7 activity stimulated by LPS/TLR4. (D) A20 inhibits IRF7 activity stimulated by Gardiquimod/TLR7.

FIG. 2.

IRF7 interacts with A20 endogenously in EBV-transformed Raji cells. One milligram of total protein from Raji cells was subjected to IP with A20 or IRF7 antibodies. (A) IP with a mixture of A20 antibodies, and Western blot analysis with the mouse monoclonal IRF7 antibody G-8. (B) IP with a mixture of IRF7 antibodies, and Western blot analysis with the mouse monoclonal A20 antibody E5-1619.

FIG. 3.

The A20 N terminus is responsible for interaction with and regulation of IRF7. (A) Scheme of expression plasmids of A20 and its mutants used in this study. (B) The A20 N terminus and its DUB activity are required for LMP1-stimulated IRF7 activity. Transfection of 293 cells and dual luciferase assay were performed as described in the legend for Fig. 1. (C and D) The A20 N terminus is responsible for interaction with IRF7. 293 cells in six-well plates were transfected with 1 μg Flag-A20 or its mutants, 1 μg Myc-IRF7 or HA-IRF7, and 0.5 μg HA-Ub or Myc-Ub. Reciprocal IP was performed with lysates collected 48 h after transfection. Western blot analysis was performed with corresponding antibodies as indicated.

FIG. 4.

A20 DUB activity is required for reduction of LMP1-stimulated IRF7 ubiquitination. 293 cells in six-well plates were transfected with plasmids as indicated. Cells were harvested 48 h after transfection for double IP with anti-IRF7. Proteins were subjected to Western blot analysis with anti-Myc or anti-IRF7. Cell lysates (5% input) were probed with anti-HA for LMP1 and anti-Flag for A20 and its mutants.

FIG. 5.

Expression of shA20 in Raji cells increases endogenous ubiquitination and activity of IRF7. (A to C). Type 3 latently EBV-infected cells (Raji) were infected with shA20 expression vectors. For luciferase assay, 2 μg IFN-α4-Luc and 1 μg Renilla luciferase were cotransfected. After 2 days, cells were collected for double IP or for luciferase assay. (A) Expression of shA20 (a mix of shA20-1 and shA20-2) increases endogenous IRF7 activity. (B) Expression of shA20 enhances endogenous ubiquitination of IRF7. (C) Endogenous A20 level is reduced in cells expressing shA20. (D) P3HR1 cells were transfected with the A20 shRNA constructs. Forty-eight hours later, cell lysates were used for Western blotting with LMP1 (clone CS1-4, Dako), A20, IRF7, and tubulin antibodies.

FIG. 6.

Regulation and function of IRFs in EBV latency. The only three IRFs with oncogenic properties, IRF7 (39, 73), -2 (72), and -4 (35, 69), as well as IRF5 (35, 40, 70), are all expressed at much higher levels in EBV type 3 latency. Both IRF4 (35, 69) and IRF7 (73) are induced by EBV LMP1, and IRF5 is induced by both TLR7 and LMP1 (16, 35), whereas IRF2 is induced through an unknown mechanism and negatively regulates the EBNA1 Q promoter in latency 3 (72). Moreover, IRF7 and likely IRF4 are activated by LMP1 (24, 38, 69). The activated IRF7 induces expression of LMP1, TAP2 and type I IFNs (39, 68, 74) but negatively regulates the EBV EBNA1 Q promoter and BART gene P1 promoter (11, 71). However, IRF5 (40) as well as A20 that is also induced by LMP1 (19, 29) negatively regulates LMP1-promoted IRF7 activity in EBV latency 3. In addition, like its role in antiviral responses (37), IRF4 may negatively regulate TLR7 activation of IRF5 in the EBV context.