Ankrd17 positively regulates RIG-I-like receptor (RLR)-mediated immune signaling

Authors

  • Yetao Wang,

    1. Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P. R. China
    2. Graduate University of Chinese Academy of Sciences, Beijing, P. R. China
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  • Xiaomei Tong,

    1. Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P. R. China
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  • Gang Li,

    1. Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P. R. China
    2. Graduate University of Chinese Academy of Sciences, Beijing, P. R. China
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  • Junhui Li,

    1. Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P. R. China
    2. Graduate University of Chinese Academy of Sciences, Beijing, P. R. China
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  • Min Deng,

    1. Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P. R. China
    2. Graduate University of Chinese Academy of Sciences, Beijing, P. R. China
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  • and Xin Ye

    Corresponding author
    1. Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P. R. China
    • Full correspondenceDr. Xin Ye, Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), 1 Beichen West Road, Chaoyang District, Beijing 100101, P. R. China Fax: +86-10-64807513 e-mail: yex@im.ac.cn

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Abstract

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), such as RIG-I, melanoma differentiation-associated gene 5 (MDA5), and virus-induced signaling adaptor (VISA), are intracellular molecules that sense diverse viral RNAs and trigger immune responses. In this study, we demonstrate that the ankyrin repeat protein ankrd17 interacts with RIG-I, MDA5, and VISA and upregulates RLR-mediated immune signaling. Overexpression of ankrd17 enhances RLR-mediated activation of IRF-3 and NF-κB and upregulates the transcription of IFN-β. It also promotes RLR signaling in response to poly (I:C), influenza virus RNA, and Sendai virus. Consistently, knockdown of ankrd17 impairs RLR signaling. Furthermore, we demonstrate that ankrd17 enhances the interaction of RIG-I and MDA5 with VISA; the ankyrin repeat domain of ankrd17 is required for its interaction with RIG-I as well as for its function in regulating the RLR pathway. Taken together, our results indicate that ankrd17 is a positive regulator of the RLR signaling pathway.

Introduction

To defend against viral infections, the host has developed various protective approaches. Recognition by pattern recognition receptors (PRRs) of pathogen-associated molecular patterns (PAMPs) initiates a series of signaling cascades that result in the induction of type I interferon (IFN-α/β) and proinflammatory cytokine production [[1]]. Viral genome RNA is an important PAMP which can be recognized by the membrane-bound toll-like receptors (TLRs) TLR3, TLR7, TLR8 and by the cytosolic RIG-I-like receptors (RLRs) RIG-I, and the melanoma differentiation-associated gene 5 (MDA5), and this recognition is critical for establishing an effective viral resistant state [[2, 3]].

As the most effective factors in innate immunity, type I IFNs induce expression of a wide range of antiviral genes through activation of the Jak-STAT pathway [[4]]. Transcriptional regulation is the primary regulatory mechanism of type I IFN expression. Regarding the IFN-β promoter region, it contains four positive regulatory domains (PRDs), PRD I, II, III, and IV. PRD I and III are also known as IFN-stimulated response elements (ISREs) and are activated by phosphorylated IFN regulatory factors (IRFs). IRF-3 and IRF-7 are well-studied transcription factors that are responsible for the activation of IFN-α and IFN-β. PRD II and IV are activated by nuclear factor κB (NF-κB) and ATF-2/c-Jun, respectively. Viral infection leads to the activation of IRF3, NF-κB, and other transcription factors which induce the expression of type I IFN and build up an antiviral state [[5]].

RLRs represent a class of cytosolic PRRs that detect exogenous RNA present in the cell. Many studies have been carried out to understand the mechanisms of RLR-mediated immune signaling that lead to the induction of type I IFNs. It is known that RIG-I and MDA5 selectively recognize different viral RNAs. Following RNA binding, the DexD/H RNA helicase domain induces an ATP- dependent conformational change within RIG-I/MDA5 and allows their N-terminal caspase recruitment domain (CARD) to interact with the N-terminal CARD domain of VISA (virus-induced signaling adaptor, also known as MAVS, Cardif, and IPS-I) [[6-9]]. VISA plays as a crucial adaptor role in the RLR-mediated antiviral response. Once RLRs bind with VISA at the outer membrane of mitochondria, other components such as mediator of IRF-3 activation (MITA, also known as STING) [10, 11], TNF-receptor associated factor 3/6 (TRAF3/6) [7, 12], receptor interacting protein 1 (RIP1) and Fas-associated death domain (FADD) [3, 13] are recruited and a VISA-centered signal transduction complex is formed that then initiates TBK1- or IKK-mediated IRF-3/7 or NF-κB activation, respectively. These signaling events finally trigger the induction of IFN-β [[14, 15]].

Numerous molecules are involved in the regulation of the RLR-signaling pathway with many interacting with critical components of the pathway to enhance or attenuate signaling. Some NLRs (NOD-like receptors) are found to act as negative regulators of the RLR pathway [[16-19]], such as NLRX1, which negatively regulates VISA activity via an interaction between its nucleotide binding domain and the CARD domain of VISA [[20, 21]]. NLRC5 inhibits both RIG-I- and MDA5-mediated NF-κB activation by binding to IKKα/β, RIG-I, and MDA5 [[22]]. WD repeat-containing protein 5 (WDR5), a VISA-interacting protein is responsible for the recruitment of the VISA-associated signaling complex and activation of IRF-3 and NF-κB [[23]]. Many ubiquitin ligases and deubiquitinases act as activators or inhibitors of the RLR pathway [[24]]. For example, Tripartite motif-containing protein 25 (TRIM25) catalyzes the ubiquitylation of RIG-I and enhances its downstream signaling. TRIM25 deficient cells are defective in producing type I IFN during viral infection [[25]]. Cylindromatosis (CYLD), a deubiquitinase, inhibits viral-induced type I IFN production by eliminating the Lys-63-linked polyubiquitylation of RIG-I [[26]]. Some viral proteins are involved in the RLR pathway. Influenza A virus NS1 interacts with VISA, RIG-I and the influenza virus RNA genome to act as a competitive inhibitor of RIG-I [[27]]. The NS3-4A protease complex of the hepatitisC virus can cleave VISA to change its mitochondrial localization and allow the virus to escape from host immune surveillance [[28]]. Understanding the mechanisms of RLR-mediated antiviral responses may help us to develop more effective strategies for immunotherapy and inflammation-related diseases.

The ankyrin repeat is one of the most common protein domains that exclusively mediates protein–protein interactions. Each ankyrin repeat contains 33 amino acid residues and exhibits a helix-turn-helix conformation. Ankyrin repeat proteins usually contain a string of such tandem repeats, which are packed in a linear array to form helix-turn-helix bundles with flexible loops. It has been reported that ankyrin repeat proteins are involved in a variety of physiological processes, such as cell cycle control, transcription regulation, and inflammatory responses [[29-31]]. As a member of the ankyrin repeat protein family, ankrd17 consists of 25 ankyrin repeats at its N terminus that are divided into two clusters by a linker region. Previously, it was found that ankrd17 is involved in cell cycle regulation [[32]]. In this study, we demonstrate that ankrd17 is a positive regulator of RLR-mediated immune signaling. Ankrd17 upregulates RLR-mediated activation of IRF-3, NF-κB, and IFN-β by enhancing the interaction between RIG-I/MDA5 and VISA. Consistent with this, knockdown of ankrd17 impairs RLR-mediated signaling. In addition, we have found that the ankyrin repeat domain of ankrd17 is required for its function in regulating the RLR pathway.

Results

Ankrd17 enhances the RLR-mediated activation of ISRE, NF-κB, and IFN-β promoters

Ankrd17 is an ankyrin repeat protein, which was previously reported to be an important regulator of the cell cycle [[32]]. In preliminary experiments looking for VISA regulatory proteins by expression cloning, we found that ankrd17 strongly interacts with VISA. Given that some ankyrin repeat proteins are involved in the innate immune response [[30]] and that ankrd17 possesses a KH domain (K homology RNA binding domain) that functions in RNA recognition, we asked whether ankrd17 is involved in RLR signaling. First, we used luciferase assays to analyze whether ankrd17 could regulate VISA-mediated immune signaling. 293T cells were cotransfected with VISA and ankrd17 mammalian expression plasmids together with ISRE-luc, NF-κB-luc, or IFN-β-luc reporters. It was found that overexpression of ankrd17 can enhance VISA-mediated activation of ISRE-luc, NF-κB-luc, and IFN-β-luc promoters (Fig. 1A, B, and C). These results demonstrate that ankrd17 coordinates with VISA to enhance expression of the ISRE, NF-κB, and IFN-β reporters. In order to check whether overexpression of ankrd17 affects cell viability, we compared this in control and ankrd17 overexpressed 293T cells using the MTT assay. As shown in Supporting Information Fig. 1A, there was no significant difference in cell viability between control and ankrd17 overexpressing cells. To determine whether ankrd17 upregulates other components involved in RLR-mediated signaling activation, 293T cells were transfected with ankrd17 plus RIG-I, MDA5, MITA, TBK1, IRF-3, or p65 together with the indicated reporters. As shown in Fig. 2A–C, ankrd17 potentiated the RIG-I-mediated activation of the ISRE, NF-κB, and IFN-β promoters. In addition, we found that ankrd17 enhanced MDA5-mediated activation (Fig. 2D) and promoted MITA-mediated activation (Fig. 2E) of the ISRE reporter; however, ankrd17 did not enhance activation of the ISRE reporter induced by TBK1 or IRF-3 (Fig. 2F and G). Ankrd17 increased p65-induced NF-κB activation (Fig. 2H). These results suggest that ankrd17 is involved in RLR signaling and may function upstream of TBK1.

Figure 1.

Ankrd17 enhances VISA-mediated ISRE, NF-κB and IFN-β activation. (A–C) 293T cells were transfected with pCMV myc-Ankrd17 and pCMV FLAG-VISA together with the indicated luciferase reporter and pCMV β-gal as an internal control (relative to β-gal). The luciferase assays were performed 24 h after transfection. Data are shown as the mean + SD and are representative of one of three independent experiments (n = 3). Data were analyzed by independent sample t-test. *p < 0.05,**p < 0.01.

Figure 2.

Ankrd17 enhances RIG-I-, MDA5-, and MITA-mediated RLR signaling.(A–C) 293T cells were transfected with pCMV myc-ankrd17 and pCMV FLAG-RIG-I together with the indicated luciferase reporter and pCMV β-gal as an internal control. (D–H) 293T cells were transfected with pCMV myc-ankrd17 with MDA5, MITA, TBK1, IRF-3 or p65 expression plasmids and the indicated luciferase reporters. All the luciferase assays were performed 24 h after transfection. Data are shown as the mean + SD and are representative of one of three independent experiments (n = 3). Data were analyzed by independent sample t-test *p < 0.05,**p < 0.01.

Ankrd17 promotes poly (I:C)- and Sendai virus (SeV)-mediated activation of IFN-β

To analyze whether ankrd17 was involved in poly (I:C)-mediated activation of the RLR pathway, we transfected 293T cells with different amounts of poly (I:C) and either pCMV myc-ankrd17 or control plasmid together with an IFN-β reporter. As shown in Fig. 3A, the IFN-β promoter was activated by poly (I:C) in a dose-dependent manner as previously reported [[33]] and this activation was significantly enhanced by the overexpression of ankrd17. Interestingly, ankrd17 boosted the IFN-β promoter about 2.5-fold as compared with the control when 0.5 μg or less of poly (I:C) was transfected. This suggests that enhancement of IFN-β promoter expression by ankrd17 correlated with the stimulus intensity up to a certain level. Next, we infected the cells with SeV and analyzed the activity of the IFN-β promoter reporter in ankrd17-overexpressing, as well as control, cells. Consistent with results obtained using poly (I:C)-treated cells, ankrd17 significantly enhanced SeV-triggered IFN-β-luc activation (Fig. 3B). Interestingly, we also found that endogenous ankrd17 was upregulated by SeV infection (Fig. 3C).

Figure 3.

Ankrd17 elevates poly (I:C)- and SeV-triggered IFN-β promoter activation. (A) 293T cells were transfected with the indicated amount of poly (I:C) and pCMV myc-ankrd17 together with an IFN-β reporter for 24 h. The cell lysates were harvested for the luciferase assay. Data are representative of one of three independent experiments. (B) 293T cells were transfected with an IFN-β reporter and pCMV myc-ankrd17 for 24 h and then infected with SeV for 6 h. The luciferase assay was performed accordingly. Data are shown as the mean ± SD and are representative of one of three independent experiments (n = 3). Data were analyzed by independent sample t-test.*p < 0.05. (C) 293T or HCT116 cells were mock treated or infected with SeV for 7 h, then cells were lysed and ankrd17 detected with an anti-ankrd17 antibody in a western blot. β-actin serves as a loading control. Data are representative of one of two independent experiments with similar results.

Ankrd17 upregulates the transcription of IFN-β and ISG54 induced by RIG-I or MITA

To confirm the results obtained, we transfected 293T cells with RIG-I or MITA expression plasmids together with pCMV myc-ankrd17, then performed real time PCR (RT-PCR) to examine the expression of IFN-β and its downstream gene ISG54. Consistent with the results in Fig. 1–3, transcription of IFN-β and ISG54 was enhanced in ankrd17-overexpressing, as compared with control, cells (Fig. 4A and B).

Figure 4.

Ankrd17 enhances the transcription of IFN-β and ISG54. 293T cells were transfected with RIG-I or MITA expression plasmids and pCMV myc-ankrd17 or control plasmid as indicated for 24 h. Then, total RNA was prepared and subjected to real time PCR (RT-PCR) using primers to detect the indicated transcripts. β-actin serves as a loading control. Data are representative of one out of three independent experiments with similar results.

Ankrd17 promotes influenza virus RNA-induced activation of IFN-β and enhances the RLR-mediated antiviral response

Next, we wondered whether ankrd17 also promotes RLR signaling in response to RNA virus. To address this question, we infected 293T cells with influenza A virus WSN and extracted the RNA, which contains the influenza A virus RNA genome which is recognized specifically by RIG-I, resulting in triggering of antiviral immunity [[34, 35]]. The RNA was then transfected together with pCMV myc-ankrd17 and the IFN-β reporter into 293T cells. The results show that overexpression of ankrd17 significantly enhanced activation of the IFN-β promoter by the virus RNA (Fig. 5A). We also analyzed the transcription of IFN-β induced by virus RNA using RT-PCR and found that the expression of ankrd17 promoted this (Fig. 5B). These data indicate that ankrd17 promotes RLR signaling in response to virus RNA. To examine whether ankrd17 could elevate VISA- or RIG-I-mediated antiviral responses, we transfected 293T cells with VISA or RIG-I expression plasmids in the presence or absence of ankrd17 and then infected the cells with influenza virus. We analyzed the levels of virus matrix protein M1 by immunoblotting. As shown in Fig. 5C, the amount of M1 protein was greatly reduced in ankrd17/VISA or ankrd17/RIG-I cotransfected cells as compared with that in cells transfected with VISA or RIG-I alone. These results indicate that ankrd17 promotes RLR signaling in response to RNA virus infection.

Figure 5.

Ankrd17 promotes influenza virus RNA-induced IFN-β activation and enhances the RLR-triggered antiviral response. (A) 293T cells were transfected with mock, RNA from 293T cells, or RNA from influenza A virus WSN infected 293T cells together with an IFN- β luciferase reporter in the presence or absence of pCMV myc-ankrd17 for 24 h. The cell lysates were harvested for the luciferase assay. Data are shown as the mean + SD and are representative of one out of three independent experiments (n = 3). Data were analyzed by independent sample t-test. *p < 0.05,**p < 0.01. (B) 293T cells were transfected with RNA from 293T cells, RNA from 293T cells infected with WSN and pCMV myc-ankrd17 as indicated for 16 h. Total RNA was extracted and subjected to RT-PCR with primers for IFN-β or β-actin as internal control. Data are representative of one of three independent experiments with similar results. (C) 293T cells were transfected with the indicated plasmids for 24 h, then infected with influenza A virus WSN for 7 h. The cells were lysed and subjected to immunoblotting with the indicated antibodies. Data are representative of one of two independent experiments with similar results.

Knockdown of ankrd17 impairs VISA-mediated activation of ISRE promoter

To further confirm the functional significance of ankrd17 in regulating the RLR pathway, we generated a stable HCT116 cell line (HCT116 si-ankrd17) in which expression of ankrd17 was reduced significantly as compared with control HCT116 cells (HCT116 si-control) (Fig. 6A). In order to know whether knockdown of ankrd17 affects cell viability, we compared the cell viability of HCT116 si-control and HCT116 si-ankrd17 by the MTT assay. As shown in Supporting Information Fig. 1B, there was no significant difference in cell viability between control and ankrd17 knockdown cells. The knockdown and control cells were then transfected with VISA and an ISRE reporter. The data show that the luciferase activity in HCT116 si-ankrd17 cells is much lower than in HCT116 si-control cells (Fig. 6B). We further transfected the cells with an IFN-β-luc reporter followed by infection with SeV. As shown in Fig. 6C, luciferase activity in the infected ankrd17 knockdown cells was lower than that in the infected control cells. These results indicate that knockdown of ankrd17 impairs the VISA- and SeV-induced signaling pathway.

Figure 6.

Ankrd17 knockdown impaired VISA- and SeV-triggered ISRE-luc and IFN-β activation. (A) HCT116 si-ankrd17 and HCT116 si-control cell lines were generated as described in the Materials and methods. The cell lysates were harvested and subjected to immunoblot with an ankrd17 antibody. The experiment was repeated twice independently with similar results. (B) The indicated cells were transfected with pCMV FLAG-VISA or control and ISRE luciferase reporter plasmid for 24 h. The cell lysates were harvested for the luciferase assay. (C) The indicated cells were transfected with an IFN-β reporter for 24 h and infected with SeV for 6 h. The cell lysates were harvested for luciferase assay. (B, C) Data are shown as the mean + SD and are representative of one out of three independent experiments (n = 3). Data were analyzed by independent sample t-test. *p < 0.05,**p < 0.01.

Ankrd17 interacts with RIG-I, MDA5, and VISA

In order to establish the subcellular localization of ankrd17 in 293T cells, we performed cell fractionation experiments. As shown in Fig. 7A, ankrd17 was observed both in the nucleus and cytoplasm. To understand the mechanism of ankrd17 in the regulation of RLR signaling, we asked whether ankrd17 could interact with other components in the RLR pathway. In order to address this question, we transfected 293T cells with FLAG-tagged RIG-I or MDA5 expression plasmids together with pCMV myc-ankrd17. The cell lysates were subjected to coimmunoprecipitation analysis. The data reveal that ankrd17 can interact with either RIG-I or MDA5. (Fig. 7B and C). We also examined if there were any interactions between ankrd17 and MITA, TRAF3/6, IRF-3, or p65; the interaction with VISA serving as a positive control. As shown in Fig. 7D, there was no detectable interaction between andkrd17 and MITA, IRF3, or p65, whereas there was a weak interaction between TRAF6 and ankrd17. Although previous results indicated that ankrd17 can promote MITA-mediated IFN-β induction (Fig. 2E and 4A), we did not detect an interaction between MITA and ankrd17. We also examined the interaction between endogenous ankrd17 and RIG-I or VISA. The data demonstrate that endogenous ankrd17 can interact with both RIG-I and VISA (Fig. 7E).

Figure 7.

Ankrd17 interacts with RIG-I, MDA5, and VISA. (A) The cell fractionation procedure is described above the blot. The indicated subcellular fractions as well as total cell lysate were analyzed by western blot using an anti-ankrd17 antibody. (B–D) 293T cells were transfected with the indicated plasmids for 24 h. The cell lysates were harvested and subjected to immunoprecipitation with FLAG antibody followed by immunoblotting with a myc antibody. (E) The lysates of 293T cells were immunoprecipitated with an RIG-I antibody followed by immnunoblotting with an ankrd17 antibody (upper panel), or immunoprecipitated with an ankrd17 antibody and then immunoblotted with a VISA antibody (lower panel). Data are representative of one of two independent experiments with similar results. * indicates nonspecific band.

The ankyrin repeat domain of ankrd17 interacts with RIG-I and promotes the RLR pathway

To analyze whether the ankyrin repeat domain of ankrd17 is important for its function, we generated ankrd17C, a truncated form of ankrd17 in which all ankyrin repeats were deleted (Fig. 8A). The coimmunoprecipitation data show that ankrd17C is not able to interact with RIG-I, although it can still bind to VISA (Fig. 8B). This indicates that the ankyrin repeat domain of ankrd17 is indispensable for the binding of ankrd17 with RIG-I. We generated two more ankrd17 truncated forms, ankrd17A and ankrd17B in which the first seven ankyrin repeats or the first ankyrin repeat cluster were deleted, respectively (Fig. 8A). The coimmunoprecipitation data indicated that the second ankyrin repeat cluster of ankrd17 is required for its interaction with RIG-I since ankrd17B but not ankrd17C is able to interact with RIG-I (Fig. 8C). Then, we compared the ability of ankrd17 and its mutants to promote VISA-mediated RLR signaling. The data showed that ankrd17A and ankrd17B can also enhanced VISA-mediated activation of ISRE, NF-κB, and IFN-β. Ankrd17C did not enhance VISA-mediated activation of the ISRE and IFN-β reporters and weakly promoted activation of the NF-κB reporter (Fig. 8D–G). These results suggest that the integrity of ankyrin repeat domain is critical for the function of ankrd17 in promoting RLR signaling.

Figure 8.

The ankyrin repeat domain of ankrd17 is required for its interaction with RIG-I and its function in RLR signaling. (A) The scheme of full-length ankrd17 and its mutants. Nuclear export sequence (NES), nuclear localization sequence (NLS), K homology RNA-binding domain (KH). (B, C) 293T cells were transfected with the indicated plasmids for 24 h. The cell lysates were harvested and subjected to immunoprecipitation with an anti-FLAG antibody followed by immunoblotting with an anti-myc antibody. The total cell lysates (TCL) were blotted with the indicated antibodies. Data are representative of one of three independent experiments with similar results. (DG) 293T cells were transfected with pCMVmyc-ankrd17 (FL) or its mutants (ankrd17A, B, and C) with pCMV FLAG-VISA together with indicated luciferase reporter plasmids for 24 h. Then cell lysates were harvested for luciferase assay (D–F) and immunoblotted with indicated antibodies (G). (D–F) Data are shown as the mean + SD and are representative of one of three independent experiments (n = 3).

Ankrd17 enhances the interaction of RIG-I and MDA5 with VISA

The results presented in Fig. 7 and 8 imply that ankrd17 may be involved in mediating the interaction of RIG-I and VISA. To test this hypothesis, we expressed FLAG-tagged RIG-I and HA-tagged VISA with or without myc-tagged ankrd17 in 293T cells and then performed immunoprecipitation analysis. The results show that overexpression of ankrd17 greatly enhanced the interaction between RIG-I and VISA (Fig. 9A). The data in Fig. 2D indicate that ankrd17 also promoted MDA5-mediated activation of the ISRE reporter. We wondered whether ankrd17 can also enhance the interaction between MAD5 and VISA since MAD5 is structurally similar to RIG-I. We replaced FLAG-RIG-I with FLAG-MDA5 and repeated the experiment. As expected, expression of ankrd17 also greatly improved the interaction between MDA5 and VISA, whereas the interaction between MITA and VISA did not change in ankrd17-overexpressed cells (Fig. 9B). In addition, ankrd17 also did not enhance the interaction between TRAF3/6 and VISA (data not shown). Therefore, we propose that ankrd17 may upregulate RLR signaling by enhancing the interaction of RLRs with VISA.

Figure 9.

Ankrd17 enhances the interaction of RLRs with VISA. (A, B) 293T cells were transfected with the indicated plasmids for 24 h. The cell lysates were harvested and immunoprecipited with a FLAG antibody followed by immunoblotting with an HA antibody. The whole cell lysates were blotted with anti-HA, anti-myc and anti-FLAG. Data are representative of one of three independent experiments with similar results.

Discussion

The sensing of cytosolic virus RNA depends on RLRs. RIG-I specifically recognizes 5′-triphosphate (5′-ppp)-containing RNA virus genomes, whereas MDA5 recognizes relatively long double-stranded viral RNAs [[36, 37]]. As RLRs bind to cytosolic virus RNA, they will be recruited to the adaptor protein VISA to trigger downstream signaling pathways that lead to the production of proinflammatory cytokines and type I IFNs [[6]]. Therefore, understanding how RLR-mediated innate immune responses are regulated is of great significance not only in developing more effective antiviral strategies but also in the treatment of inflammation-associated diseases.

The RLR pathway-mediated induction of type I IFNs by virus RNA seems to represent a ubiquitous mechanism existing in most cell types [[38]]. Many proteins have been found to be involved in the RLR pathway, acting as effective regulatory factors [[34]]. In regard to RLRs and VISA, ankrd17 is widely expressed in all examined human cells [[32, 38]], and our experiments suggest that ankrd17 can be physically associated with RIG-I and VISA. Overexpression of ankrd17 enhanced RIG-I-, MDA5-, VISA-, and MITA-mediated activation of ISRE, NF-κB, and IFN-β. Conversely, as compared with mock treated cells, VISA-mediated activation of ISRE was severely impaired in ankrd17 knockdown cells. Therefore, the ankrd17-mediated RLR-signaling enhancement may also widely exist in most kind of cells. Interestingly, MITA has also been reported to play an important role in virus DNA-mediated cell antiviral responses [[39]]. In our study, we observed that overexpression of ankrd17 enhanced MITA-mediated ISRE-luc activation and IFN-β transcription, whereas ankrd17 knockdown resulted in impaired ISRE-luc activation induced by MITA (data not shown). This suggests that ankrd17 may be involved in viral DNA-triggered immune responses, although we did not detect an interaction between MITA and ankrd17.

As widely used defense mechanisms against microbial invasion, the TLR and RLR pathways share many key molecules, such as TBK1, IKKs, TRAF3/6, IRF-3/7, and NF-κB and even some regulatory factors, such as GSK3β [[2], [16], [18], [40]]. Our preliminary data indicate that ankrd17 also potentiates TIR-containing adaptor inducing IFN-β (TRIF)-induced IFN-β-luc activation (data not shown) and interacts with TRAF6. Whether ankrd17 participates in TLR3/4 or other TLR-mediated signaling is an intriguing subject for further study.

Structurally, ankrd17 is a typical ankyrin repeat protein containing 25 ankyrin repeats which is further divided into two clusters by a linker peptide. According to our knowledge, the ankyrin repeat is one of the most widely existing protein domains that exclusively mediate protein–protein teractions [[29]]. The ankyrin repeat motifsof ankrd17 seem not to be required for its association with VISA since the truncated form of ankrd17 that lacks the ankyrin repeats (ankrd17C) strongly interacts with VISA, whereas the second ankyrin repeat cluster of ankrd17 containing ten ankyrin repeats is indispensable for its interaction with RIG-I. We also found that ankrd17C did not enhance VISA-mediated ISRE-luc and IFN-β-luc activation. These data suggest that the integrity of the ankyrin repeat domain of ankrd17 is critical for its function in the RLR pathway. Inconsistent to our initial assumption, we found the truncated form Ankrd17C which contains the KH domain did not enhance the VISA-mediated activation on ISRE and IFN-β reporters as shown in Fig. 8. So the data indicated that the ankyrin repeat but not KH domain of ankrd17 is required for its enhancing effect on RLR pathway. Whether KH domain of Ankrd17 could bind viral and host RNAs and possesses other functions still needs further investigation.

VISA is regarded as a downstream adaptor of RIG-I and MDA5 and is considered as an RLR binding protein through a CARD–CARD interaction [[34]]. Indeed, we detected weak binding of RIG-I and MDA5 with VISA by coimmunoprecipitation in an overexpression system; however, consistent with another report [[8]], we did not observe an endogenous interaction between RIG-I and VISA, even in virally infected cells. It is still not clear how the interaction between RIG-I and VISA takes place in physiological conditions and whether other molecules are involved in this process. Our results show that the interactions between RIG-I and VISA as well as MDA5 and VISA were apparently enhanced by ankrd17. However, we did not find an obvious effect of ankrd17 on the interactions between VISA and MITA, TRAF3, or TRAF6. These observations indicate ankrd17 may be specifically involved in the interactions between RLRs and VISA.

Ankyrin repeat proteins widely exist in different kinds of organisms. The assembly of multiple ankyrin repeat motifs renders ankyrin repeat proteins highly versatile in protein–protein interactions. Many of these interactions and their functions have been well investigated, for example, 53BP2-p53, INK4-CDK6, IκBα-NF-κB [[29]], of which the IκBs are considered to be a family of structurally related and evolutionarily conserved immune regulators [[31, 41]]. There are five IκBs (IκBα, IκBβ, IκBγ, IκBɛ, and Bcl-3) in mammals and one (Cactus) in the fly. These IκB proteins bind to NF-κB dimers by their ankyrin repeat domain to keep NF-κB in the cytoplasm and to sequestrate them from their transcriptional targets [[42]]. Here, we identified that the ankyrin repeat protein ankrd17 functions as a positive regulator by enhancing the interaction of RIG-I or MDA5 with VISA, although the detailed mechanism regarding how ankrd17 regulates the RLR-signaling pathway still needs to be further investigated. Based on these observations, we propose that there may be more ankyrin repeat proteins involved in immune signaling with the ankyrin repeats forming a modular architecture that mediates the interactions with their target proteins.

Materials and methods

Plasmids and antibodies

Ankrd17 (full-length), ankrd17A (aa464-2352), ankrd17B (aa726-2352), and ankrd17C (aa1164-2352) were each cloned into pCMV-myc. pCMV FLAG VISA, RIG-I, IRF-3, and MITA were provided by Hong-Bing Shu (College of Life Sciences, Wuhan University, China). myc-Tagged TBK1 was generated by cloning TBK1 cDNA into pCMV-myc. ISRE, NF-κB, and IFN-β promoter luciferase reporter plasmids were from Hong Tang (Chinese Academy of Science, China), pCMV FLAG TRAF3 and TRAF6 were provided by Xu Z (Chinese Academy of Science). pCMV FLAG IKK-β was provided by Geraldine Brosnan (Viral Immune Evasion Group School of Biochemistry and Immunology, Trinity College, Dublin, Ireland), pCMV FLAG MDA5 was provided by Dr. Jan Rehwinkel (Cancer research UK, London Research Institute, UK). pCMV FALG p65 was provided by Chee-Kwee Ea (California Institute of Technology, USA).

The following antibodies were used: mouse monoclonal anti-FLAG, HRP-conjugated antimouse or rabbit IgG, mouse anti-FLAG M2 agarose (sigma), rabbit anti-ankrd17 as described previously [[32]], mouse anti-ankrd17 (Abnova), mouse anti-myc antibody (9E10), mouse anti-VISA, and rabbit anti-RIG-I (Santa Cruz Biotechonolgy).

Cell culture and virus infection

293T and HCT116 cells were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (PAA), 10 mM HEPES buffer (pH 7.4) in 37°C with 5% CO2. For virus infection, 293T cells were plated on 10 cm dish and infected with WSN virus at a multiplicity of infection of 1 for 16 h and then the virus was harvested.

RNA preparation and RT-PCR

Total RNA of 293T cells, virus infected or transfected 293T cells was extracted using TRIzol reagent (Invitrogen). For RT-PCR, the cDNA was synthesized by AMV reverse transcriptase (Promega) followed by PCR with the indicated primers. Gene-specific primers were as follows: ISG54 forward, 5′-AAT GCC ATT TCA CCT GGA ACT TG-3′, ISG54 reverse, 5′-GTG ATA GTA GAC CCA GGC ATA-3′; IFN-β forward, 5′-CAC GAC AGC TCT TTC CAT GA-3′, IFN-β reverse, 5′-AGC CAG TGC TCG ATG AAT CT-3′; RIG-I forward, 5′-TCC TTT ATG AGT ATG TGG GCA-3′, RIG-I reverse, 5′-TCG GGC ACA GAA TAT CTT TG-3′; MITA forward; 5′-ACC AGA GCA CAC TCT CCG GTA CCT-3′, MITA reverse, 5′-GCT CAC TGC ACC CCG TAG CAG GTT G-3′.

Generation of an ankrd17-knockdown cell line

293T cells were transfected with pSuper shRNA-ankrd17 or pSuper shRNA-control as a negative control for 48 h. The supernatants were harvested and used to infect HCT116 cells in the presence of 5 μg/mL of polybrene for 48 h. Then, the HCT116 cells were selected with puromycin (1 μg/mL) for 3 weeks and named either HCT116 si-ankrd17 or HCT116 si-control for pSuper shRNA-ankrd17 or pSuper shRNA-control transfected cells, respectively.

Transfection and luciferase assay

293T cells or HCT116 si-ankrd17 or HCT116 si-control cells were transfected with the indicated plasmids or RNA for 24 h. The cell lysates were harvested for luciferase assay on Glomax-2020 luminometer (Promega) according to the manufacturer's instructions. The raw data were analyzed by independent sample t-test using SPSS statistical software.

Immunoprecipitation

293T cells were transfected with plasmid for 24 h, and lysed in lysis buffer (1% TritonX-100, 150 mM NaCl, 20 mM HEPES (pH7.5), 10% glycerol, 1 mM EDTA) containing protease inhibitor (Roche). The lysates were immunoprecipitated with the corresponding antibodies at 4°C for 3 h and subjected to immunoblot with the indicated antibodies.

Cell viability assay

293T cells and HCT116 cells were seeded and cultured for indicated time. Then 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT) solution (5 mg/mL) were added and incubated at 37°C for 3 h. The supernatant was removed and DMSO was added to dissolve formazan particles. The absorbance at 570 nm was measured with spectrophotometer. The percentage of cell viability was calculated accordingly.

Acknowledgments

This work was supported by The Ministry of Science and Technology of China (2012CB519003, 2009ZX10004-101, 2011CB504705) and Chinese Academy of Sciences Innovation projects (KSCX2-YW-R-198, KSCX2-EW-J-6). X.Y. is a principal investigator of the Innovative Research Group of the National Natural Science Foundation of China (Grant No. 81021003).

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Abbreviations
CARD

caspase recruitment domain

IRF

IFN regulatory factor

ISRE

IFN-stimulated response element

KH domain

K homology RNA binding domain

MDA5

melanoma differentiation-associated gene 5

MITA

mediator of IRF-3 activation

NLR

NOD-like receptor

PRD

positive regulatory domain

RIG-I

retinoic acid-inducible gene-I

RLR

RIG-I-like receptor

SeV

Sendei virus

TRAF

TNF-receptor associated factor

VISA

virus-induced signaling adaptor

Ancillary