BinCARD2 as a positive regulator of interferon response in innate immunity

https://doi.org/10.1016/j.bbrc.2019.02.029Get rights and content

Highlights

  • BinCARD isoform2 (BinCARD2) is a one of CARD proteins involved in apoptosis and inflammation.

  • BinCARD2 knock down suppresses innate immune responses induced by 3pRNA, poly(I:C) and vesicular stomatitis virus (VSV).

  • BinCARD2 colocalizes with MAVS on the mitochondrial membrane.

  • BinCARD2 associates with MAVS with its TM (transmembrane) domain.

  • BinCARD2 modulates MAVS oligomerization in innate immunity.

Abstract

Innate immunity is a system that recognizes primarily and excludes pathogenic microorganism. MAVS/IPS-1/Cardif/Visa functions as an adapter protein for RIG-I like receptors (RLRs) and plays a key role in the production of antiviral proteins, interferons (IFNs), for RNA viruses. However, the activation mechanism is not fully understood. Here, we show that BinCARD isoform2 (BinCARD2), carrying CARD domain structure like MAVS, functions in innate immune response. Knockdown of BinCARD2 reduced the RLR ligand-induced expression of IFN-β mRNA and activation of the IFNB promoter. The activation of the IFNB promoter by overexpression of MAVS or TBK1 was suppressed by silencing of BinCARD2, but no effect on IFNB promoter activation by overexpression of TRIF or constitutive activated IRF-3. Furthermore, we confirmed that BinCARD2 protein associated with MAVS but not TBK1 by immunoprecipitation and colocalized with MAVS. Accordingly, we investigated whether BinCARD2 was involved in MAVS activation and showed that siBinCARD2 did not affect RIG-I/MAVS binding but impaired the MAVS oligomerization. Moreover, we infected A549 cells with vesicular stomatitis virus (VSV) and found that induction of IFN-β and IL-6 mRNA after VSV infection was decreased by BinCARD2 knockdown. Thus, these data may suggest that BinCARD2 associates with MAVS to positively modulate the oligomerization in the RIG-I like receptors pathway and activates innate immune response.

Introduction

The immune system is a system to exclude non-self including invading pathogen based on recognition of self and non-self. Vertebrate immunity is divided into innate immunity and acquired immunity. Innate immune system is a basic defense mechanism which recognizes structures and pathogen-associated molecular patterns (PAMPs) of saccharides, lipids, proteins and nucleic acid derived from microorganism to exclude pathogens and infected cells at early stages of infection. Pathogen recognition receptors (PRRs) played an important role in this mechanism [1]. In infection of RNA viruses including influenza virus, hepastis C virus (HCV) and measles virus, intracellular viral RNA sensor RLRs (RIG-I like receptors) including RIG-I (retinoic acid-inducible gene-I) and MDA5 (melanoma differentiation-associated gene 5) recognize genomic RNA of invaded RNA virus as PAMPs [[1], [2], [3], [4]]. Activated RLRs induce production of type Ⅰ interferon (IFN) and inflammatory cytokines via oligomerization of adaptor molecule MAVS (mitochondrial antiviral signaling protein, IPS-1/Cardif/Visa) [[5], [6], [7], [8]]. These cytokines inhibit virus production and activate immunocompetent cells and adaptive immune system. The adoptive immune response has antigen-specificity and exhibit effective antiviral property [1,4,9]. MAVS is essential to inflammatory cytokines production [[5], [6], [7], [8], [9]] and target of viral immune evasion [10,11]. These reports indicate that MAVS has an important role in RLR pathway.

MAVS is an adaptor protein which has CARD (caspase activation and recruitment domain) in N terminal and single-pass TM (transmembrane) domain in C terminal. CARD domain of MAVS is involved in binding with activated RLRs including RIG-I and MDA5 [12,13], which induce MAVS oligomerization [[13], [14], [15]]. The activated MAVS recruits various signaling molecules, including TBK-1 (TANK-binding kinase1) and interacts each other. It inducts activation of transcription factors, IRF-3 (interferon regulatory factor3) and NF-κB (Nuclear factor-kappa B), and production of IFNs and inflammatory cytokines [16,17]. On the other hand, TM domain is related to localization of MAVS on mitochondrial membrane, which is essential to IFN responses [5,18].

From domain database analysis (Prosite), we focused on BinCARD isoform2 (BinCARD2) similar to MAVS in structure. BinCARD2 is one of two isoforms transcript from C9orf89 [19], which has CARD domain in N terminal and TM domain in C terminal. BinCARD isoform1 (BinCARD1) does not have TM domain. BinCARD1 inhibits NF-κB activation by interaction with BCL10 (B-cell lymphoma/leukemia 10) essential for activation of NF-κB in activated T cells [20], however, the function of BinCARD2 is still unknown. Recently, BinCARD2 localization on mitochondria was reported [19]. In this study, we hypothesized that BinCARD2 resembled to MAVS in domain structure had a role in innate immunity and investigated BinCARD2 function.

Human embryonic kidney cells 293T (HEK293T), human cervix epitheloid carcinoma (HeLa), human lung carcinoma (A549) were obtained from American Type Culture Collection and maintained in DMEM (Dulbecco's modified eagle medium, Nissui) containing 10% fetal bovine serum (FBS, Invitrogen).

The cDNA for mouse wild type human BinCARD2 (encoded by C9orf89), deletion mutants, BinCARD2ΔCARD and BinCARD2ΔTM, was obtained by RT-PCR of total RNA from A549 cells, HA-tagged MAVS, Flag-tagged RIG-I and TBK1 was obtained total RNA from mouse embryonic fibroblasts or HEK 293T cell as templates. Then the cDNA was cloned into a pTA2 vector with the Target Clone-Plus-TA cloning kit (Toyobo). For Flag- and HA-tagged proteins, cDNA was cloned into the XhoI and NotI sites of the pCXN2-Flag or pIRM-3HA vector (Supplementary Table 3). The TRIF (Toll/IL-1 receptor domain-containing adapter inducing IFN-beta) expression vector [21] and IRF-3/5D expression vector [22] were generously provided by Professor Seya (Hokkaido University) and Kubota Nuclear Research Institute (National Institute of Infectious Diseases), respectively. FuGENE6 (Roche) reagent was used for gene transfer with lipid transfection. Chemically synthesized 21-nucleotide siRNA, including control siRNA (siPerfect Negative control), was obtained from Sigma (Supplementary Table 1). In this experiments, siBinCARD2#2 was used as siBinCARD2. Cells were transfected with 50 nM siRNA in 2.0 μl Lipofectamine 2000 or Lipofectamine RNAiMAX (Invitrogen).

3pRNA was amplified double stranded DNA, sense strand (5′-TAATACGACTCACTATAGGGAAACTAAAAGGGAGAAGTGAAAGTG-3 ′) and antisense strand (5′-CACTTTCACTTCTCCCTTTTAGTTTCCCTATAGTGGTCGTATTA-3′) as a template with MEGAscript Kit (Ambion) at 37 °C for 24 h [23] according to the attached protocol. Poly (I: C) was purchased from GE Healthcare Co., Ltd. Stimulation of various cells was carried out using OPTI-MEM (Invitrogen) and Lipofectamine 2000 (Invitrogen) according to the attached protocol and adjusting the final concentration to 1.0 μg/ml.

Total RNA was analyzed by quantitative RT-PCR with the appropriate primers (Supplementary Table 2) and the ReverTra Ace Moloney murine leukemia virus reverse transcriptase with point mutations (Toyobo), SYBR Premix Ex Taq II reagent mixture (TAKARA) and a StepOnePlus real-time PCR system (Applied Biosystems). Data were normalized to the expression of GAPDH for each sample.

HEK293T cells seeded on 24-well plates were transiently cotransfected with luciferase reporter plasmids (100 ng each of p-125 Luc, p-55C1BLuc (provided by T. Fujita) and pNF-κB-Luc (Promega)), together with increasing doses of expression vector or control vector. As an internal control, 10 ng renilla luciferase reporter plasmid (pRL-TK (TOYO INK)) was transfected simultaneously. Then, 48 h after transfection, cells were stimulated with 3pRNA or poly(I:C) and luciferase activity was measured with the Dual-Luciferase Reporter Assay system (Promega) and Centro LB 960 luminometer (Berthold).

HeLa cells were plated in glass slide glass chamber (Iwaki) and transfected with HA-tagged BinCARD2 and Flag-tagged MAVS expression vector at concentration of 0.5 μg. At 24 h after transfection, the cells were stimulated with 3pRNA for 24 h and fixed with 4% paraformaldehyde at room temperature (RT) for 10 min. Following washing with PBS, the cells were permeabilized by incubation in 0.2% (wt/vol) Triton x-100 (Wako) and blocked by incubation in 2% (wt/vol) bovine serum albumin (A6003; Sigma) in PBS. After blocking treatment, the cells were incubated with mouse anti-HA antibody (HA-Tag (6E2) Mouse mAb # 2367, Cell Signaling) and rabbit anti-human MAVS antibody (MAVS Antibody # 3993, Cell Signaling) at RT for 1 h. Flag-tagged MAVS and HA-tagged BinCARD2 were stained with anti-mouse IgG antibody conjugated with Alexa Fluor 594 dye-labeled chicken and anti- rabbit IgG antibody conjugated with Alexa Fluor 488 dye-labeled chicken (Life Technologies) by incubation at RT for 1 h. Subsequently, the glass slide was mounted with VECTASHIELD H-1000 (Vector) and observed with a confocal laser microscope Fluoview FV 1000 (Olympus).

HEK 293T were seeded in 10 cm dish and expression vector and siRNA were introduced 24 h after. HEK293T cells were lysed with lysis buffer (1% NP-40, 50 mM Tris-HCl, pH7.5, 150 mM NaCl, 1 mM EDTA) to which 2 μg/ml Leupeptin, 1 μM APMSF, 1 μM Na3VO4 were added. Mouse antibody against HA-tag (Anti-HA-tag mAb M132-3, MBL), or rabbit anti-Flag antibody (Anti-Flag antibody produced in rabbit F 7425, Sigma), and the mixture was added to whole cell lysate. After 16 h incubation at 4 °C, Protein G-Sepharose 4 Fast Flow (GE Healthcare) were added. The mixture was incubated at 4 °C for 1 h with gentle shaking. Beads were then washed four times with lysis buffer, heated at 100 °C for 3 min and centrifuged. The immune-complexes in supernatant were eluted with SDS-PAGE sample buffer, and the precipitated proteins analyzed by Western blot.

Total proteins were extracted with lysis buffer and measured the absorbance with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). After addition of 2 × SDS buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.04% bromophenol blue, 10% 2-mercaptoethanol) to 20 μg proteins, samples were heated for 3 min at 100 °C. After 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the samples were developed and transferred to a polyvinylidene difluoride (PVDF) membrane Immobilon-P (Pore size 0.45 μm: Millipore). The transferred PVDF membrane was immersed in (5% skim milk or 2% BSA TBS-T containing 0.6% TBS) solution for 1 h at RT. After reacting the primary antibody at room temperature for 1 h, the antibody was washed with TBS-T for 30 min, followed by reacting the secondary antibody at RT for 1 h, and then washed with TBS-T for 30 min. Chemiluminescence emission was detected using Pierce Western Blotting Substrate Plus (Thermo SCIENTIFIC) and protein band intensities were assessed using LAS 4000 mini (GE healthcare).

Cells were infected with 5.0 MOI of vesicular stomatitis virus (VSV; Indiana serotype) for 1 h in an infection-medium (serum-free MEM containing amino acids and trypsin) at 37 °C. Virus solution adjusted to have 1 × 106 pfu (plaque forming unit) of VSV was added to OPTI-MEM and incubated at 37 °C. and 5% The cells were harvested 8 h after adding DMEM medium containing 10% FBS and quantitative RT-PCR was carried out. In the VSV infection experiment, the molecular biological defense of Hokkaido University Genetic Disease Research Institute We conducted in the P2 laboratory of the field.

Statistical allays were performed with Microsoft Excel. Differences between control and experimental groups were evaluated with the Student's t-test.

Section snippets

Results

BinCARD2 is reported to be a protein which have CARD domain in N terminal and TM domain in C terminal (19). The domain structure of BinCARD2 like MAVS (Fig. 1A) led us to speculate BinCARD2 involvement in intracellular RNA detection system. Thus, we examined whether BinCARD2 was related to the innate response for RLR ligands, especially IFN-β expression by RIG-I ligand. First, we made three types of siRNA which knockdown BinCARD2 (Supplementary Table 1, Supplementary Fig. 1A) and investigated

Discussion

It is known that CARD domain is widely seen in proteins involved in apoptosis and inflammation, however, function of BinCARD2 CARD domain is still unclear. Some CARD proteins participate in signal transduction via CARD-CARD interaction. MAVS, one of CARD proteins involved in inflammation, has CARD domain in N terminal and TM domain in C terminal. MAVS is known to localize on mitochondrial outer membrane and its CARD interacts with CARD of RIG-I or MDA5 activated by viral RNA detection [1,2,24].

Acknowledgements

This work was supported by grants from Japanese Ministry of Education, Culture, Sports, Science and Technology, grant-in-aid for research (A) (25253030). The authors declare no conflict of interest.

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