ABSTRACT
Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that induces type I interferon-mediated host-protective innate immunity against viral infection. Although cylindromatosis (CYLD) has been shown to negatively regulate innate antiviral response by removing K-63-linked polyubiquitin from RIG-I, the regulation of its expression and the underlying regulatory mechanisms are still incompletely understood. Here we show that RIG-I activity is regulated by inhibition of CYLD expression mediated by the microRNA miR-526a. We found that viral infection specifically upregulates miR-526a expression in macrophages via interferon regulatory factor (IRF)-dependent mechanisms. In turn, miR-526a positively regulates virus-triggered type I interferon (IFN-I) production, thus suppressing viral replication, the underlying mechanism of which is the enhancement of RIG-I K63-linked ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic miR-526a expression inhibits the replication of EV71 virus. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and suggest a novel mechanism for the evasion of the innate immune response controlled by EV71.
IMPORTANCE RNA virus infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. In turn, miR-526a positively regulates virus-triggered type I IFN production and inhibits viral replication, the underlying mechanism of which is the enhancement of RIG-I K-63 ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein; cells with overexpressed miR-526a were highly resistant to EV71 infection. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of the innate immune response controlled by EV71.
INTRODUCTION
EV71 is a positive-stranded RNA virus which belongs to the picornavirus family (1) and is the causative agent of hand-foot-and-mouth disease (HFMD) in young children and infants. The genome of EV71 is approximately 7.5 kb in length and contains a single open reading frame encoding a polyprotein precursor, which is processed into structural (VP1, VP2, VP3, and VP4) and nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins during viral infection (2). Despite the protective role of type I interferon (IFN-I) in EV71 infection, EV71 inoculation is unable to elicit production of these interferons. Most members of the picornavirus family, including poliovirus, rhinovirus, echovirus, and encephalomyocarditis virus, use strategies to inhibit IFN-I induction by interfering with melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-inducible gene I (RIG-I) (3–5) or by restricting IFN secretion through repression of the cellular secretory pathway (6). Recent studies revealed that the 3C protease of EV71 associated with RIG-I and cleaved TRIF (TIR-domain-containing adapter-inducing interferon beta) and IRF7 (interferon regulatory factor 7) (7, 8); moreover, EV71 inhibited IFN-I-induced ISGs (interferon stimulating genes) in host cells by reducing IFNAR1 (type I interferon receptor 1) levels in host cells (9). However, additional work is required to understand the mechanisms by which EV71 is able to escape from innate antiviral responses.
IFN-I, as the first line of host immune response, is critical in mediating antiviral defense. The host senses viral and bacterial pathogen invasion by recognition of pathogen-associated molecular patterns with pattern recognition receptors, including membrane-bound TLRs (Toll-like receptors) (10, 11) and cytosolic sensory molecules, such as the multidomain-containing NOD (nucleotide-binding oligomerization domain) proteins, RIG-I, and MDA-5 helicases (12–14). Both RIG-I and MDA-5 contain caspase recruitment domains (CARDs) that interact with the CARD domain-containing protein mitochondrial antiviral signaling (MAVS) upon binding to uncapped RNA, resulting in MAVS association with IκB kinase (IKK) proteins. While MAVS association with IKKα/β activates NF-κB (nuclear factor-κ gene binding), its association with TBK1 (TANK-binding kinase 1) as well as IKKε leads to the activation of IRF-3/IRF-7; coordinated activation of the NF-κB and IRF pathways further results in the assembly of a multiprotein enhancer complex that drives the expression of IFN-β and the IFN-mediated antiviral immunity (15–19).
RIG-I signaling is negatively regulated at multiple levels. Previous reports showed that the ubiquitination status of RIG-I is controlled by CYLD, a tumor suppressor originally identified as a genetic defect in familial cylindromatosis (20). Indeed, CYLD was shown to interact with the CARDs of RIG-I and to remove K63-linked polyubiquitin chains from RIG-I, which inhibits downstream signaling. DC (dendritic cells) lacking CYLD constitutively polyubiquitinate RIG-I and show enhanced activity of TBK1 and IKKε, suggesting that CYLD regulates basal RIG-I activity by modulating its K63-polyubiquitin status (21). CYLD also acts as a negative regulator of NF-κB and Jun N-terminal kinase signaling pathways by removing Lys-63-linked polyubiquitin from NEMO (NF-κB essential modulator), IKKε, TRAF2 (TNF receptor associated factor 2), or BCL3 (B-cell CLL/lymphoma3) (22–25). These findings thus establish CYLD as a critical regulator of antiviral innate immune response.
MicroRNAs (miRNAs), an abundant class of highly conserved noncoding RNA oligonucleotides (18 to 25 nucleotides [nt] long), suppress gene expression by binding to the 3′ untranslated region (UTR) of target mRNAs. MiRNAs play key roles in the regulation of diverse biological processes. Recently, a role for miRNAs in the regulation of innate immune responses in monocytes and macrophages was proposed (40). Direct roles of miRNAs in innate immune responses were discovered in a study that identified miR-146a as a negative-feedback regulator in RLR signaling by targeting IL-1R-associated kinase (IRAK) 1 and TNF receptor-associated factor 6 (TRAF6) (26). Further reports showed that both miR-155 and miR-132 were induced in a monocyte cell line treated with the TLR4 ligand lipopolysaccharide (LPS) (27). Given the important roles of the RIG-I signaling pathway in the innate antiviral immune response, identifying more miRNAs that can regulate RIG-I-dependent IFN-I production is of vital importance. In fact, many viruses have evolved strategies to interfere with these innate signaling events and hence inhibit IFN-β production. However, to date, there are few reports about the regulation of RIG-I signaling pathway by miRNAs, especially during EV71 infection.
In the present study, we found that miR-526a was significantly upregulated in macrophages upon viral infection in an IRF-dependent manner. Then we demonstrated that miR-526a feedback positively regulated vesicular stomatitis virus (VSV)-triggered IRF3 activation by suppressing CYLD expression and subsequent RIG-I ubiquitination. Furthermore, we found that miR-526a upregulation was blocked by EV71 3C protease, whereas ectopic miR-526a expression inhibited the replication of EV71. Thus, the present study demonstrates for the first time that miR-526a is a positive feedback regulator of the RIG-I signaling and that EV71 targets miR-526a to suppress RIG-I-dependent IFN-I production.
MATERIALS AND METHODS
Cell culture and transfection.293T, Vero, RD, MDCK, and THP-1 cells were cultured in Dulbecco's modified Eagle medium (DMEM) and RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen), 100 U/ml penicillin, and 100 mg/ml streptomycin. Vectors and epitope tagging of Flag-tagged IRF3, IRF7, p65, MDA-5, RIG-I, N-RIG-I, MAVS, and TBK1 were expressed by cloning the respective genes into the pcDNA3-Flag vector. The miR-526a expression plasmid was expressed by cloning miR-526a into the Pires 2-EGFP vector. Small interfering RNA (siRNA) oligonucleotides were ordered from GenePharma and transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The sequences of primers for small interfering RNAs used are shown in Table 1.
Sequences of RNAi oligonucleotides used in the present study
miRNA mimics and inhibitors.MiR-526a mimics (double-stranded RNA [dsRNA] oligonucleotides) and miR-526a inhibitors (single-stranded chemically modified oligonucleotides) from GenePharma were used for the overexpression and inhibition of miR-526a activity, respectively. Negative-control mimics and inhibitors (GenePharma) were transfected as matched controls. Primer sequences were as follows: miR-526a mimics, 5′-CUCUAGAGGGAAGCACUUUCUG-3′ (sense) and 5′-GAAAGUGCUUCCCUCUAGAGUU-3′ (antisense); miR-526b mimics, 5′-CUCUUGAGGGAAGCACUUUCUGU-3′ (sense) and 5′-AGAAAGUGCUUCCCUCAAGAGUU-3′ (antisense); control mimics, 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); miR-526a inhibitor, 5′-CAGAAAGUGCUUCCCUCUAGAG-3′ (chemically modified by 2′-Ome), and control inhibitor, 5′-CAGUACUUUUGUGUAGUACAA-3′.
Viruses and viral plaque assay.The construction of the green fluorescent protein-encoding Newcastle disease virus (NDV-GFP) used in this study was previously described (28) and was obtained from Cheng Cao (Beijing Institute of Biotechnology). Vesicular stomatitis virus (VSV) and herpes simplex virus (HSV) were also obtained from Cheng Cao. VSV and NDV-GFP were propagated in chicken embryo fibroblasts and chicken embryos, respectively, and titrated in MDCK cells. HSV was propagated and titrated in Vero cells. EV71 viral strain GDV-103 (purchased from China Center for Type Culture Collection [CCTCC]) was grown in RD cells and was propagated and titrated in RD cells.
Luciferase reporter assays. (i) CYLD 3′ UTR luciferase reporter assay.Four CYLD 3′ UTR luciferase reporter constructs were made by amplifying the human CYLD mRNA 3′ UTR sequence by PCR and cloning into the pGL3-cm plasmid. The 293T cells were cotransfected with 200 ng of luciferase reporter plasmid (cm-1 to cm-4), 4 ng of pRL-TK-Renilla luciferase (pRL) plasmid, and various RNAs (final concentration, 20 nM). After 36 h, luciferase activities were measured using the dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions. Data were acquired by determining the ratios of firefly luciferase activity to that of pRL luciferase.
(ii) IFN-β, NF-κB, and IRF3 luciferase reporter assay.Luciferase reporter plasmids (200 ng) containing promoters for IFN-β, NF-κB (IFN-β-luc, NF-κB-luc) and IRF3-luciferase plasmids (Gal4-IRF3 and UAS-luc) were cotransfected with 4 ng of pRL plasmid into 293T cells. After various treatments, luciferase activities were measured as described above (29).
RNA quantification.Total RNA from cells was extracted with TRIzol reagent (Invitrogen) following the manufacturer's instructions. For the quantification of miR-526a and miR-526b, RNA was reverse transcribed using the TaKaRa microRNA reverse transcription kit and miRNA-specific stem-loop primers (Table 2). Similarly, U6A small nuclear RNA was quantified using its reverse primer for the reverse transcription reaction (Table 2). Real-time quantitative PCR (RT-PCR) analysis was performed using the multicolor real-time PCR detection system (IQ5; Bio-Rad) and SYBR RT-PCR kits (TaKaRa). RT-PCR primer sequences for miR-526a, miR-526b, and U6A are listed in Table 3, and the relative expression level of miRNAs was normalized to that of U6A by the 2−ΔΔCT threshold method (30). RT-PCR mixtures were incubated in a 96-well plate at 94°C for 2 min, followed by 40 cycles of 94°C for 20 s and 60°C for 30 s. All reverse transcriptase reactions, including no-template controls and reverse transcription-negative controls, were run in duplicate. Sequences of RT-PCR primers for other genes used in the study are listed in Table 3. Data were normalized to the level of β-actin expression in each sample as described above.
Sequences of RT primers used in the present study
Sequences of RT-PCR oligonucleotides used in the study
Immunoprecipitation and immunoblotting.Cells were harvested in cell lysis buffer (50 mM Tris-HCl [pH 7.5], 10 mM sodium fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 mg/ml pepstatin A) containing 1% Nonidet P-40. Whole-cell lysates was subjected to immunoprecipitation with anti-Flag agarose beads (Sigma-Aldrich). Immunoblot analysis was performed with anti-hemagglutinin (HA) and anti-Flag (Sigma-Aldrich). The prepared samples were detected with anti-VSV G (Santa Cruz Biotechnology), anti-CYLD, -IRF3, -IRF3-p, -IKKα, -IKKβ, and -IRF7 (Epitomic), anti-IKKα/β-p (Cell Signaling Technology), anti-IκB-p and -IκB (Sigma-Aldrich), and anti-MDA-5 and -IFNAR1(Abcam) antibodies.
Flow cytometry.293T cells were cotransfected with miR-526a mimics or miR-526a inhibitors followed by NDV-GFP infection. At 24 h postinfection, cells were subjected to flow-cytometric analysis on a FACSCalibur system, and data were analyzed with CellQuest software (both from BD Biosciences). The mean fluorescence intensity and percent green-fluorescing cells were determined.
Assay of IFN-β secretion from THP-1 cells.To assay for IFN-β secretion, THP-1 cells (1 × 106 cells/ml) were infected with VSV. The culture medium was then used to quantify IFN-β with an AlphaLISA IFN-β kit following the manufacturer's instructions (PerkinElmer Life Sciences).
In vivo ubiquitination assay.Samples were harvested after transfection and infection, and whole-cell lysates (WCL) were prepared in a 1% NP-40 lysis buffer supplemented with 0.1% protease inhibitor cocktail (Sigma-Aldrich) and the deubiquitinase inhibitor N-ethylmaleimide (10 mM; Sigma-Aldrich). Protein-protein interactions were disrupted by sonication (3 pulses of 10 s) using the 550 Sonic Dismembrator (Fisher Scientific Inc.) followed by boiling for 10 min in 1% SDS. WCL (250 to 500 μg) were immunoprecipitated with 1 μg of anti-Flag antibody. Polyubiquitination was detected using a monoclonal anti-HA antibody (Sigma-Aldrich).
Statistical analysis.The difference between two groups was statistically analyzed by a two-tailed Student's t test. All data are the averages of triplicates and are representative of results from at least 3 independent experiments.
RESULTS
Viral infection upregulates miR-526a expression through the IRF pathway.To identify miRNAs selectively involved in the regulation of innate immune response against viral infection, we constructed 168 miRNAs of unknown function by using the Pires 2-EGFP vector as a backbone and screened for IFN-β luciferase activity in 293T cells transfected with a cDNA construct from a sublibrary for 168 miRNAs together with an IFN-β luciferase reporter followed by VSV infection. MiR-526a was identified as 1 out of 21 potent IFN-β inducers among 168 miRNAs that we analyzed (data not shown). We thus wanted to investigate whether miR-526a expression was regulated by VSV challenge in human immune cells. To this end, the expression of miR-526a from VSV-infected THP-1 cells was determined at different times postinfection. Figure 1A indicates that miR-526a is a VSV infection-responsive gene in THP-1 monocytes. Its induction was upregulated quickly from 2 to 36 h and peaked at 16 h after VSV challenge, concomitant with the kinetics of VSV replication in the culture supernatants of infected cells (Fig. 1B). However, miR-526b was not induced (Fig. 1C). Furthermore, VSV induced miR-526a expression in a dose-dependent manner (Fig. 1D). MiR-526a was also upregulated in THP-1 transfected with poly(I·C) (MDA-5 agonist), poly(dA·dT), or cyclic bis(3′5′)-diguanylic acid (c-di-GMP) or infected with NDV (Fig. 1E). The expression of miR-526a could be induced by LPS (a TLR4 agonist) or HSV infection to a much lesser extent than VSV infection (Fig. 1E), indicating that miR-526a might participate in the cytosolic innate immune response triggered by RNA virus.
Viral infection upregulates miR-526a expression through the IRF pathway. THP-1 cells were infected with VSV (multiplicity of infection [MOI] = 1) or LPS (0.1 μg/ml) for the indicated times. The expression of miR-526a (A) and miR-526b (C) was measured by RT-PCR and normalized to the expression of U6A in each sample. (B) The titers of VSV harvested from the infected cells were determined at different times postinfection. THP-1 cells were infected with VSV at different MOIs for 16 h (D) or various stimuli as indicated (E and F), and the expression of miR-526a was measured as described for panel A. (G) THP-1 cells were transfected with Flag-tagged IRF3, IRF7, or p65, and miR-526a expression was measured 24 h later as described for panel A. (H and I) THP-1 cells were transfected with the indicated siRNA oligonucleotides (10 nM) for 48 h and then infected with VSV at an MOI of 0.1 for 16 h. MiR-526a expression was measured as described for panel A. Cell-based studies were performed at least three independent times with comparable results. Data are means ± standard deviations (SD). Student's t test was used for statistical analysis. *, P < 0.05, #, P < 0.01, and **, P < 0.01 compared to time zero samples or matched controls.
The C-terminal regulatory domain of RIG-I recognizes RNA carrying 5′-triphosphates (3pRNA) (31). To selectively assess the role of miR-526a in cytosolic signaling pathway, THP-1 cells were treated with the RIG-I agonist 3pRNA or poly(I·C). As shown in Fig. 1F, miR-526a was upregulated by poly(I·C) to a lesser extent than 3pRNA. Therefore, miR-526a may participate mainly in the RIG-I signaling pathway.
We then assessed the contribution of the IRF pathway and the NF-κB pathway to the regulation of miR-526a expression. VSV-induced upregulation of miR-526a expression was significantly increased in cells transfected with the IRF3 or IRF7 expression plasmid but was unchanged in p65-overexpressing cells (Fig. 1G). Consistent with the regulatory role of IRF3/7 in miR-526a expression, VSV-induced upregulation of miR-526a expression was attenuated in siIRF7- and siIRF3-treated macrophages (Fig. 1H). MDA-5, IFNAR, MAVS, and RIG-I knockdown cells also showed defective miR-526a induction in response to VSV infection, whereas VSV-induced miR-526a upregulation was intact in p65 knockdown cells (Fig. 1I), further demonstrating the involvement of the IRF3/7 pathway in the regulation of miR-526a expression.
MiR-526a positively regulates VSV-triggered IFN-I production.To further determine whether virus-induced miR-526a expression could affect the innate immune response, we investigated the role of miR-526a in IFN-I production using miR-526a mimics and miR-526a inhibitors. MiRNA mimics are double-stranded RNAs synthesized to simulate naturally occurring mature microRNAs, while miRNA inhibitors are chemically modified antisense single-stranded RNAs that are widely employed in miRNA loss-of-function studies. As shown in Fig. 2A and B, transfection of miR-526a mimics (miR-526a M) increased miR-526a expression about 6-fold in macrophages, whereas miR-526a inhibitors (miR-526a I) decreased its expression level over 70%. Increasing amounts of miR-526a mimics were cotransfected with the expression construct for RIG-I 2CARD (N-RIG-I), MAVS, TBK1, or MDA-5 into 293T cells together with an IFN-β luciferase reporter (IFN-β-luc); miR-526b mimics were used as negative miRNA controls. Thirty-six hours after transfection, the luciferase activity was measured and normalized based on pRL luciferase activity. As shown in Fig. 2C and D, overexpression of miR-526a, but not miR-526b, potently activated the IFN-β promoter mediated by RIG-I, MDA-5, and its downstream molecules. We next investigated the function of miR-526a in VSV-induced IFN-β production. Consistent with the stimulating effect of miR-526a on the IFN-promoter, miR-526a enhanced IFN-promoter activity substantially in response to VSV infection (Fig. 2E and F). Similar enhancement of VSV-induced activation of NF-κB and IRF3 luciferase reporters by miR-526a was also observed (Fig. 2F). Congruence results showed that miR-526a overexpression increases VSV-triggered IFN-β production at both the mRNA and protein levels (Fig. 2G and H). We went further to investigate the effect of miR-526a on VSV-induced production of proinflammatory cytokines and chemokines and found that miR-526a significantly enhanced VSV-induced production of IL-8 (interleukin 8), ISG56 (IFN-stimulated gene 56), ISG54 (IFN-stimulated gene 54), and CXCL-2 (chemokine ligand 2) in macrophages (Fig. 2I). Taken together, these results demonstrate that miR-526a positively regulates VSV-triggered IFN-I production.
MiR-526a positively regulates VSV-triggered type I IFN production. (A and B) THP-1 cells were transfected with miR-526a mimics (miR-526a M) or control mimics (MCtrl) (A) or with miR-526a inhibitors (miR-526a I) or control inhibitors (ICtrl) (B). The final RNA concentration was 5 nM, and the miR-526a expression was measured by RT-PCR. (C and D) The indicated amounts of miR-526a or miR-526b mimics were cotransfected with the indicated plasmid together with IFN-β-luc, as well as pRL as an internal control. The luciferase activity was measured 36 h later and normalized for transfection efficiency. (E) 293T cells were transfected with control mimics, miR-526a mimics, or miR-526b mimics. After 12 h, cells were transfected with IFN-β-luc and infected with VSV at an MOI of 1 for the indicated times. The luciferase activity was measured and normalized for transfection efficiency. (F) Transfection of 293T cells with increasing amounts of miR-526a mimics or control mimics. After 12 h, the cells were transfected with IFN-β-luc, IRF3-luc, or NF-κB-luc; 24 h later, the cells were infected with VSV at an MOI of 1 for another 16 h. The luciferase activity was then measured and normalized for transfection efficiency. (G and H) THP-1 cells were transfected with miR-526a mimics or control mimics and infected with VSV at an MOI of 1 for 16 h. IFN-β mRNA (G) and protein (H) levels were analyzed by RT-PCR and AlphaLISA, respectively. (I) THP-1 cells were transfected with miR-526a mimics (5 nM) and infected with VSV at an MOI of 1 for 16 h. RT-PCR was performed using the primers specific for IFN-responsive genes, including ISG56, ISG54, IL-8, and CXCL-2. Cell-based studies were performed at least three independent times, with comparable results. Data are means ± SD. Student's t test was used for statistical analysis. **, P < 0.01 compared to the corresponding control.
MiR-526a suppresses viral replication.Consistent with the positive effect of miR-526a on IFN-I production, overexpression of miR-526a inhibitors attenuated IFN-promoter activity substantially in response to VSV infection (Fig. 3A and B). Similar reduction of VSV-induced activation of NF-κB and IRF3 luciferase reporters by miR-526a inhibitors was also observed (Fig. 3A). Congruence results showed that miR-526a inhibits VSV-triggered IFN-β production at both the mRNA and protein levels (Fig. 3C and D).
MiR-526a suppresses viral replication. (A) 293T cells were transfected with increasing amount of miR-526a inhibitors. After 12 h, cells were transfected with IFN-β-luc, IRF3-luc, or NF-κB-luc for 24 h and infected with VSV at an MOI of 1 for 16 h. The luciferase activity was measured and normalized for transfection efficiency. (B) 293T cells were transfected with control inhibitors or miR-526a inhibitors. After 12 h, cells were transfected IFN-β-luc for 24 h and infected with VSV at an MOI of 1 for the indicated times. The luciferase activity was measured and normalized for transfection efficiency. (C and D) THP-1 cells were transfected with control inhibitors or miR-526a inhibitors for 48 h and infected with VSV at an MOI of 1 for the indicated times. IFN-β mRNA (C) and protein (D) levels were analyzed by RT-PCR or AlphaLISA. (E) THP-1 cells were transfected with control mimics or miR-526a mimics together with control inhibitors or miR-526a inhibitors, respectively, for 48 h followed by VSV infection. After 16 h, the level of VSV G protein was monitored by immunoblotting using anti-VSV G antibody. α-Tubulin was used as an equal loading control. (F) 293T cells were transfected with miR-526a mimics or miR-526a inhibitors. After 48 h, cells were infected with NDV-GFP, and images were taken at 16 h postinfection. (G) As described for panel F, NDV-GFP was quantified by FACS. NDV-GFP expression was defined as the product of the percent GFP-positive cells and the geometric mean of the fluorescence index (MFI). (H) 293T cells were transfected with miR-526a mimics or miR-526b mimics. After 48 h, cells were infected with NDV-GFP. The level of NDV replication was monitored by immunoblotting using anti-GFP antibody. α-Tubulin was used as an equal loading control. Numbers below certain Western blots indicate relative levels determined by software-based quantification of the representative results shown. Cell-based studies were performed at least three independent times with comparable results. Data are means ± SD. Student's t test was used for statistical analysis. **, P < 0.01 compared to the corresponding control.
We then investigated the biological significance of virally induced-miR-526a upregulation by measuring VSV G protein levels in the whole-cell lysates of the infected macrophages and found that miR-526a mimics inhibits VSV replication, whereas miR-526a inhibitors rescue the suppressed VSV replication (Fig. 3E). Furthermore, another RNA virus, NDV-GFP, was also tested in this study. By quantification of NDV-GFP with fluorescence-activated cell sorting (FACS) and immunofluorescent analysis, we found that miR-526a mimics inhibit NDV-GFP replication by approximately 50%, whereas miR-526a inhibitors reverse the suppression of NDV replication (Fig. 3F and G). While a similar reduction of NDV-GFP protein by miR-526a mimics was also observed, miR-526b mimics failed to exert any effect on NDV replication (Fig. 3H). These results provide biological evidence that miR-526a acts as a positive regulator of RIG-I-mediated type I IFN signaling and thereby modulates the innate antiviral cellular response.
MiR-526a targets CYLD.To characterize the mechanism associated with miR-526a function, we used a web-based miRNA target prediction program TargetScanHuman (http://www.targetscan.org) to search for potential miR-526 targets. Among the possible targets of miR-526a (some of the predicative targets are listed in Table 4), we focused on CYLD, USP8 (ubiquitin-specific protease 8), and PKR (protein kinase R), which could contribute to the antiviral phenotype observed. Result showed that only CYLD mRNA expression was significantly downregulated by miR-526a overexpression (Fig. 4A). CYLD mRNA has 4 putative miR-526a target sites. To certify the possibility that CYLD is regulated by miR-526a, we constructed reporter plasmids by cloning 4 fragments of the human CYLD 3′ UTR containing prediction sites to the 3′ UTR region of the firefly luciferase gene in the pGL3-cm vector and named them cm-1 to cm-4. By cotransfection of the reporter plasmids and internal control pRL plasmids into 293T cells, we observed that miR-526a downregulated luciferase gene expression in cm-2 and cm-3 plasmids (representing site 1 and site 2 in Fig. 4B; Fig. 4C). Mutations in the two sites of cm-2 and cm-3 completely abolished the ability of miR-526a to inhibit luciferase activity (Fig. 4D). Furthermore, transfection of miR-526a mimics decreased CYLD protein expression (Fig. 4E), whereas miR-526a inhibitors rescue the suppressed CYLD expression (Fig. 4F). To investigate whether the suppression of CYLD by miR-526a is functionally relevant, we analyzed the CYLD expression in response to VSV infection. Concomitant with enhanced miR-526a expression induced by VSV challenge, CYLD expression was reduced at both the mRNA (Fig. 4G) and protein (Fig. 4H) levels in the presence of viral infection. Moreover, virus-induced downregulation of CYLD was inhibited by the expression of miR-526a inhibitors (Fig. 4G and I); however, virus-induced downregulation of MAVS was unchanged by miR-526a inhibitors (Fig. 4J). Together, these findings suggest that endogenous human CYLD is a novel target of miR-526a, and miR-526a acts as a positive regulator of innate antiviral response by suppressing CYLD expression.
miR-526a targets associated with immunity in humans, as predicted by TargetScan
MiR-526a targets CYLD. (A) 293T cells were transfected with miR-526a mimics or control mimics. After 48 h, PKR, USP8, and CYLD expression levels were analyzed by RT-PCR. (B) Human CYLD might be the molecular target of miR-526a. Shown is a sequence alignment of miR-526a and its target sites in the 3′ UTR of CYLD, which was downloaded from TargetScan (http://www.targetscan.org). Red letters are mutated sequences. (C) 293T cells were cotransfected with luciferase reporter plasmids cm-1 to cm-4 containing predicted sites of the human CYLD 3′ UTR. After 36 h, the luciferase activity was measured and normalized for transfection efficiency. (D) 293T cells were cotransfected with the WT or CYLD 3′ UTR luciferase reporter plasmid containing mutations in luciferase reporter plasmids cm-2 and cm-3. After 36 h, the luciferase activity was measured and normalized for transfection efficiency. (E) 293T cells were transfected with increasing amounts of miR-526a or miR-526b mimics for 48 h, and the CYLD level was detected by immunoblotting using anti-CYLD antibody. α-Tubulin was used as equal loading control. (F) 293T and THP-1 cells were transfected with miR-526a mimics or miR-526a inhibitors, and CYLD level was detected by immunoblotting using anti-CYLD antibody. α-Tubulin was used as equal loading control. (G and J) THP-1 cells were transfected with control inhibitors or miR-526a inhibitors. After 48 h, CYLD (G) and MAVS (J) expression levels were detected by RT-PCR. (H) THP-1 cells were infected with VSV at an MOI of 1 for the indicated times. The expression of CYLD was measured by immunoblotting using anti-CYLD antibody. α-Tubulin was used as an equal loading control. (I) THP-1 cells were transfected with control mimics or miR-526a mimics together with control inhibitors or miR-526a inhibitors following VSV infection at an MOI of 1 for 12 h. CYLD expression was detected by immunoblotting using anti-CYLD antibody. α-Tubulin was used as an equal loading control. Numbers below certain Western blots indicate relative levels determined by software-based quantification of the representative results shown. Cell-based studies were performed at least three independent times, with comparable results. Data are means ± SD. Student's t test was used for statistical analysis. **, P < 0.01 compared to the corresponding control.
MiR-526a regulates antiviral response through enhancement of K63-linked RIG-I ubiquitination.The removal of Lys-63-linked polyubiquitination chains from RIG-I by CYLD has been shown to inactivate the RIG-I-mediated antiviral response. As miR-526a targeted CYLD for degradation, we tested whether RIG-I is a target for miR-526a by transfecting 293T cells with full-length RIG-I in the presence of miR-526a mimics. As shown in Fig. 5A, RIG-I underwent increased polyubiquitination in a miR-526a mimic dose-dependent manner. Using a ubiquitin mutant with only one lysine at position 63 available for conjugation (HA-Ub K63), we further revealed that VSV infection leads to increased K63-linked polyubiquitination of RIG-I, which is enhanced by miR-526a mimics but abrogated by miR-526a inhibitors (Fig. 5B). In particular, VSV-induced MDA-5 ubiquitination levels were nearly unchanged by miR-526a mimics or miR-526a inhibitors (Fig. 5C). Furthermore, miR-526a inhibitors failed to exert its suppressive effect on RIG-I ubiquitination in siCYLD cells (Fig. 5D). These data indicated that miR-526a specifically targets CYLD expression and the subsequent RIG-I ubiquitination.
MiR-526a regulates the IFN-I innate immune response through enhancement of K63-linked RIG-I ubiquitination. (A) Cells expressing Flag-RIG-I, HA-Ub, and increasing doses of miR-526a mimics were immunoprecipitated with an anti-Flag antibody and analyzed by immunoblotting with an anti-Flag antibody. (B and C) Flag-RIG-I (B) or Flag-MDA-5 (C) was cotransfected with HA-Ub K63; miR-526a mimics together with control inhibitors or miR-526a inhibitors were infected with VSV at an MOI of 1 for 16 h. Samples were subjected to immunoprecipitation with an anti-Flag antibody and analyzed by immunoblotting with an anti-Flag antibody. (D) Flag-RIG-I was cotransfected with HA-Ub K63. miR-526a inhibitors together with siCYLD oligonucleotides (10 nM) were infected with VSV at an MOI of 1 for 16 h. Samples were subjected to immunoprecipitation with an anti-Flag antibody and analyzed by immunoblotting with an anti-Flag antibody. (E) 293T cells were transfected with miR-526a together with control inhibitors or miR-526a inhibitors for 48 h and infected with VSV (MOI = 1) for 16 h. The levels of CYLD, IKKα, IKKβ, IKKα/β-p, IRF3, IRF3-p, IκB, and IκB-p were monitored by immunoblotting. α-Tubulin was used as equal loading control. (F and G) 293T cells were transfected with increasing amount of miR-526a mimics for 24 h and infected with VSV (MOI = 1) for 16 h (F) or transfected with poly(I·C) for 24 h (G). IRF3 and IRF3 phosphorylation was analyzed by immunoblotting with anti-IRF3 and IRF3-p antibodies. (H) THP-1 cells were transfected with miR-526a mimics or miR-526a inhibitors followed by VSV infection for 12 h. The levels of IRF3 or IRF3-p at the indicated times were monitored by immunoblotting. α-Tubulin was used as an equal loading control. (I) THP-1 cells were transfected with miR-526a mimics or control mimics, and the levels of CYLD, RIG-I, MAVS, and TBK1 were monitored by immunoblotting. α-Tubulin was used as an equal loading control. Numbers below certain Western blots indicate relative levels determined by software-based quantification of the representative results shown. Similar results were obtained in three independent experiments.
To better understand how miR-526a activates RIG-I signaling, IRF3 phosphorylation level was analyzed with miR-526a overexpression. As illustrated in Fig. 5E, miR-526a strongly stimulated IRF3 phosphorylation, IκB phosphorylation, and IKKε phosphorylation induced by VSV. However, addition of miR-526a inhibitors drastically reduced the signals for IRF3 activation and NF-κB activation. Similarly, IRF3 phosphorylation induced by VSV infection (Fig. 5F and H) or poly(I·C) transfection (Fig. 5G) was significantly enhanced by miR-526a mimics. Conversely, miR-526a inhibitors blocked the activation of IRF3 induced by VSV infection (Fig. 5H). Meanwhile, miR-526a mimics reduced the amount of CYLD protein, whereas the levels of RIG-I and TBK1 proteins remained unchanged (Fig. 5I). These observations suggest that enhancement of K63-linked RIG-I ubiquitination by miR-526a may be a mechanism by which miR-526a activates the IFN-I innate immune response.
Downregulation of miR-526a by EV71 3C protease impairs the innate immune response.Since miR-526a upregulation induced by viral infection was partially mediated by IRF7, which can be disrupted by EV71 3C protease (8), we investigated the expression kinetics of miR-526a in response to EV71 infection. Interestingly, EV71 infection reduced miR-526a expression, concomitant with the declined IRF7 expression (Fig. 6A and B). Since EV71 3C protease induced IRF7 cleavage, we evaluated whether EV71 3C affects the expression of miR-526a. As shown in Fig. 6C and D, ectopically expressed EV71 3C blocked the upregulation of miR-526a induced by VSV infection in a dose-dependent manner. Finally, we assessed whether miR-526a is functionally linked to EV71 infection. Specifically, miR-526a mimic-treated 293T cells were infected with EV71. As shown in Fig. 6E, miR-526a mimics reduced the production of VP1 over 90% at 8 h postinfection. VP1 mRNA expression was also significantly reduced by miR-526a overexpression (Fig. 6F). These results suggest that control of IRF7-regulated miR-526a expression by the 3C protein may represent a novel viral mechanism to escape cellular responses.
Downregulation of miR-526a by EV71 3C impairs IFN-I production. (A and B) 293T cells were infected with EV71 at an MOI of 1 for the indicated times. MiR-526a expression was detected by RT-PCR (A), and IRF7 was detected by immunoblotting (B). (C) 293T cells were transfected with different amounts of Flag-3C followed by VSV infection at an MOI of 1. Cells were harvested at the indicated times. MiR-526a expression was monitored by RT-PCR. (D) 293T cells were transfected with increasing amounts of Flag-3C followed by infection with VSV at an MOI of 1. MiR-526a expression was monitored by RT-PCR. (E and F) 293T cells were transfected with control mimics or miR-526a mimics followed by infection with EV71 at an MOI of 1 for the indicated times. The level of VP1 was detected by immunoblotting (E) or RT-PCR (F). Numbers below certain Western blots indicate relative levels determined by software-based quantification of the representative results shown. Cell-based studies were performed at least three independent times, with comparable results. Data are means ± SD. Student's t test was used for statistical analysis. **, P < 0.01 compared to the corresponding control.
DISCUSSION
Various molecules have been shown to regulate the induction of IFN-I by targeting distinct components of the virus-triggered signaling pathways (32–34). Here, we report a novel positive feedback regulator in RIG-I signaling at the level of miRNAs. First, we found that RNA viral infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. Second, we showed that miR-526a positively regulates viral-triggered IFN-I production and that the inducible miR-526a expression suppresses virus replication. Third, we also demonstrated that miR-526a targets human CYLD, which regulates IFN signaling by removing K63-linked RIG-I ubiquitination. Fourth, the present study showed that the miR-526a level is reduced by EV71 infection or 3C expression, which correlates with a decreased IRF7 level in host cells. Finally, cells with overexpressed miR-526a were highly resistant to EV71 infection. These findings not only indicate that miR-526a is a positive regulator of the RIG-I-mediated innate antiviral response but also suggest a novel mechanism for the evasion of innate immune control by EV71.
CYLD is a known deubiquitinase, originally identified as a genetic defect in familial cylindromatosis (20). Previous reports indicated that CYLD inhibits IRF3 signaling and subsequent IFN-β production by removing Lys-63-linked polyubiquitin chains of RIG-I (21, 35). Here we report that miR-526a triggers stronger K63-linked RIG-I ubiquitination, coincident with enhanced IRF3 signaling and NF-κB signaling. Our findings and those in a previous report (35) suggest that CYLD is significantly downregulated by VSV. While the molecular mechanism regarding virus-induced CYLD downregulation remains elusive, our results indicate that miR-526a modulates CYLD protein availability at both the mRNA and protein levels, revealing the possible mechanism of infection-triggered CYLD downregulation.
Besides its important role in RIG-I signaling, CYLD also acts as a negative regulator of NF-κB, Wnt/β-catenin signaling, and Jun N-terminal kinase signaling pathways. Our findings may have implications for the involvement of miR-526a in the development and progression of other human disorders, such as multiple myeloma and colon and hepatocellular carcinomas, linked to loss of CYLD (36–38). MiR-526a may thus play an even more profound role in host antiviral response and cancer. Since viruses also play a role in the pathogenesis of several tumor types, miRNAs influencing viral-host interactions have important implications not only for infectious diseases but also for carcinogenesis. Therefore, miR-526a may have the potential to become a novel drug target in virally induced infectious or malignant diseases.
TargetScan was applied in this study to identify potential miR-526a targets in the human genome. The minimum free energy (MFE) of the miRNA-target duplex was determined to predict the miRNA target sites. Low-MFE values between the miRNAs and the target sites reveal energetically more probable hybridizations between the miRNAs and the target genes. The possible targets of miR-526a in the human genome that may contribute to the antiviral effect are listed in Table 4. Vita software was applied to identify the potential miR-526a targets in the EV71 genome. A previous report (41) showed two potential miR-296-5p targets (nt 2115 to 2135 and nt 2896 to 2920) in the EV71 genome which were validated by luciferase reporter assays and Western blotting; therefore, miR-296 and miR526b were also analyzed in parallel. Table 5 gives the statistics of miRNA targets on viruses with different predictive parameters. For instance, if the MFE cutoff is set at <−20 kcal/mol, and the score cutoff is set at 120, the numbers of targeted viruses for miR-526a, miR-526b, and miR-296 are 3, 7, and 14, respectively. Although our data showed a role for the miR-526a-CYLD-RIG-I axis in regulating antiviral responses, we cannot exclude the possibility that miR-526a may have other possible targets in humans or viruses that could contribute to the antiviral phenotype observed. Besides that, miR-526a is a member of the miR-515 family, which includes a number of members that have high degree of homology. In fact, stem-loop RT analyses that are specific for mature miRNAs and can discriminate among related miRNAs that differ by as little as one nucleotide were used for better specificity and efficiency in this study (39). However, as we did not assess the expression levels of pri- and pre-miR-526a, which are dissimilar to other family members, we still cannot exclude the possibility of other miRNAs from the -515 family regulating the innate immune signaling pathway. Such a possibility needs to be addressed in our future studies. In addition, detection of the miR-526a precursors in the future will also clarify if IRF3/7 regulates miR-526a expression directly on the miRNA gene promoter or just as an indirect effect mediated by the products of other IRF3/7-regulated genes that might influence miRNA.
miR-526a targets on EV71 predicted by Vita
Taken together, the results of present study identified a novel positive regulator of the RIG-I signaling pathway and proposed a new mechanism for the evasion of innate immune control by EV71. The viral infection was first sensed by RIG-I, which in turn initiated IFN-I production against the infection; the simultaneously activated IRF3 and IRF7 upregulated the expression of miR-526a, which then enhanced the innate antiviral immune response by suppressing CYLD expression and subsequent RIG-I activation. EV71-encoded 3C protein targeted miR-526a downregulation to suppress the IFN signaling pathway by IRF7 cleavage, which finally led to the extensive replication of and persistent infection with the invading virus.
ACKNOWLEDGMENTS
This work was supported in part by the Basic Research Program of China (2012CB518900), the National Natural Science Foundation of China (31170029, 31270911, 31300637, 31100960, and 31270800). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
FOOTNOTES
- Received 14 May 2014.
- Accepted 8 July 2014.
- Accepted manuscript posted online 23 July 2014.
- Address correspondence to Xiao Yang, xyang{at}nic.bmi.ac.cn, or Hui Zhong, towall{at}yahoo.com.
C.X, X.H., Z. Zheng, and Z. Zhang contributed equally to this work.
REFERENCES
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