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LRRC25 inhibits type I IFN signaling by targeting ISG15‐associated RIG‐I for autophagic degradation

2017; Springer Nature; Volume: 37; Issue: 3 Linguagem: Inglês

10.15252/embj.201796781

1460-2075

Yang Du, Tianhao Duan, Yan‐Chun Feng, Qingxiang Liu, Meng Lin, Jun Cui, Rong‐Fu Wang,

Inflammasome and immune disorders

Article29 December 2017free access Source DataTransparent process LRRC25 inhibits type I IFN signaling by targeting ISG15-associated RIG-I for autophagic degradation Yang Du Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Tianhao Duan Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yanchun Feng Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Qingxiang Liu Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Meng Lin Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Jun Cui Corresponding Author [email protected] orcid.org/0000-0002-8000-3708 Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Rong-Fu Wang Corresponding Author [email protected] orcid.org/0000-0002-8834-2763 Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX, USA Department of Microbiology and Immunology, Weill Cornell Medical College, Cornell University, New York, NY, USA Institute of Biosciences and Technology, College of Medicine, Texas A & M University, Houston, TX, USA Search for more papers by this author Yang Du Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Tianhao Duan Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yanchun Feng Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Qingxiang Liu Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Meng Lin Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Jun Cui Corresponding Author [email protected] orcid.org/0000-0002-8000-3708 Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Rong-Fu Wang Corresponding Author [email protected] orcid.org/0000-0002-8834-2763 Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX, USA Department of Microbiology and Immunology, Weill Cornell Medical College, Cornell University, New York, NY, USA Institute of Biosciences and Technology, College of Medicine, Texas A & M University, Houston, TX, USA Search for more papers by this author Author Information Yang Du1,2,‡, Tianhao Duan1,2,‡, Yanchun Feng1,2,‡, Qingxiang Liu2, Meng Lin2, Jun Cui *,1,2 and Rong-Fu Wang *,3,4,5 1Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China 2Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China 3Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, TX, USA 4Department of Microbiology and Immunology, Weill Cornell Medical College, Cornell University, New York, NY, USA 5Institute of Biosciences and Technology, College of Medicine, Texas A & M University, Houston, TX, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +86 20 39943429; E-mail: [email protected] *Corresponding author. Tel: +1 713 441 7359; E-mail: [email protected] EMBO J (2018)37:351-366https://doi.org/10.15252/embj.201796781 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The RIG-I-like receptors (RLRs) are critical for protection against RNA virus infection, and their activities must be stringently controlled to maintain immune homeostasis. Here, we report that leucine-rich repeat containing protein 25 (LRRC25) is a key negative regulator of RLR-mediated type I interferon (IFN) signaling. Upon RNA virus infection, LRRC25 specifically binds to ISG15-associated RIG-I to promote interaction between RIG-I and the autophagic cargo receptor p62 and to mediate RIG-I degradation via selective autophagy. Depletion of either LRRC25 or ISG15 abrogates RIG-I-p62 interaction as well as the autophagic degradation of RIG-I. Collectively, our findings identify a previously unrecognized role of LRRC25 in type I IFN signaling activation by which LRRC25 acts as a secondary receptor to assist RIG-I delivery to autophagosomes for degradation in a p62-dependent manner. Synopsis Upon RNA virus infection, LRRC25 recognizes ISG15-associated immune receptor RIG-I and facilitates its degradation via p62-mediated selective autophagy, thereby limiting RIG-I-dependent type I interferon signaling. LRRC25 negatively regulates type I interferon signaling upon RNA virus infection. LRRC25 promotes autophagic degradation of RIG-I in a p62-dependent manner. ISG15 serves as a signal for LRRC25-mediated RIG-I degradation. The interaction between RIG-I, ISG15 and LRRC25 forms a negative feedback loop to avoid prolonged immune activation upon viral infection. Introduction The innate immune responses, triggered by pathogen-associated molecular patterns (PAMPs), are the first line of defense against invading viruses. In virus-infected cells, viral RNAs can be detected by pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), RLRs, as well as several sensors of DNA (O'Neill, 2006; Takaoka et al, 2007; Unterholzner et al, 2010; Loo & Gale, 2011; Zhang et al, 2011; Sun et al, 2013). As the major members of RLRs family, RIG-I and MDA5 (melanoma differentiation-associated gene 5) play pivotal roles in sensing cytosolic viral RNAs (Loo & Gale, 2011). Both RIG-I and MDA5 are composed of a conserved DEAD box helicase/ATPase domain, two caspase-recruiting domains (CARDs), and a C-terminal regulatory domain (CTD). Upon binding with viral RNAs via their CTD domains, RIG-I and MDA5 use their CARDs to activate downstream adaptor MAVS (also known as IPS-1, VISA, or CARDIF) (Kawai et al, 2005; Meylan et al, 2005; Seth et al, 2005; Xu et al, 2005). MAVS then triggers the signal cascades to initiate type I interferons (IFNs) production, as well as the downstream expression of multiple IFN stimulated genes (ISGs) and inflammation cytokines. Due to its critical role in type I IFN activation, the activity of RIG-I must be tightly regulated to protect the host from uncontrolled immune response such as autoimmune disease. Several negative regulators of RIG-I have been reported to inhibit type I IFN signaling through the control of the RIG-I degradation (Arimoto et al, 2007; Chen et al, 2013; Zhao et al, 2016). RNF125 is the first reported E3 ligase, which conjugates ubiquitin to RIG-I to mediate the degradation of RIG-I through proteasome pathway (Arimoto et al, 2007). Siglec-G also promotes RIG-I degradation through enhancing K48-linked ubiquitination of RIG-I by recruiting E3 ubiquitin ligase c-Cbl (Chen et al, 2013). Recently, it has been shown that another E3 ligase CHIP associates with RIG-I and promotes RIG-I for proteasomal degradation (Zhao et al, 2016). Besides, it has been reported that RIG-I can also be conjugated by ISG15, which results in the degradation of RIG-I (Kim et al, 2008). However, the mechanisms underlying degradation of RIG-I dependent on ISG15 remain to be explored. In particular, RIG-I in general is degraded through ubiquitination-mediated proteasome pathway, and it has not been reported that RIG-I could be degraded through other degradation systems (such as lysosome and autolysosome). Leucine-rich repeat (LRR) domain is conserved in a variety of prokaryotic and eukaryotic proteins and displays significant functions in innate immunity (Ng et al, 2011). In mammals, the functions of LRR-containing proteins, including NOD-like receptors (NLRs) and TLRs in innate immune responses, are well characterized (Inohara et al, 2005; Takeuchi & Akira, 2010). Besides NLRs and TLRs, the functions of other LRR-containing proteins in innate immunity remain to be determined. In this study, we identified LRRC25 as the negative regulator of RLR-mediated type I IFN signaling. Ectopic expression of LRR-containing protein 25 (LRRC25) inhibited the phosphorylation of endogenous IRF3 and antiviral response following RLR ligand stimulation, whereas knockdown or knockout of LRRC25 had the opposite effects. Upon RNA virus infection, LRRC25 specifically interacts with ISG15-associated RIG-I to mediate RIG-I degradation via p62-dependent selective autophagy. Depletion of ISG15 abrogates RIG-I-p62 interaction as well as the autophagic degradation of RIG-I. Our findings provide new insights into the tight regulation of type I IFN signaling through its crosstalk with selective autophagy pathway and uncover a negative feedback loop to regulate RIG-I-mediated type I IFN signaling. Results LRRC25 inhibits RLR-mediated type I IFN signaling pathway To identify the possible LRRC proteins that engage in type I IFN signaling pathway, we screened 22 candidate genes encoding LRRC proteins and found that LRRC25 significantly inhibited the ISRE activation induced by RIG-I CARD domains (RIG-I (N)) (Fig 1A). It has been reported that poly(I:C)-low molecular weight (LMW) is a specific ligand for RIG-I, but not for MDA5 (Kato et al, 2008; Takeuchi & Akira, 2010). We then analyzed the expression of LRRC25 in THP-1 cells upon challenge with vesicular stomatitis virus with enhanced GFP (VSV-eGFP), intracellular (IC) poly(I:C) (LMW), or IFN-β and found that LRRC25 protein level was up-regulated by all these treatments (Fig 1B–D). However, qPCR analysis showed that the mRNA level of LRRC25 was not changed by these treatments (Fig EV1A). These results suggest that LRRC25 protein can be stabilized by the activation of type I IFN signaling. Furthermore, we found that type I IFN signaling stabilized LRRC25 by blocking its proteasome-dependent degradation, since the proteasome inhibitor MG132, but not the lysosome inhibitor NH4Cl, could stabilize LRRC25 and diminish the difference of LRRC25 protein level with or without RIG-I (N) overexpression (Figs 1E and EV1B). In addition, we found that ectopic expression of RIG-I (N) could not block the proteasome degradation of TBK1 mediated by USP38 (Lin et al, 2016), indicating the specific stabilization of LRRC25 mediated by RIG-I-type I IFN axis (Fig EV1C). To further test the role of LRRC25 in type I IFN signaling, we showed that LRRC25 inhibited ISRE-luc and IFN-β-luc activities after treatment with IC poly (I:C) LMW or infection with Sendai virus (SeV) (Figs 1F and G, and EV1D). However, LRRC25 had no effect on TLR3- or cGAS-mediated type I IFN activation (Fig EV1E and F), suggesting that LRRC25 specifically inhibits RLR-induced type I IFN signaling pathway. Similarly, Myc-LRRC25 significantly decreased the phosphorylation of endogenous IRF3 after treated with IC poly(I:C) LMW or infected with SeV (Fig 1H). To demonstrate a link between attenuated type I IFN response and antiviral immunity caused by LRRC25, we transfected empty vector and LRRC25 into 293T cells and subsequently infected the cells with VSV-eGFP. LRRC25 rendered the cells more susceptible to viral infection and enhanced the replication of VSV-eGFP at different time points (Fig 1I and J). Collectively, these data suggest that ectopic expression of LRRC25 markedly inhibits the type I IFN response and antiviral immunity. Figure 1. LRRC25 inhibits RLR-mediated type I IFN signaling pathway A. HEK293T cells were transfected with a control plasmid or plasmids expressing 22 LRRCs along with RIG-I (N) and a reporter plasmid carrying the ISRE promoter (ISRE-Luc). 24 h after transfection, cells were analyzed for ISRE-luc activity. B–D. THP-1 cells were treated with VSV-eGFP (MOI = 0.1), intracellular (IC) poly(I:C) low molecular weight (5 μg/ml), or IFN-β (10 ng/ml) for indicated time points. Cell lysates were used for immunoblot analysis with the indicated antibodies. E. HEK293T cells were transfected with plasmids for LRRC25, together with an empty vector or RIG-I (N) for 24 h. Before harvesting, the cells were treated with DMSO or MG132 (5 μM) for 4 h. Cell lysates were used for immunoblot analysis with the indicated antibodies. F, G. HEK293T cells were transfected with plasmids for Myc-LRRC25, plus an ISRE-luc (F) or an IFN-β-luc (G) reporter plasmid. After 12 h, cells were treated with IC poly(I:C) LMW (5 μg/ml) or SeV (MOI = 0.1) for 24 h or 14 h, respectively, and analyzed for ISRE-luc and IFN-β-luc activity. H. HEK293T cells were transfected with an empty vector or Myc-LRRC25. After 12 h, cells were left untreated or treated with IC poly(I:C) LMW (5 μg/ml) or SeV (MOI = 0.1) for 24 h or 14 h, respectively. Before harvesting, the cells were treated with MG132 (5 μM) for 4 h. Protein extracts were analyzed by immunoblot using the indicated antibodies. I. HEK293T cells were transfected with an empty vector (EV) or Myc-LRRC25. 24 h post-transfection, cells were infected with VSV-eGFP (MOI = 0.001) for the indicated time points and subjected to phase-contrast (PH) and fluorescence microscopy analyses. Scale bar, 80 μm. J. Flow cytometry analyses of 293T cells in (I). Numbers at the top-right corner indicate the percentage of cells expressing eGFP. Bar: population of GFP-positive cells. Data information: In (B–E, H–J), data are representative of three independent experiments. In (A, F, G), data are mean values ± SEM (n = 3). **P < 0.01, ***P < 0.001 (Student's t-test). Source data are available online for this figure. Source Data for Figure 1 [embj201796781-sup-0002-SDataFig1.tif] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. LRRC25 has no effect on the type I IFN signaling pathway mediated by TLR3 or c-GAS, related to Fig 1 THP-1 cells were treated with VSV-eGFP (MOI = 0.1), intracellular (IC) poly(I:C) low molecular weight (5 μg/ml) or IFN-β (10 ng/ml) for indicated time points. Total RNA was extracted for qPCR analysis for LRRC25, IFN-β, and IFIT2. HEK293T cells were transfected with plasmids for LRRC25, together with an empty vector or RIG-I (N) for 24 h. Before harvesting, the cells were treated with DMEM or NH4Cl (10 mM) for 6 h. Cell lysates were used for immunoblot analysis with the indicated antibodies. HEK293T cells were transfected with Flag-TBK1, together with an empty vector, Flag-RIG-I (N), or Myc-USP38 for 24 h. Before harvesting, the cells were treated with DMSO or MG132 (10 μM) for 6 h. Cell lysates were harvested and used to perform immunoblot analysis with the indicated antibodies. The expression of LRRC25 in Fig 1F and G was analyzed by IB analysis. HEK293T cells were transfected with an empty vector (no wedge) or increasing amounts (wedge) of vector for LRRC25, along with vectors for cGAS and STING. 24 h post-transfection, the cells were analyzed for ISRE activity by a reporter gene assay, and the expressions of cGAS, STING, and LRRC25 were analyzed by IB analysis. HEK293T cells were transfected with an empty vector or plasmid for LRRC25, plus plasmids for TLR3 and ISRE-luc reporter, followed by no treatment or treatment with poly (I:C) (10 μg/ml). After 24 h, cell lysates were analyzed for ISRE-luc activity. Data information: In (B–E), data are representative of three independent experiments. In (A, E, F), data are mean values ± SEM (n = 3). Download figure Download PowerPoint LRRC25 deficiency enhances antiviral responses To determine the physiological function of LRRC25 during RNA virus infection, we designed two LRRC25-specific small interfering RNA (siRNA) to knock down the expression of LRRC25. Both of them could efficiently knock down endogenous LRRC25 (Fig EV2A). To determine the effects of LRRC25 knockdown on ISRE-luc activity, we showed that knockdown of endogenous LRRC25 increased the ISRE-luc activity stimulated by IC poly(I:C) (Fig EV2B). Next, we tested the effect of LRRC25 knockdown on the replication of VSV-eGFP and found that LRRC25 knockdown substantially inhibited viral infection compared to those of cells treated with scrambled siRNA (Fig EV2C and D). Click here to expand this figure. Figure EV2. LRRC25 deficiency enhances antiviral immune responses, related to Fig 2 THP-1 cells were transfected with control or LRRC25-specific siRNAs. Cell lysates were harvested and used to perform immunoblot analysis with the indicated antibodies. HEK293T cells were transfected with control or LRRC25-specific siRNAs, together with an ISRE-luc reporter plasmid. After 24 h, the cells were treated with IC poly(I:C) (5 μg/ml) for 24 h. The cells were analyzed for ISRE activity by a reporter assay, and the expression of LRRC25 was analyzed by IB analysis. THP-1 cells were transfected with control or LRRC25-specific siRNAs for 24 h, and then, the cells were infected with VSV-eGFP (MOI = 0.01) for the indicated time points and subjected to phase-contrast (PH) and fluorescence microscopy analyses. Scale bar, 80 μm. Flow cytometry analyses of THP-1 cells in (C). Numbers at the top-right corner indicate the percentage of cells expressing eGFP (infected cells). LRRC25 KO THP-1 and LRRC25 KO HEK293T cells were generated by the CRISPR/Cas9 system. The sequences of target sgRNA are as indicated. Data information: In (A–D), data are representative of three independent experiments. In (B), data are mean values ± SEM (n = 3). ***P < 0.001 (Student's t-test). Download figure Download PowerPoint To further substantiate these findings, we used CRISPR/Cas9 system to generate LRRC25 knockout (KO) THP-1 and 293T cells, respectively. The deletion of LRRC25 was confirmed at the DNA and protein levels (Figs 2A and EV2E). We found that the phosphorylation of IRF3 (p-IRF3) in LRRC25 KO THP-1 cells was higher than that in control cells after VSV-eGFP infection (Fig 2B). We next sought to address whether the enhanced IRF3 phosphorylation by LRRC25 deficiency promotes type I IFN and ISG expressions. Using qPCR analysis, we showed that LRRC25 KO markedly increased mRNA abundance of IFN-β following VSV infection (Fig 2C). Consistent with these observations, we found that VSV infection resulted in increased production of IFN-β in LRRC25 KO THP-1 cells compared to control cells (Fig 2D). Consistently, LRRC25 KO also resulted in higher expression of IFIT1 and IFIT2 after infection with VSV-eGFP (Fig 2E). To further investigate whether the elevated IFN response is correlated with enhanced antiviral immunity, we infected LRRC25 KO and control THP-1 cells with VSV-eGFP. The percentage of GFP+ cells increased with the extended response time, but it was markedly inhibited by LRRC25 deficiency in LRRC25 KO THP-1 cells (Fig 2F and G). We next isolated human peripheral blood mononuclear cells (PBMCs) and knocked down endogenous LRRC25 to evaluate the physiological importance of LRRC25 during influenza A (H1N1) infection. As expected, we found that knockdown of endogenous LRRC25 increased the phosphorylation of endogenous IRF3 after H1N1 infection in PBMCs (Fig 2H). Furthermore, qPCR analysis showed that the deficiency of LRRC25 highly enhanced the transcription of IFN-β, IFIT1, and IFIT2 upon H1N1 infection in PBMCs (Fig 2I and J). Taken together, these results suggest that LRRC25 deficiency strongly potentiates the type I IFN activation and antiviral immunity. Figure 2. LRRC25 deficiency enhances antiviral responses A. Protein extracts of control, LRRC25 KO THP-1, and LRRC25 KO HEK293T cells were used to perform immunoblot analysis with the indicated antibodies. B. Control or LRRC25 KO THP-1 cells were infected with VSV-eGFP (MOI = 0.1) for indicated time points. Cell lysates were harvested and analyzed by immunoblot. C, D. Control or LRRC25 KO THP-1 cells were infected with VSV-eGFP (MOI = 0.01) for indicated time points and subjected to qPCR analysis for IFN-β (C) or ELISA analysis (D). E. Control or LRRC25 KO THP-1 cells were infected with VSV-eGFP (MOI = 0.01) for indicated time points and subjected to qPCR analysis for IFIT2 and IFIT1. F. Control or LRRC25 KO THP-1 cells were infected with VSV-eGFP (MOI = 0.01) for 0–18 h and subjected to phase-contrast (PH) and fluorescence microscopy analyses. Scale bar, 40 μm. G. Flow cytometry analyses of THP-1 cells in (F). Numbers at the top-right corner indicate the percentage of cells expressing eGFP. H. PBMCs were transfected with control or LRRC25-specific siRNAs for 24 h, and then, the cells were infected with influenza A/Puerto Rico/8/34 (H1N1) (PR8) (MOI = 5) for the indicated time points. Cell lysates were harvested and used to perform immunoblot analysis with the indicated antibodies. I, J. PBMCs were transfected with control or LRRC25-specific siRNAs for 24 h, and then, the cells were infected with H1N1 (MOI = 5) for the indicated time points and subjected to qPCR analysis for IFNB (I), IFIT2, and IFIT1 (J). Data information: In (A, B, F–H), data are representative of three independent experiments. In (C–E, I, J), data are mean values ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test). Source data are available online for this figure. Source Data for Figure 2 [embj201796781-sup-0003-SDataFig2.tif] Download figure Download PowerPoint LRRC25 interacts with RIG-I Since LRRC25 specifically inhibited RLR-mediated type I IFN signaling, we next sought to determine the molecular targets for LRRC25. We co-transfected 293T cells with RIG-I (N), MAVS, TBK1, IKKi, and IRF3, together with increasing amounts of LRRC25 plus the ISRE luciferase reporter, and found that LRRC25 markedly inhibited activation of ISRE-luc induced by RIG-I (N), but had weak or no inhibition of ISRE-luc reporter activity induced by MAVS, TBK1, IKKi, or IRF3 (Figs 3A and EV3A). Furthermore, we found that LRRC25 markedly inhibited RIG-I-mediated ISRE-luc activation by IC poly(I:C) stimulation (Fig EV3B), suggesting that LRRC25 may block type I IFN signaling through active RIG-I. In addition, we observed that LRRC25 also inhibited ISRE-luc activation induced by MDA5, a RLR recognizing high molecular weight RNA fragments (Loo & Gale, 2011) (Fig EV3C). Figure 3. LRRC25 interacts with RIG-I A. HEK293T cells were transfected with an empty plasmid (no wedge) or increasing amounts (wedge) of plasmid for LRRC25, plus an ISRE-luc reporter and plasmids for RIG-I (N), MAVS, TBK1, IKKi or IRF3. 24 h post-transfection, cell lysates were analyzed for ISRE-luc activity. B. HEK293T cells were transfected with Flag-RIG-I and Myc-LRRC25. After 12 h, cells were left untreated or treated with IC poly(I:C) LMW (5 μg/ml) for 24 h. Cell lysates were immunoprecipitated using anti-Flag, followed by immunoblots using the indicated antibodies. C, D. THP-1 cells (C) or PBMCs (D) were infected with VSV-eGFP (MOI = 0.1) for indicated time points, and cell lysates were immunoprecipitated using anti-RIG-I, followed by immunoblots using anti-LRRC25. E. The structure of RIG-I and its mutants (top). HEK293T cells were transfected with deletion mutants of RIG-I, along with HA-LRRC25 for 24 h. Before harvesting, the cells were treated with MG132 (5 μM) for 4 h. Cell lysates were immunoprecipitated using anti-Flag, followed by immunoblots using the indicated antibodies (bottom). F. The structure of LRRC25 and its mutants (upper). HEK293T cells were transfected with deletion mutants of LRRC25, along with Flag-RIG (N). 24 h post-transfection, cell lysates were harvested and immunoprecipitated using anti-Flag, followed by immunoblots using the indicated antibodies (lower). Data information: In (B–F), data are representative of three independent experiments. In (A), data are mean values ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. LRRC25 cannot interact with MAVS, related to Fig 3 The expressions of RIG-I (N), MAVS, TBK1, IKKi, IRF3, and LRRC25 in Fig 3A were analyzed by IB analysis. HEK293T cells were transfected with an empty plasmid or increasing amounts of plasmid for LRRC25, plus an ISRE-luc reporter and plasmids for RIG-I. After 12 h, cells were left untreated or treated with IC poly(I:C) (5 μg/ml) for 24 h. Cell lysates were analyzed for ISRE-luc activity. HEK293T cells were transfected with an empty plasmid (no wedge) or increasing amounts (wedge) of plasmid for LRRC25, plus an ISRE-luc reporter and plasmid for MDA5. 24 h post-transfection, cell lysates were analyzed for ISRE-luc activity. HEK293T cells were transfected with Flag-LRRC25. After 12 h, the cells were left untreated or treated with IC poly(I:C) LMW (5 μg/ml) for 24 h. Cell lysates were immunoprecipitated using anti-Flag, followed by immunoblots using the indicated antibodies. HEK293T cells were transfected with Flag-RIG-I (N), Flag-MDA5 (N), Flag-MAVS (N), and HA-LRRC25 for 24 h. Before harvesting, the cells were treated with DMSO or MG132 (5 μM) for 4 h. Cell lysates were immunoprecipitated using anti-Flag, followed by immunoblot using the indicated antibodies. Data information: In (A, D, E), data are representative of three independent experiments. In (B, C), data are mean values ± SEM (n = 3). *P < 0.05, ***P < 0.001 (Student's t-test). Source data are available online for this figure. Download figure Download PowerPoint Co-immunoprecipitation (co-IP) and immunoblot analyses further showed that LRRC25 interacted with RIG-I after IC poly(I:C) treatment (Fig 3B). To examine the physiological relevance of these findings, we infected THP-1 cells with VSV-eGFP and found that LRRC25 strongly associated with RIG-I after viral infection (Fig 3C). By contrast, LRRC25 did not associate with MAVS by IC poly(I:C) treatment (Fig EV3D). To further assess whether LRRC25 interacts with RIG-I in primary cells, we isolated PBMCs and then challenged them with VSV-eGFP and found that RIG-I strongly interacted with LRRC25 after VSV-eGFP infection in PBMCs (Fig 3D). Collectively, these data suggest that LRRC25 interacts with RIG-I after viral infection. To distinguish which domain of RIG-I was involved in the interaction with LRRC25, we constructed full-length (FL) RIG-I, truncated RIG-I (N) and RIG-I lacking CARDs (RIG-I (ΔN)), and analyzed their ability to interact with LRRC25. We found that LRRC25 strongly interacted with RIG-I (N), but not with RIG-I (ΔN) (Fig 3E). In addition, we evaluated the interaction between LRRC25 and the CARDs of MDA5 and MAVS. We found that LRRC25 could interact with CARDs of RIG-I and MDA5, but not with CARD domain of MAVS (Fig EV3E), suggesting that the interaction between LRRC25 and CARDs of RLRs is specific. In addition, using truncated LRRC25 fragments, we found that all of the LRRC25 domains can interact with RIG-I (N) (Fig 3F). Taken together, these results suggest that LRRC25 strongly interacts with the CARD domain of RLRs. LRRC25 targets RIG-I for autophagic degradation We next sought to determine how LRRC25 inhibits type I IFN signaling through its interaction with RIG-I. When we transfected 293T cells with plasmids encoding RIG-I (N) and LRRC25, we found that the protein levels of RIG-I (N) were reduced with increasing LRRC25 protein expression (Fig 4A). However, LRRC25 had no effects on the mRNA level of RIG-1 (N) (Fig 4B), indicating that LRRC25 destabilized RIG-I (N) at the protein level.