3C Protease of Enterovirus D68 Inhibits Cellular Defense Mediated by Interferon Regulatory Factor 7

ABSTRACT

Human enterovirus 68 (EV-D68) is a member of the EV-D species, which belongs to the EV genus of the Picornaviridae family. Over the past several years, clusters of EV-D68 infections have occurred worldwide. A recent outbreak in the United States is the largest one associated with severe respiratory illness and neurological complication. Although clinical symptoms are recognized, the virus remains poorly understood. Here we report that EV-D68 inhibits innate antiviral immunity by downregulation of interferon regulatory factor 7 (IRF7), an immune factor with a pivotal role in viral pathogenesis. This process depends on 3C^pro, an EV-D68-encoded protease, to mediate IRF7 cleavage. When expressed in host cells, 3C^pro targets Q167 and Q189 within the constitutive activation domain, resulting in cleavage of IRF7. Accordingly, wild-type IRF7 is fully active. However, IRF7 cleavage abrogated its capacity to activate type I interferon expression and limit replication of EV-D68. Notably, IRF7 cleavage strictly requires the protease activity of 3C^pro. Together, these results suggest that a dynamic interplay between 3C^pro and IRF7 may determine the outcome of EV-D68 infection.

IMPORTANCE EV-D68 is a globally emerging pathogen, but the molecular basis of EV-D68 pathogenesis is unclear. Here we report that EV-D68 inhibits innate immune responses by targeting an immune factor, IRF7. This involves the 3C protease encoded by EV-D68, which mediates the cleavage of IRF7. These observations suggest that the 3C^pro-IRF7 interaction may represent an interface that dictates EV-D68 infection.

INTRODUCTION

Enterovirus D68 (EV-D68) was first isolated from children with lower respiratory tract infections in California, USA, in 1962 and belongs to the species Enterovirus D within the Enterovirus genus, Picornaviridae (1). A global upsurge of EV-D68 infections in patients with respiratory tract infections (RTIs) has been observed in recent years (2–21). In 2014, a large outbreak of EV-D68 infections occurred in the United States, which raised public health concern owing to severe respiratory illness and neurological complications (22–30).

Although linked to clinical disease, EV-D68 remains poorly characterized. EV-D68 is structurally similar to other enteroviruses (31). The virus possesses a genome approximately 7.4 kb in size, with the capacity to encode a large precursor that is processed into structural proteins (VP1, VP2, VP3, and VP4) and nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) (17). Viral infection initiates with sialic acids of the epithelial cells (32). In this process, the virus preferentially binds to α2,6-linked sialic acids rather than to α2,3-linked sialic acids (33). In addition, EV-D68 is able to infect leukocyte cells (34). As such, active replication of EV-D68 is thought to trigger cytokine responses (35).

It is well established that the pattern-recognition receptors (PRRs) initiate innate antiviral immunity through activation of interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), and/or nuclear factor-κB (NF-κB) (36). This leads to the induction of type I interferons (IFNs) and inflammatory cytokines (37). IRF3 is a major player in the early phase of IFN induction, whereas IRF7 is critical in the late phase because its expression requires IFN derived from the initial infection (38, 39). Once activated, IRF7 cooperates with IRF3 to mediate antiviral responses. Recently, we reported that 3C^pro of EV-D68 perturbs the Toll-like receptor 3 (TLR3) pathway that controls cytokine expression (35). Nonetheless, whether EV-D68 targets other immune factors is unknown.

In this study, we found that EV-D68 suppresses expression of type I IFNs through cleavage of IRF7 in infected cells. This activity requires a functional viral 3C^pro. Furthermore, we show that IRF7 cleavage occurs at two sites located in the constitutive activation domain (CAD), resulting in inactive IRF7 fragments. Together, these results suggest that control of IRF7 by 3C^pro may be a viral mechanism that contributes to EV-D68 disease.

MATERIALS AND METHODS

Cell lines and viruses.

293T (CRL-11268; ATCC) cells and HeLa (CCL-2; ATCC) cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT), 100 U/ml penicillin, and 100 U/ml streptomycin at 37°C in a 5% CO₂ humidified atmosphere. Human monocytic THP1 (TIB-202; ATCC) cells were cultured in RPMI 1640 media supplemented with 10% FBS. EV-D68 infection was carried out as described previously (35). Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were cultured in RPMI 1640 media supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 U/ml), glutamine (2 mM), HEPES (5 mM), and sodium pyruvate (0.5 mM).

Plasmids.

Plasmids pEGFPC1, pEGFP-3C and pEGFP-3C variants, pCMV6-Flag-Myc-IRF7, pGL3-IFN-β–Luc, IFN-α4–Luc, and pRL-SV40 have been described elsewhere (35, 40). The IRF7 mutants were constructed by using Pfu DNA polymerase (Stratagene, La Jolla, CA). Plasmid pCDNA3.1-V5-IRES-EV71-2A was a gift from Zhendong Zhao (Institute of Pathogen Biology, Chinese Academy of Medical Sciences, China) and was described before (41). To construct pCDNA3.1-V5-IRES-EV-D68-2A, the sequences of EV71 were replaced with that of EV-D68. All variants were confirmed by subsequent sequencing.

Antibodies and reagents.

Antibodies against Flag, Myc, green fluorescent protein (GFP), V5, and β-actin were purchased from Sigma (St. Louis, MO). Rabbit antibodies against TBK1 and IRF7 (target amino acids near the N terminus of human IRF7) were purchased from Cell Signaling Technology (Danvers, MA). Rabbit antibody against IRF7 that recognizes the C terminus of human IRF7 was purchased from Novus Biologicals (Littleton, CO). Rabbit antibody against IRF3 was purchased from Epitomics (Burlingame, CA). Mouse anti-EV-D68 has been described before (35). IRDye 800-labeled IgG and IRDye 680-labeled IgG secondary antibodies were purchased from Li-Cor Biosciences (Lincoln, NE). Rupintrivir was purchased from Santa Cruz (Santa Cruz, CA).

Reporter assays.

293T cells were seeded in 24-well plates at a cell density of 3 × 10⁵ cells per well. At 16 h after plating, cells were transfected with a control plasmid or a plasmid expressing IRF7, IRF7 mutants, and 3C^pro or its variants along with pGL3-IFN-β–Luc, IFN-α4–Luc, and pRL-SV40 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). At 24 h after transfection, cells were harvested and cell lysates were used to determine luciferase activities using a dual-luciferase reporter system (Promega, Madison, WI) according to the manufacturer's instructions. The firefly luciferase activities were normalized to the Renilla luciferase activities.

Quantitative real-time reverse transcription-PCR (RT-PCR).

Total RNA was extracted using TRIzol reagent (Invitrogen) and treated with DNase I (Pierce, Rockford, IL). Aliquots of RNA were reverse transcribed to cDNA using a Superscript cDNA synthesis kit (Invitrogen) according to the manufacturer's instructions. Samples were then subjected to quantitative real-time PCR analysis using primers specific for detection of IFN-β, IFN-α1, interferon-stimulated gene 56 (ISG56), ISG54, interleukin-6 (IL-6), IL-8, RANTES, and IFN-γ-inducible protein 10 (IP10) using a SYBR green kit (TaKaRa Bio, Otsu, Japan), according to the manufacturer's instructions. Expression of IFN-β, IFN-α1, ISG56, ISG54, IL-6, IL-8, RANTES, and IP10 mRNA was normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA expression. The sequences of primers have been described elsewhere (42, 43).

Western blot analysis.

Cells were pelleted by centrifugation and lysed in buffer containing 150 mM NaCl, 25 mM Tris (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, and 1 mM EDTA with protease inhibitor cocktail (Roche, Indianapolis, IN). Aliquots of cell lysates were electrophoresed on 12% SDS-PAGE gels and transferred to a nitrocellulose membrane (Pall, Port Washington, NY). The membranes were blocked with 5% nonfat dry milk, and then proteins on the membrane were incubated with the indicated primary antibodies at 4°C overnight. This was followed by incubation with the corresponding IRD Fluor 800-labeled IgG or IRD Fluor 680-labeled IgG secondary antibody (Li-Cor Inc., Lincoln, NE). After washing, the membranes were scanned with an Odyssey infrared imaging system (Li-Cor, Lincoln, NE) at a wavelength of 700 or 800 nm, and the molecular sizes of the developed proteins were determined by comparison with prestained protein markers (Fermentas, Hanover, MD).

RNA interference (RNAi).

To generate control or IRF7-knockdown cell lines, THP1 and HeLa cells were seeded onto 24-well plates. The next day, cells were infected with lentiviruses expressing scrambled or IRF7-specific short hairpin RNA (shRNA) (GenePharma) at a multiplicity of infection (MOI) of 100. After 72 h, cells were selected by the use of 1 μg/ml puromycin for 1 week. The sequence of short hairpin RNA targeting IRF7 was 5′-CCAAGAGCTGGTGGAATTC-3′ as described previously (44).

Study approval.

This study was approved by the ethical review committee of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences. The methods were carried out in accordance with the approved guidelines.

RESULTS

EV-D68 infection does not stimulate the type I IFN response in HeLa cells.

Innate antiviral immunity plays a pivotal role in controlling virus infection (36). To assess the impact of EV-D68, we investigated the cytokine responses in virus-infected cells. As such, HeLa cells were infected with EV-D68. At different time points postinfection, the cells were examined for the expression of cytokines or IFN-stimulated genes by quantitative real-time RT-PCR analysis. In parallel, Sendai virus was included as a control. As illustrated in Fig. 1A, Sendai virus significantly stimulated IFN-β expression, which diminished as virus infection progressed, and, with a different kinetics or magnitude, also stimulated expression of ISG56, ISG54, IL-8, RANTES, and IP10 (panels B to G). Under these experimental conditions, EV-D68 barely induced expression of IFN-β, IFN-α1, ISG56, ISG54, RANTES, and IP10 (panels A to F), although EV-D68 replicated in infected cells (panel H). However, EV-D68 triggered a rapid induction of IL-8 expression which peaked at 4 h postinfection and declined thereafter. These results suggest that EV-D68 infection negatively modulates the type I IFN response.

FIG 1.

Innate immune responses are not activated in EV-68-infected HeLa cells. HeLa cells were infected with EV-D68 at an MOI of 2. In parallel, Sendai virus (Sev) was included as a control. At different time points (hours) postinfection, total levels of RNA extracted from cells and expression of IFN-β (A), IFN-α1 (B), ISG54 (C), ISG56 (D), RANTES (E), IP10 (F), IL-8 (G), EV-D68, and GAPDH genes were evaluated by quantitative real-time PCR using SYBR green. Results are expressed as increases in mRNA levels relative to those seen in cells collected at 0 h and were normalized by using the GAPDH housekeeping gene.

EV-D68 promotes the cleavage of IRF7 in infected cells.

Previous studies suggest that IRF7 is essential for type I IFN induction (45). As IRF7 protects against picornavirus infection (45), we hypothesized that EV-D68 may inhibit the type I IFN response by targeting IRF7. To evaluate this, we analyzed protein levels in EV-D68-infected cells. Specifically, cells were mock infected or infected with increasing doses of EV-D68. At 24 h after infection, cell lysates were subjected to Western blot analysis. As illustrated in Fig. 2A, in HeLa cells infected with EV-D68, expression of IRF3, TBK1, and β-actin exhibited little change, but the level of IRF7 expression decreased as the infection dose of EV-D68 increased. This coincided with the appearance of VP1 expression. Similarly, this phenotype was observed in THP-1 cells (Fig. 2B) and PBMCs (Fig. 2E). Moreover, a cleaved fragment (about 40 kDa) of IRF7, representing the C terminus, was detectable in PBMCs (Fig. 2E, lane 6). When ectopically expressed Flag-Myc-tagged IRF7 (Flag-Myc was added to the C terminus of IRF7) in 293T cells, this cleaved IRF7 fragment was also seen in EV-D68-infected cells (Fig. 2F, lane 6). Therefore, EV-D68 induced IRF7 cleavage in infected cells. Notably, small interfering RNA (siRNA) knockdown of IRF7 increased viral replication (Fig. 2C and D). Moreover, ectopic expression of IRF7 decreased viral replication (Fig. 2G and H). We conclude that the interaction between IRF7 and EV-D68 determines the outcome of viral infection.

FIG 2.

Effects of EV-D68 infection on IRF7. (A) HeLa cells were mock infected or infected with an increasing dose of EV-D68. At 24 h after infection, cell lysates were subjected to Western blot analysis with antibodies against IRF7 (target amino acids near the C terminus of human IRF7), IRF3, TBK1, VP1, 3C, and β-actin. (B) THP1 cells were mock infected or infected with an increasing dose of EV-D68. At 24 h after infection, cell lysates were subjected to Western blot analysis with antibodies against IRF7, IRF3, TBK1, VP1, and β-actin. (C) Control or IRF7-knockdown HeLa cells were infected with EV-D68 (MOI = 1) for 24 h. Cell lysates were subjected to Western blot analysis with antibodies against IRF7, VP1, and β-actin. (D) Control or IRF7-knockdown THP1 cells were infected with EV-D68 (MOI = 2) for 24 h. Cell lysates were subjected to Western blot analysis with antibodies against IRF7, VP1, and β-actin. (E) Peripheral blood mononuclear cells isolated from healthy donors were mock infected or infected with an increasing dose of EV-D68. At 24 h after infection, cell lysates were subjected to Western blot analysis with antibodies against IRF7, IRF3, TBK1, VP1, 3C, and β-actin. (F) 293T cells were transfected with pCMV6-Flag-Myc-IRF7 for 24 h. Cells were then mock infected or infected with an increasing dose of EV-D68. At 24 h after infection, cell lysates were analyzed by Western blotting using antibodies against Myc (C terminal of IRF7), VP1, 3C, and β-actin. (G) 293T cells were transfected with a control plasmid or with increasing amounts (50 ng, 200 ng, and 500 ng; wedge) of pCMV6-Flag-Myc-IRF7. At 24 h after transfection, cells were infected with EV-D68 (MOI = 2) for 24 h. Cell lysates were subjected to Western blot analysis with antibodies against Flag (C terminal of IRF7), VP1, and β-actin. (H) 293T cells were treated as described for panel G. Total RNA was extracted, and the viral RNA levels of EV-D68 were evaluated by quantitative real-time PCR using SYBR green. Results are expressed as viral RNA levels relative to GAPDH RNA levels. *, P < 0.05; **, P < 0.01. In panels E and F, arrows denote EV-D68-induced cleavage fragments.

Expression of 3C^pro induces cleavage of IRF7 in 293T cells.

EV-D68 encodes two proteases, 2A^pro and 3C^pro. To determine whether viral proteases are responsible for the cleavage of IRF7, we performed additional assays. 293T cells were transfected with 2A^pro or 3C^pro along with IRF7 and subjected to Western blot analysis. As indicated in Fig. 3A, 3C^pro cleaved IRF7, producing a fragment of 40 kDa identical to that seen with EV-D68-infected cells. Furthermore, an antibody that recognizes the N terminus of human IRF7 detected two fragments of approximately 20 kDa. Such cleavage correlated with the extent of 3C^pro expression (Fig. 3B). In reporter assays, 3C^pro of EV-D68 also inhibited IRF7-stimulated IFN-β (Fig. 3C) and IFN-α (Fig. 3D) promoter activation in a dose-dependent manner. On the other hand, when expressed, 2A^pro did not produce any visible cleavage product of IRF7 (Fig. 3E). Consistently, 2A^pro had no inhibitory effect on promoter activation of IFN-β and IFN-α by IRF7 (Fig. 3F and G). These results suggest that 3C^pro of EV-D68 mediates IFR7 cleavage and subsequently inhibits its activity.

FIG 3.

The 3C protease of EV-D68 cleaves IRF7. (A) 293T cells were transfected with plasmids encoding Myc-Flag-IRF7 along with GFP or GFP-3C. At 24 h after transfection, cells were lysed and analyzed by Western blotting with antibodies against IRF7 (targeting the N terminus of human IRF7), Flag, GFP, and β-actin. (B) 293T cells were transfected with Flag-IRF7 and a control plasmid or increasing amounts (5 ng, 10 ng, 20 ng, and 50 ng; wedge) of plasmids expressing GFP-3C. (C and D) At 24 h after transfection, cells were analyzed by Western blotting. Effects of 3C^pro of EV-D68 on IFN-β (C) or IFN-α4 (D) promoter activation are indicated. 293T cells were transfected with IRF7 and GFP-3C along with IFN-β–Luc or IFN-α4–Luc; pRL-SV40 was used as an internal control. At 24 h after transfection, cell lysates were assayed for luciferase activities. (E) 293T cells were transfected with Myc-Flag-IRF7 along with a control plasmid or pCDNA3.1-V5-IRES-2A. At 24 h after transfection, cells were lysed and analyzed by Western blotting with antibodies specific for Flag, V5, and β-actin. (F and G) Effects of 2A^pro of EV-D68 on IFN-β (F) or IFN-α4 (G) promoter activation. 293T cells were transfected with IRF7 and IRES-2A along with IFN-β–Luc or IFN-α4–Luc; pRL-SV40 was used as an internal control. At 24 h after transfection, cell lysates were assayed for luciferase activities. ***, P < 0.001. In panels A and B, arrows denote 3C^pro-induced cleavage fragments.

3C protease activity is crucial for cleavage and inhibition of IRF7.

To probe the nature of 3C^pro-mediated IRF7 cleavage, we determined whether its proteolytic activity is involved. Rupintrivir is an inhibitor of 3C^pro with a broad spectrum of activity against picornaviruses (46). As such, we examined its effect on IRF7 cleavage in transfected cells. As indicated in Fig. 4A, expression of 3C^pro resulted in a cleaved IRF7 fragment in the absence of rupintrivir (lane 2). However, when cells were treated with rupintrivir, 3C^pro of EV-D68 failed to mediate the cleavage of IRF7 (lane 4), suggesting a role of protease activity. As 3C^pro of EV-D68 bears a catalytic triad consisting of Cys147, His40, and Glu71, we also carried out mutational analysis. As shown in Fig. 4B, wild-type 3C^proof EV-D68 effectively induced expression of a cleaved IRF7 product. However, neither the H40D nor the C147A variant exerted IRF7 cleavage as measured by Western blotting (top panel). Immunoprecipitation assays revealed that both the H40D and C147A variants associated with IRF7 (lower panel). These data indicate that 3C^pro forms a complex with IRF7. Notably, wild-type 3C^pro and IRF7 were not coimmunoprecipitated, presumably due to cleavage of full-length IRF7 (Fig. 4B). In reporter assays, wild-type 3C^pro inhibited IFN-β and IFN-α promoter activation mediated by IRF7 whereas the H40D and C147A variants had no inhibitory effect (Fig. 4C and D). Hence, the protease activity of 3C^pro of EV68 is essential to inhibit IRF7 function.

FIG 4.

(A) Effect of the protease inhibitor rupintrivir on IRF7 cleavage. 293T cells were transfected with Flag-IRF7 along with GFP or GFP-3C. Four hours after transfection, cells were incubated with the protease inhibitor rupintrivir (1 μM) for 24 h. Cell lysates were then processed for Western blot analysis. (B) Interaction of 3C^pro variants of EV-D68 with IRF7. 293T cells were transfected with Flag-IRF7 and GFP or GFP-3C variants as indicated. Cell lysates were immunoprecipitated (IP) with anti-Flag antibody. Immunoprecipitates and aliquots of cell lysates were subjected to Western blot analysis (WB) with antibodies against Flag, GFP, and β-actin. (C and D) Effects of 3C^pro variants on IRF7-mediated IFN-β and IFN-α4 promoter activation. 293Tcells were transfected with plasmids encoding IRF7 and IFN-β–Luc (C) or IFN-α4–Luc (D), along with GFP or GFP-3C variants. pRL-SV40 was included as an internal control. Twenty-four hours after transfection, cells were harvested to determine luciferase activities. ***, P < 0.001. In panels A and B, arrows denote 3C^pro-induced cleavage fragments.

3C^pro cleaves IRF7 at Gln-167 and Gln-189.

Since IRF7 cleavage produced two 20-kDa fragments representing the N terminus and one 40-kDa fragment representing the C terminus (Fig. 5A), we inferred that the IRF7 cleavage sites might be located in the N-terminal domain. Therefore, we focused on the sites with a signature sequence (AXXQ↓G/S) of 3C protease. We constructed a series of mutants in which Q was replaced with A or R. As illustrated in IRF7 cleavage assays, the Q167A mutation blocked appearance of the lower 20-kDa fragment (Fig. 5B, lane 4), the Q189R mutation blocked the appearance of the upper 20-kDa fragment (Fig. 5B, lane 6), and the Q167A/Q189R combined mutation blocked the appearance of both (Fig. 5B, lane 8). These two cleavage sites were verified by the antibodies targeting the C terminus of human IRF7 plasmids (Fig. 5B). Q167 and Q189 lie close to each other within the protein, which might account for the fact that these two cleavage fragments could not be distinguished from each other by the antibodies targeting the C terminus in the gel. 3C^pro predominantly mediated IRF7 cleavage at Q189 because the cleavage fragment from Q167A was more abundant than that from Q189R (Fig. 5B, lanes 4 and 6). In reporter assays, the Q167A/Q189R mutant was able to activate the IFN-β and IFN-α promoters and reverse the inhibition of GFP-3C (Fig. 5C and D). Collectively, these data suggest Q167 and Q189 as 3C^pro cleavage sites within IRF7.

FIG 5.

3C^pro of EV-D68 cleaves IRF7 at two specific sites of the N-terminal domain. (A) Schematic diagrams of IRF7 denoting the cleavage sites and fragments for 3C^pro of EV-D68. (B) Cleavage analysis of wild-type IRF7 or 3C^pro-resistant mutants. 293T cells were transfected with wild-type IRF7 or IRF7 mutants with alanine or arginine substitutions for glutamine, along with GFP (lanes 1, 3, 5, and 7) or GFP-3C (lanes 2, 4, 6, and 8), as indicated. Twenty-four hours after transfection, cell lysates were subjected to Western blot analysis with antibodies against IRF7, Flag, GFP, and β-actin. (C and D) Effects of EV-D68 3C^pro on IFN-β or IFN-α4 promoter activation mediated by wild-type IRF7 or 3C^pro-resistant mutants. 293T cells were transfected with wild-type IRF7 or the Q167A/Q189R mutant of IRF7 along with IFN-β–Luc (C) or IFN-α4–Luc (D). pRL-SV40 was used as a control. Twenty-four hours after transfection, cell lysates were assayed for luciferase activities. **, P < 0.01. (E and F) Effects of putative IRF7 cleavage fragments on IFN-β or IFN-α4 promoter activation. 293T cells were transfected with IRF7 or Myc-Flag-IRF3 (full length and fragments consisting of amino acids 1 to 167 and 190 to 503) along with IFN-β–Luc (E) or IFN-α4–Luc (F). pRL-SV40 was used as a control. Twenty-four hours after transfection, cell lysates were assayed for luciferase activities. (G) 293T cells were treated as described for panel G. Total RNA was extracted, and the viral RNA levels of EV-D68 were evaluated by quantitative real-time PCR using SYBR green. Results are expressed as viral RNA levels relative to the GAPDH RNA level. (H) 293T cells were transfected with pCMV6 vector or with IRF7 (500 ng) (full length and fragments consisting of amino acids 1 to 167 and 190 to 503). Twenty-four hours after transfection, cells were infected with EV-D68 (MOI = 2) for 24 h. Cell lysates were subjected to Western blot analysis with antibodies against Myc, VP1, and β-actin. **, P < 0.01.

3C^pro-mediated cleavage inactivates IRF7.

To test whether IRF7 cleavage mediated by 3C^pro has a functional consequence, we generated IRF7 variants with site-specific mutations or deletions (Fig. 5A). In reporter assays, full-length IRF7 activated the IFN-β (Fig. 5E) and IFN-α (Fig. 5F) promoters. When cotransfected with IRF3, IRF7 had an enhanced activity (Fig. 5E). In contrast, the 1-to-167 and 190-to-503 fragments lost the ability to activate the IFN-β (Fig. 5E) and IFN-α (Fig. 5F) promoters. Intriguingly, the 1-to-167 fragment suppressed IRF3-mediated IFN-β promoter activation and the 190-to-503 fragment lost the synergist effect with IRF3 (Fig. 5E). Consistently, in 293T cells, ectopic expression of full-length IRF7 significantly inhibited EV-D68 replication, whereas the 1-to-167 and 190-to-503 fragments failed to inhibit viral replication (Fig. 5G and H).

DISCUSSION

EV-D68 is a human pathogen that primarily causes respiratory illness (17). Asthma or wheezing appears to be a risk factor for severe disease (30, 47). Although the underlying events are largely unknown, aberrant immunity is speculated to mediate viral pathogenesis (17). In this study, we provide evidence that EV-D68 inhibits the expression of type I IFN-, chemokine-, and interferon-stimulated genes in infected cells. In doing so, the 3C protease mediates IRF7 cleavage to facilitate viral replication. Intriguingly, EV-D68 stimulates the expression of inflammatory cytokines (e.g., IL-8). These results are consistent with the notion that EV-D68 differentially modulates cytokine responses, which may affect viral replication or infection.

Although actively replicated in infected cells, EV-D68 induced little expression of endogenous type I IFN, ISGs, and chemokines. This is quite different from Sendai virus, which stimulated robust antiviral immunity in infected cells. These results are in line with the model that EV-D68 potently suppresses the type I IFN response. MDA-5 is generally thought to detect picornaviruses (48–50). However, evidence suggests a pivotal role for TLR3 and RIG-I as well (51–55). We recently reported that EV-D68 perturbs TLR3 via proteolytic cleavage of the TIR-domain-containing adapter-inducing interferon-β (TRIF) adaptor (35). As multiple pathogen recognition sensors exist, this alone is insufficient to explain the effective silencing of IFN expression mediated by EV-D68. In support of this, we noted that EV-D68 infection reduced the expression of IRF7 in epithelial cells, cells of a human macrophage cell line, and PBMCs. Indeed, viral protease 3C, rather than 2A, mediated the proteolytic cleavage of IRF7. The precise relation of 3C to TRIF and IRF7 in EV-D68 infection has yet to be established. It is likely that dual targeting of these immune factors may render EV-D68 an advantage to negate antiviral immunity.

The 3C protease of EV-D68 adopts a chymotrypsin-like fold with two β-barrel domains that harbor the catalytic triad of H40, E71, and C147 (56). We demonstrated that H40D or C147A substitution in the catalytic triad eliminated its capacity to cleave IRF7. In correlation, the 3C mutants failed to block IFN induction. Interestingly, H40D or C147A substitution had no effect on the physical interaction between 3C^pro and IRF7. While the IRF7 binding site(s) is undefined, these data argue that the 3C protease may recruit IRF7 via a separate region or motif that directs IRF7 to the catalytic site. As the result of IRF7 cleavage, EV-D68 3C^pro interrupts the type I IFN response. This is reminiscent of the 3C protease encoded by EV71 that causes hand-foot-and-mouth disease and neurological complications (40). Although it does contain amino acid sequence variations, the 3C protease of EV-D68 is 53% identical to its counterpart from EV71 (56). The fact that both EV-D68 and EV71 target IRF7 likely implies a convergent evolution of viral evasion strategy. It also suggests the importance of IRF7 signaling in the control of enteroviruses.

IRF7 is a master regulator of the IFN immune response to virus infections (45). Our data suggest that replication of EV-D68 is functionally linked to IRF7. Knockdown of IRF7 enhanced viral replication. Conversely, overexpression of IRF7 reduced viral replication. Previous work suggests that, in addition to the N-terminal DNA-binding domain, IRF-7 contains a constitutive activation domain (CAD), a virus-activated domain, an inhibitory domain, and a signal response domain (39). Upon viral infection, these domains work coordinately to regulate the activity of IRF7. We observed that 3C^Pro of EV-D68 cleaved IRF7 at two sites (Gln167 and Gln189) within the CAD, resulting in the amino-terminal domain (fragment 1 to 167) and the carboxyl-terminal domain (fragment 190 to 503). Consequently, IRF7 cleavage may uncouple the DNA binding domain from the activation domain. Alternatively, it may simply destroy the constitutive activation domain, resulting in nonfunctional IRF7. It should be pointed out that IRF7 cleavage fragments also exerted a dominant negative effect on IRF3-mediated gene expression. As such, IRF7 cleavage by EV-D68 adds another layer of viral control, which may provide an additional benefit for EV-D68 infection.

Type I IFN is the first line of host defense against virus infection. It is, therefore, not surprising that enteroviruses have evolved various evasion strategies (57). For example, 3C^pro from coxsackievirus B3 (CVB3), enterovirus 71 (EV71), and poliovirus (PV) cleaves RIG-I (58). On the other hand, 2A^pro from these viral pathogens targets mitochondrial antiviral-signaling protein (MAVS) and melanoma differentiation-associated protein 5 (MDA5). Moreover, 3C^pro of EV71 and CVB3 cleaves TRIF (43, 59). The present study further revealed that EV-D68 negatively regulates IRF7 through the viral 3C protease. It is notable that the 3C^pro proteases of enteroviruses are closely related phylogenetically (56). These observations support the view that enteroviruses likely share common mechanisms to inhibit IFN induction, although elucidation of the relative contribution of each one in viral replication awaits further investigation. Given that IRF7 deficiency predisposes humans to severe viral illness (60), we speculate that the interplay of EV-D68 3C^pro and IRF7 may represent an interface that determines EV-D68 replication or pathogenesis in vivo. Additional work is required to test this hypothesis.