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. 2011 Feb 4;41(3):354-65.
doi: 10.1016/j.molcel.2010.12.029.

Linear ubiquitin assembly complex negatively regulates RIG-I- and TRIM25-mediated type I interferon induction

Kyung-Soo Inn  1 , Michaela U Gack, Fuminori Tokunaga, Mude Shi, Lai-Yee Wong, Kazuhiro Iwai, Jae U Jung
Affiliations

Linear ubiquitin assembly complex negatively regulates RIG-I- and TRIM25-mediated type I interferon induction

Kyung-Soo Inn et al. Mol Cell. .

Abstract

Upon detection of viral RNA, retinoic acid-inducible gene I (RIG-I) undergoes TRIM25-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that the linear ubiquitin assembly complex (LUBAC), comprised of two RING-IBR-RING (RBR)-containing E3 ligases, HOIL-1L and HOIP, independently targets TRIM25 and RIG-I to effectively suppress virus-induced IFN production. RBR E3 ligase domains of HOIL-1L and HOIP bind and induce proteasomal degradation of TRIM25, whereas the NZF domain of HOIL-1L competes with TRIM25 for RIG-I binding. Consequently, both actions by the HOIL-1L/HOIP LUBAC potently inhibit RIG-I ubiquitination and antiviral activity, but in a mechanistically separate manner. Conversely, the genetic deletion or depletion of HOIL-1L and HOIP robustly enhances virus-induced type I IFN production. Taken together, the HOIL-1L/HOIP LUBAC specifically suppresses RIG-I ubiquitination and activation by inducing TRIM25 degradation and inhibiting TRIM25 interaction with RIG-I, resulting in the comprehensive suppression of the IFN-mediated antiviral signaling pathway.

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Figures

Figure 1
Figure 1. HOIL-1L/HOIP interacts with TRIM25 and RIG-I
(A–B) Interactions between TRIM25 and HOIL-1L or HOIP. 293T were transfected with V5-TRIM25 together with (A) HA-HOIL-1L or (B) Flag-HOIP, followed by co-IP and IB. (C) HOIL-1L interacts with RIG-I. 293T cells transfected with GST-RIG-2CARD and V5-HOIL-1L or vector were used for GST-pull down (PD) assay and IB. (D) HOIP requires HOIL-1L to interact with RIG-I. GST-RIG-2CARD and Flag-HOIP were transfected with increasing amounts of HOIL-1L, followed by IP with anti-Flag. (E) Endogenous HOIL-1L/HOIP interacts with RIG-I and TRIM25. WT MEFs were mock-infected or infected with SeV for 6 h and treated with MG132 for 4 h before harvest, followed by IP with control IgG, anti-RIG-I or anti-TRIM25. (F) Co-localization of HOIL-1L/HOIP with TRIM25. HeLa cells were mock-treated or infected with SeV for 10 h, followed by fixation and staining with anti-TRIM25, anti-HOIL-1L or anti-HOIP antibody. Co-localization between TRIM25 and HOIL-1L, Pearson’s correlation=0.871 and Mander’s overlap=0.885; co-localization between TRIM25 and HOIP, Pearson’s correlation=0.702 and Mander’s overlap=0.955 (G) TRIM25 SPRY domain is responsible for HOIL-1L/HOIP interaction. HA-HOIL-1L or Flag-HOIP was transfected with TRIM25 deletion mutants, followed by co-IP. (H) Binding ability of HOIL-1L or HOIP mutants. Deletion mutants of HOIL-1L or HOIP were transfected with TRIM25 or GST-RIG-I 2CARD as indicated. Cells were treated with MG132 for 6 h, followed by Co-IP and IB. (I) RIG-I interaction with HOIL-1L/HOIP in TRIM25−/− MEFs. WT and TRIM25−/− MEFs were infected with SeV for 10 h, followed by co-IP using control IgG or anti-RIG-I. (J) In vitro interaction between RIG-I-2CARD and HOIL-1L. Bacterially purified GST fusion proteins of RIG-I-2CARD, HOIL-1L ΔRBR-V5 and HOIP ΔRBR-Flag were used for in vitro binding, followed by co-IP and IB as indicated. See also Figure S1.
Figure 2
Figure 2. HOIL-1L/HOIP complex negatively regulates RIG-I mediated IFN-β signaling
(A–B) Inhibition of RIG-I-2CARD induced IFN-β and NF-κB promoter activities by HOIL-1L/HOIP. 293T cells were transfected with RIG-I-2CARD with increasing amounts of HOIL-1L, HOIP, or both together with IFN-β (A) or NF-κB promoter (B). (C) Inhibition of SeV induced IFN-β promoter activity by HOIL-1L/HOIP. IFN-β promoter activities were measured from 293T cells transfected with full-length RIG-I and HOIL-1L and/or HOIP. At 24 h after transfection, cells were mock-infected or infected with SeV (40 HAU/ml) for 10 h before luciferase assay. (D-F) RIG-I-2CARD, TRIM25, HOIL-1L and HOIP plasmids were transfected together with IFN-β (D), ISRE (E), or NF-κB (F) reporter plasmids into 293T. All luciferase assays were performed at least three times and graphs show the mean ± SD. Values are normalized by pRL-TK Renilla.
Figure 3
Figure 3. HOIL-1L/HOIP depletion increases IFN-β production and anti-viral response
(A) WT, HOIL-1L−/−, HOIP knock-down MEF (HOIP KD), HOIL-1L−/−-HOIP KD MEFs were established as described in the Experimental Procedures. Depletion and reduction of HOIL-1L and HOIP, respectively, were confirmed by IB. (B) Enhanced IFN-β promoter activity in HOIL-1L/HOIP depleted MEFs. MEFs were infected with SeV (50 HAU/ml) for 10 h and then subjected to dual-luciferase assay. Experiments were performed in triplicate and graph shows the mean ± SD. (C) Increased IFN-β production in HOIL-1L/HOIP depleted MEFs. MEFs were infected with SeV (50 HAU/ml) for 12 h and supernatants were subjected to mouse IFN-β ELISA. Experiments were performed in triplicate and graph shows the mean ± SD. (D) Suppression of IFN-β promoter activity in HOIL-1L−/− MEFs by complementation of HOIL-1L. HOIL-1L was co-transfected with reporter plasmid as indicated. Virus infection and luciferase assay were performed similarly to (A). Experiments were performed in triplicate and graph shows the mean ± SD. (E–F) Viral replication in HOIL-1L/HOIP depleted MEFs. MEFs were infected with VSV-eGFP (M.O.I=0.02). Supernatants were taken at 48 h p.i. and subjected to plaque assay. (G) VSV-eGFP replication in 293T expressing vector alone or HOIL-1L.
Figure 4
Figure 4. HOIL-1L/HOIP LUBAC inhibits TRIM25-mediated RIG-I ubiquitination
(A) Inhibition of RIG-I ubiquitination by HOIL-1L/HOIP. 293T cells transfected with GST-RIG-I-2CARD and TRIM25 together with HOIL-1L or HOIP were subjected to GST-PD and IB. (B) Inhibition of endogenous RIG-I ubiquitination by HOIL-1L/HOIP. 293T cells transfected with HA-ubiquitin with or without HOIL-1L/HOIP were mock-infected or infected with SeV for 10 h, followed by IP with anti-RIG-I. (C) RIG-I ubiquitination in HOIL-1L/HOIP depleted MEF. WT, HOIL-1L−/−, HOIP-KD and HOIL-1L−/−-HOIP-KD MEFs infected with SeV were used for IP and IB. Anti-ubiquitin (αUb) antibody and anti-K63-ubiquitin chain specific antibody (αK63) were used to detect RIG-I ubiquitination. (D) Decreased interaction between RIG-I and MAVS by HOIL-1L/HOIP expression. 293T cells transfected with Flag-RIG-I-2CARD, GST-MAVS-CARD-proline-rich domain (PRD), V5-HOIL-1L and/or Myc-HOIP were used for co-IP and IB. See also Figure S2.
Figure 5
Figure 5. HOIL-1L/HOIP LUBAC induces TRIM25 ubiquitination and degradation
(A) Increment of TRIM25 levels in HOIL-1L/HOIP depleted MEFs. WT, HOIL-1L−/−, HOIP KD, HOIL-1L−/−-HOIP KD MEFs were mock-infected or infected with SeV for 12 h. Cell lysates were subjected to IB. (B) Decreased endogenous TRIM25 levels upon HOIL-1L/HOIP overexpression. Endogenous TRIM25 levels were analyzed by IB and densitometry. IBs show representative data, and graph shows the averages of triplicate. (C) Reduction of TRIM25 by HOIL-1L/HOIP expression. At 36 h after the transfection with V5-TRIM25 and GST together with HOIL-1L and HOIP as indicated, 293T cells were mock-treated or treated with MG132 for 6 h and were subjected to IB. (D) TRIM25 ubiquitination induced by HOIL-1L/HOIP. 293T cells were transfected with V5-TRIM25, HOIL-1L and HOIP and MG132 treatment for IP. (E) Endogenous TRIM25 ubiquitination upon SeV infection. MEFs were mock-infected or infected with SeV for 10 h and cell lysates were subjected to IP using anti-TRIM25 antibody. (F) TRIM25 ubiquitination levels in HOIL-1L/HOIP depleted MEFs. MEFs were infected with SeV and treated with MG132, followed by IP with anti-TRIM25 and IB with anti-Ub or anti-TRIM25. (G) TRIM25 ubiquitination by HOIL-1L/HOIP RINGCS mutants. 293T cells were transfected with V5-TRIM25 and WT or RINGCS mutants of HOIL-1L or HOIP, followed by IP with anti-V5 and IB with anti-Ub or anti-V5. (H) In vitro ubiquitination of TRIM25 by LUBAC. Purified GST-TRIM25 RINGCS was subjected to an in vitro ubiquitination with baculovirus-purified HOIL-1L/HOIP LUBAC, followed by IB with anti-GST antibody. See also Figure S3.
Figure 6
Figure 6. HOIL-1L/HOIP LUBAC inhibits the interaction between RIG-I and TRIM25
(A) Inhibition of RIG-I ubiquitination by HOIL-1L/HOIP RINGCS mutants. 293T cells were transfected with GST-RIG-I-2CARD and TRIM25 together with HOIL-1L, HOIP, or RINGCS mutants and treated with MG132, followed by GST-PD. (B) Inhibition of RIG-I-2CARD and TRIM25 interaction by HOIL-1L/HOIP. 293T cells were transfected with GST-RIG-I-2CARD and TRIM25 together with HOIL-1L and/or HOIP as indicated, followed by GST-PD. (C) Increased interaction between RIG-I and TRIM25 in HOIL-1L/HOIP depleted MEFs. MEFs were mock-infected or infected with SeV (50 HAU/ml) for 12 h and subjected to co-IP using control IgG or anti-RIG-I. (D) Time-course analysis of endogenous RIG-I and TRIM25 interaction. At different time points after SeV infection, the same amounts of proteins from WT MEFs were subjected to co-IP using anti-TRIM25. (E) In vitro competition assay. Bacterially purified GST-RIG-I-2CARD and GST-TRIM25 were incubated with increasing amounts of GST-HOIL-1L ΔRBR-V5 or GST-HOIP ΔRBR-Flag, followed by IP with anti-RIG-I. (F) Inhibition of RIG-I and TRIM25 interaction by LUBAC. Biotin-labeled GST-TRIM25 were added to GST-RIG-I coated wells with increasing amounts of GST-HOIL-1L ΔRBR-V5/GST-HOIP ΔRBR-Flag (LUBAC) or unlabeled GST-TRIM25. Bound biotin-labeled GST-TRIM25 was detected using streptavidin-HRP. Experiments were performed in triplicate and graph shows the mean ± SD. See also Figure S4.
Figure 7
Figure 7. Roles of the NZF and RBR domains of HOIL-1L in RIG-I mediated IFN-β signaling
(A) Inability of HOIL-1L NZFCS mutant to bind RIG-I. 293T cells were transfected with GST-RIG-I-2CARD together with HA-HOIL-1L WT or NZFCS mutant, followed by GST-PD and IB with indicated antibodies. (B) Inability of HOIL-1L NZFCS mutant to interfere the RIG-I-TRIM25 interaction. 293T cells were transfected with GST-RIG-I-2CARD and TRIM25 together with HA-HOIL-1L WT or NZFCS mutants and treated with MG132, followed by GST-PD and IB. (C) Inability of HOIL-1L NZFCS mutant to inhibit RIG-I ubiquitination. 293T cells were transfected with GST-RIG-I-2CARD and TRIM25 together with HOIL-1L WT or NZFCS mutant as indicated, followed by GST-PD and IB. (D) Effect of HOIL-1L WT or NZFCS mutant on RIG-I-mediated IFN-β promoter activity. IFN-β promoter activities were determined from 293T cells transfected with RIG-I-2CARD, and TRIM25 together with HOIL-1L WT or NZFCS mutant. Experiments were performed in triplicate and graph shows the mean ± SD. (E-F) Complementation of HOIL-1L−/− MEFs. HOIL-1L−/− MEFs were infected with recombinant retrovirus containing HOIL-1L WT, RINGCS, or NZFCS mutant, followed by the puromycin antibiotic selection. (E) Complemented MEFs were transfected with IFN-β reporter, followed by mock-infection or SeV infection and luciferase assay. Experiments were performed in triplicate and graph shows the mean ± SD. (F) Complemented MEFs were mock-infected or infected with SeV (50 HAU/ml) and supernatants were subjected to IFN-β ELISA. Experiments were performed in triplicate and graph shows the mean ± SD. (G) Hypothetical model for LUBAC-mediated inhibition of RIG-I and TRIM25 pathway. See also Figure S5.
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