NLRC5 Regulates Enterovirus 71 Infection Through an IFN-β-Dependent Pathway

TL;DR

This study shows how NLRC5 helps control Enterovirus 71 infection by regulating immune responses and limiting viral replication.

Contribution

The study reveals a dual role of NLRC5 in suppressing EV71 replication and modulating immune responses via an IFN-β-dependent pathway.

Findings

EV71 infection increases NLRC5 expression through the RIG-I-IRF3-mediated IFN-β pathway, promoting MHC-I molecule expression.

NLRC5 suppresses EV71 replication and limits inflammation by downregulating IFN-β pathway mediators and binding to the viral genome.

EV71 activates the NLRC5-dependent MHC-I response in an IFN-β-dependent manner.

Abstract

During viral infection, NLR family CARD domain-containing protein 5 (NLRC5) participates in innate immunity through multiple mechanisms. These include regulating type I interferon and related immune factor expression, as well as modulating immune cell functions, such as cytotoxic T lymphocytes (CTLs) and macrophages, thereby promoting antiviral defence and maintaining immune homeostasis. Our study demonstrates that (1) Enterovirus 71 (EV71) infection upregulates NLRC5 expression through the RIG-I-IRF3-mediated IFN-β pathway, which in turn promotes MHC-I molecule expression and (2) NLRC5 suppresses EV71 replication and simultaneously restrains excessive inflammatory responses by fine-tuning IFN-β production through a negative feedback loop. This loop operates via two distinct mechanisms, namely, direct downregulation of key IFN-β pathway mediators (e.g., RIG-I and IRF3) and binding to the 5′UTR of the EV71 genome to inhibit viral replication, thereby indirectly dampening the IFN-β signal. Furthermore, we show that EV71 activates the NLRC5-dependent MHC-I response in an IFN-β-dependent manner. Collectively, these results elucidate the dual role of NLRC5 during EV71 infection, offering novel insights into viral pathogenesis and highlighting potential targets for antiviral drug development.

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1. Introduction

EV71 primarily infects children under three and is the major causative agent of hand, foot, and mouth disease (HFMD). The typical clinical symptoms include scattered herpetic lesions and painful ulcers on the oral mucosa, as well as maculopapular rashes on the hands, feet, and buttocks that gradually develop into vesicles [1]. EV71 is mainly transmitted through the digestive tract, respiratory droplets, and direct contact. Although generally self-limiting, with most patients recovering within one to two weeks, a small proportion of young children can rapidly develop severe complications such as brainstem encephalitis, encephalomyelitis, neurogenic pulmonary edema, and other severe central nervous system (CNS) complications, which can be fatal [2]. In March 2008, an outbreak of EV71-associated HFMD occurred in Fuyang, Anhui Province, China, which resulted in 23 pediatric deaths, rapidly spread nationwide, and became a significant public health threat and economic burden. By May of the same year, the disease was classified as a Category C infectious disease in China [3,4]. In addition to China, large-scale EV71-related HFMD outbreaks have been reported in the United States, Canada, and several Asia-Pacific countries [5]. A pathogen analysis of HFMD-related fatalities in China from 2008 to 2014 revealed that EV71 was responsible for 90.22% in fatality cases [6]. Therefore, effective prevention and treatment strategies against EV71 are of great clinical importance.

Although China approved the first inactivated vaccine for the prevention of severe EV71 infection in 2015 [7], its broad applicability remains challenging due to the high mutation rate of RNA viruses and limited cross-protection against different prevalent strains. Furthermore, considering the limited vaccination coverage and the limited availability of specific antiviral drugs for EV71 infection, an in-depth investigation into the pathogenic mechanisms of EV71 is essential for antiviral therapy [8].

Upon viral infection, the host deploys a range of antiviral defence mechanisms while the virus hijacks cellular components to facilitate its replication and evade immune surveillance. This dynamic interplay ultimately determines the outcome of infection [9]. As the first line of defence, the innate immune system rapidly initiates immune responses during the early stages of pathogen invasion. It employs pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) from host cells, thereby inducing the production of type I interferons (IFN-I) and pro-inflammatory cytokines [10]. The NOD-like receptor (NLR) family plays a crucial role in innate immunity, with NLRC5 being a key member [11]. NLRC5 not only functions as a PRR to recognize pathogens but also regulates inflammatory responses and is critically involved in MHC-I molecule expression [12]. MHC-I-restricted CTLs were significantly elevated in mild severe HFMD group [13].

NLRC5 can bind to the promoter region of the MHC-I gene, promoting its transcription and increasing the expression level of MHC-I molecules [14]. This enhances the ability of host cells to recognize and eliminate virus-infected cells. Previous studies have shown that in A549 cells infected with respiratory syncytial virus (RSV) and ST cells infected with porcine deltacoronavirus (PDCoV), both NLRC5 and MHC-I molecules are activated, leading to the inhibition of viral replication [15,16]. Furthermore, research has revealed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can suppress MHC-I expression by targeting the STAT1-IRF1-NLRC5 axis, thereby evading immune recognition [17].

Here, we report that NLRC5 acts as a host restriction factor against EV71. We show that EV71 infection upregulates NLRC5 expression via the RIG-I–IRF3–IFN-β signal axis, which, in turn, modulates MHC-I expression. Additionally, NLRC5 exerts a negative feedback inhibitory effect on IFN-β production. This work elucidates a key aspect of EV71 pathogenesis and reveals potential targets for antiviral development.

2. Materials and Methods

2.1. Cell and Virus

Our research utilized various cell lines, including RD, Hela, HEK293T, and Vero, all sourced from the Stem Cell Bank, Chinese Academy of Sciences (CAS). The cells were inoculated into Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were then cultured in a 5% CO_2_ incubator at 37 °C. The EV71 AH strain was stored in our lab and propagated in RD cells.

2.2. Animal EV71 Infection Study

Three-day-old C57 sucking mice were challenged with the indicated dose of EV71, while the control group was injected with an equivalent volume of sterile PBS. Each neonatal mouse served as the independent experimental unit. Mice with congenital malformations or abnormal vital signs before the experiment were excluded. The sample size was determined based on previous viral infection studies in neonatal mice, with n ≥ 10 per group to ensure statistical power and a total of 40 mice in the experiment. The outcomes were assessed as follows: (1) survival status and clinical signs were recorded daily for a week; (2) body weight was measured daily; and (3) tissues were collected from the mice 3 days post-infection.

2.3. Western Blot

Cells for Western blot were lysed in 150 µL RIPA (P0013D, Beyotime, Shanghai, China) supplemented with protease inhibitor (P1005, Beyotime, Shanghai, China) and phosphatase inhibitor (GK10012, Glpbio, Montclair, CA, USA) from 6-well plates, then lysed on ice for 10 min. Following the addition of SDS-PAGE loading buffer (5×, diluted to 1×), the samples were denatured at 100 °C for 10 min. The denatured proteins were then separated on 10% or 12% SDS-polyacrylamide gels and electrotransferred onto PVDF membranes. Subsequently, the membranes were blocked with 5% skim milk for 2 h at room temperature before being probed with specific primary antibodies, such as those targeting NLRC5 (1:1000, sc-515888, Santa Cruz, CA, USA), RIG-I (1:1000, sc-376845, Santa Cruz, CA, USA), IRF3 (1:1000, sc-33641, Santa Cruz, CA, USA), phospho-STAT1 (1:1000, sc-8394, Santa Cruz, CA, USA), MHC-I (1:1000, sc32235, Santa Cruz, CA, USA), DYKDDDDK (1:1000, 66008-4-Ig, Proteintech, Wuhan, China), β-actin (1:5000, 66009-1-Ig, Proteintech, Chicago, IL, USA), and VP1 (1:1000, NZK-A14002, hbnzk, Hubei, China) overnight at 4 °C. The membrane was washed with TBST (50 mM Tris Base, 0.15 M NaCl, 2.7 mM KCl, 0.1% Tween-20 (ST2789, Shanghai, China)) 3 times, 10 min/time, and incubated with secondary antibody (1:10,000, Proteintech, Wuhan, China) for 1 h at room temperature. Following another wash with TBST, an-ultra-high sensitivity ECL (HY-K1005, MCE, Monmouth Junction, NJ, USA) was added to visualize the target proteins.

2.4. Real-Time Quantitative PCR

Total RNA extraction from the RD and Hela cells was carried out with Trizol (AG21102, AGbio, Hunan, China). The extracted RNA was reverse-transcribed into cDNA using an Evo M-MLV cDNA synthesis kit (AG11728, AGbio, Hunan, China). Then, the RT-qPCR analysis was conducted employing the SYBR Green Pro Taq HS Premix (AG11701, AGbio, Hunan, China) according to the manufacturer’s protocol. Total mRNA levels were analyzed by 2^−ΔΔCt^. The primers used in this study are provided in the Supplementary Materials.

2.5. Immunofluorescence

After infecting the Hela cells with EV71 for 12 h, the cells were washed with PBS and then fixed with 4% paraformaldehyde at 4 °C for 15 min, followed by Triton X-100 (P0096, Beyotime, Shanghai, China permeabilization at room temperature for 7 min. Subsequently, after another wash with PBS, the cells were treated with 5% bovine serum albumin (BSA) at room temperature for 1 h, followed by incubation with the NLRC5 antibody at 4 °C overnight. Relative secondary antibodies were then incubated for 1 h at 37 °C. The nucleus was stained with 4′, 6-diaminidine-2 phenylindole (DAPI, P0131, Beyotime, Shanghai, China) for 5 min at room temperature. Finally, images were obtained using a fluorescence microscope (Leica, Wet, Germany).

2.6. TCID50

Hela cells were seeded in 96-well plates (1.0 × 10^4^ cells/well). After 18 h, the cells were subjected to infection with ten-fold serial dilutions of the virus. Then, 100 µL of each dilution was added to 8 parallel wells, while untreated cells were added to the control wells. Following infection, all the cells were maintained at 37 °C in a 5% CO_2_ atmosphere and observed under a microscope for 2–3 days. The observation results were recorded, and the virus titre was determined using the Reed–Muench formula [18].

2.7. Cell Transfection

The pcDNA3.1-Flag-NLRC5 plasmid was generously provided by Li YP (Zhongshan School of Medicine, Sun Yat-sen University). The NLRC5 siRNA [19] was constructed by TsingkeBiotechnology Co., Ltd. (Nanjing, China) and then transfected into the Hela cells using Lipofectamine 3000 (L3000008-0.75 mL; Thermofisher, Waltham, MA, USA), following the manufacturer’s protocol.

2.8. Dual-Luciferase Reporter Assay

The luciferase assay was performed using a dual-luciferase reporter assay system kit (RG027, Beyotime) according to the manufacturer’s protocol. For the EV71 5′UTR luciferase reporter assay, the pRHF-EV71 5′UTR plasmid and Flag-NLRC5 plasmid (100 ng, 200 ng, 500 ng, 1000 ng) were con-transfected into 293T cells in 24-well plates using Lipofectamine 3000 (L3000008-0.75 mL; Thermo Fisher, Waltham, MA, USA), and the relative luciferase activity was determined by the dual-luciferase assays after 48 h. For the IFN-β luciferase reporter assay, the IFN-β-luc plasmid, pRL-TK, and the Flag-NLRC5 plasmid (100 ng, 200 ng, 500 ng) were con-transfected into 293T cells in 24-well plates using Lipofectamine 3000. Poly (I:C) was transfected 24 h later, and 48 h later the relative luciferase activity was determined by the dual-luciferase assays. Both firefly luciferase activity and Renilla luciferase activity were measured using a single-tube luminometer (Lumipro, Atila Biosystems, Mountain View, CA, USA).

2.9. Statistical Analysis

All statistical analyses were conducted using GraphPad Prism 8.0.2 (San Diego, CA, USA). The data shown in the graphs are the mean or the mean ± s.e.m. For the comparison between multiple groups, one-way ANOVA was used to test for statistical differences in the standard deviations (SDs) of all conditions. The t-test was used to compare two groups. p < 0.05 was considered statistically significant.

3. Results

3.1. NLRC5 Activation After EV71 Infection

We first injected 3-day-old C57 suckling mice with ultracentrifuged EV71 viral suspension at a titre of 1 × 10^9^ pfu/mL, administering 20 μL per mouse. The control group was injected with an equivalent volume of sterile PBS. Brain tissues from the infected mice were partially fixed in formalin, while the remaining samples were stored at −80 °C. Histological staining revealed that the brain tissues of the control group exhibited normal cellular morphology, whereas those of the infected group showed inflammatory cell infiltration, nuclear pyknosis, and vacuolation (Figure 1A,B).

RNA-sequence analysis of gene expression profiles indicated an upregulation of the NLR signal pathway in the brain tissues of the infected mice, with a significant increase in NLRC5 expression (Figure 1C,D). Furthermore, quantitative analysis of NLRC5 mRNA levels in the lungs, liver, and heart of the infected mice demonstrated a significant upregulation compared to the control group (Figure 1E). These findings suggest that EV71 infection in suckling mice induces an increased expression of NLRC5. Subsequently, we examined NLRC5 expression levels in EV71-infected cell models. The results demonstrated that NLRC5 was activated by EV71 infection in RD and Hela cells (Figure 1F–I).

3.2. EV71-Mediated NLRC5 Response Depends on EV71 Replication

Next, we aimed to further investigate whether NLRC5 activation depends on EV71 replication. We infected RD cells with different MOI values of the virus and collected cellular protein samples after 6 h. Western blot analysis was performed to detect NLRC5 protein expression. The results showed that with an increased MOI, the NLRC5 expression level also increased (Figure 2A). Moreover, infection with UV-inactivated or heat-inactivated virus which have been validated in previous studies failed to activate NLRC5 [20,21] (Figure 2B). These findings demonstrate that NLRC5 expression depends on a biologically active virus.

3.3. NLRC5 Protects Host Cells by Inhibiting EV71 Replication

Some studies have reported that the NLRC5 molecule plays a protective role in the body [17,22], while other studies suggest that the inflammation induced by NLRC5 may exacerbate RSV and IAV infection due to immune pathological damage [16,23]. Therefore, we aimed to further explore the biological function of NLRC5 during EV71 infection. The results showed that as NLRC5 expression increased, the expression of viral structural protein VP1 significantly decreased (Figure 3A,B). The supernatants from these experiments were subjected to TCID_50_ assays to determine viral titres, which revealed that viral replication was markedly suppressed by NLRC5 in a dose-dependent manner (Figure 3C). Next, we transfected Flag-NLRC5 or the control plasmid, then infected with EV71 24 h later, and collected protein samples at different time points. We found that the virus replication was significantly inhibited in the experimental group at 6, 12, and 24 h post-infection (Figure 3D). To further explore the mechanism by which NLRC5 inhibits EV71 replication, we constructed the EV71 dual-luciferase reporter plasmid pRHF-EV71 5′UTR, which contains the EV71 5′UTR sequence, with Rluc placed upstream and Fluc downstream. After co-transfection with different concentrations of Flag-NLRC5, the luciferase activities of Rluc and Fluc were measured using a dual-luciferase reporter assay. The results demonstrated that the IRES activity was progressively suppressed as the Flag-NLRC5 plasmid concentration increased (Figure 3E).

To further validate the functional role of NLRC5 in viral infection, we knocked down its expression using sequence-specific small interfering RNA (siRNA) targeting NLRC5. RT-qPCR analysis confirmed a significant knockdown effect, and siNLRC5-2 was selected for subsequent experiments (Figure 3F). We transfected Hela cells with different concentrations of siNLRC5 and infected them with EV71. After 12 h of infection, protein samples were collected to analyze VP1 expression. The results showed that as NLRC5 expression decreased, viral VP1 expression increased correspondingly [24] (Figure 3G). The virus titre analysis showed a gradual increase with reduced NLRC5 expression (Figure 3H). Overall, these results indicate that NLRC5 plays a protective role in cells by inhibiting EV71 replication, which aligns with previous reports [23,25].

3.4. EV71 Activates NLRC5 Through the IFN-β Signal Pathway

Our previous studies have shown that NLRC5 can be activated by interferons. In order to explore whether interferons play a key role in regulating NLRC5, we stimulated RD cells with different interferon concentrations and then analyzed the NLRC5 expression. The results showed that compared to IFN-α, IFN-β had a more pronounced effect on activating NLRC5 (Figure 4A), suggesting that IFN-β plays a crucial role in NLRC5 activation. Next, we examined intracellular interferon levels following EV71 infection in the RD and Hela cells. We found that intracellular IFN-β mRNA expression initially increased and then decreased (Figure 4B,C), which was consistent with the expression pattern of NLRC5 after infection.

The RIG-I receptor is recognized for its role in detecting aberrant viral RNA within the cytoplasm. Previous studies have shown that RNA viruses, such as respiratory syncytial virus (RSV) and porcine deltacoronavirus (PDCoV), are sensed by RIG-I, triggering interferon activation and subsequent antiviral responses [15,16]. Based on this evidence, we asked whether key components of the interferon signal pathway are modulated during EV71 infection.

To address this, we performed Western blot analysis on protein lysates from RD cells harvested at various time points after EV71 infection. We observed that RIG-I expression was elevated in a time-dependent manner following EV71 infection (Figure 4D). Given that RIG-I generally relays signals via transcription factors, such as IRF3 or IRF7, to induce downstream interferon pathways, we next measured the mRNA expression levels of IRF3 and IRF7 in both RD and Hela cells following EV71 infection. Our results demonstrated that IRF3 was temporally activated, whereas IRF7 expression remained largely unaltered (Figure 4E,F). Moreover, the temporal expression profile of the IRF3 protein in both cell types aligned with that of NLRC5 under identical experimental conditions—peaking at 6 h post-infection (hpi) in RD cells and at 12 hpi in Hela cells (Figure 4G,H).

To further validate the role of IFN-β in regulating NLRC5 expression, we infected interferon-deficient cells (Vero) with EV71 and analyzed interferon pathway-related proteins. While IRF3 expression exhibited a transient increase followed by a decline, a comparison of the expression levels of the relevant proteins between the 6 h infection group and the control group revealed that the protein signals were inconsistent, with some being upregulated and others downregulated, showing no discernible pattern, which are the downstream components of the IFN-β pathway (Figure 4I). This observation indicates that viral infection exerts a profound impact on host cells. In contrast, exogenous IFN-β stimulation robustly upregulated STAT1, NLRC5, and MHC-I in a dose-dependent manner (Figure 4J). Taken together, these results demonstrate that EV71 infection activates the RIG-I–IRF3–IFN-β axis, which, in turn, drives NLRC5 expression.

3.5. EV71 Upregulation of MHC-I Expression via the IFN-β-NLRC5 Axis

NLRC5 is a well-established transcriptional regulator of MHC-I molecules and thus a nucleocytoplasmic shuttling protein that exerts transcriptional regulatory functions upon nuclear entry [14]. However, its role in modulating MHC-I expression during EV71 infection remains unclear.

Transcriptomic profiling of brain tissues from the EV71-infected neonatal mice showed increased expression of MHC-I-related genes (Figure 5A). In addition, flow cytometry confirmed the upregulation of MHC-I in Hela cells at 12 h post-infection (Figure 5B). Consistent with these findings, RT-qPCR analysis of RD cells infected with EV71 for 6 h and Hela cells infected for 12 h revealed that MHC-I expression was elevated alongside NLRC5 activation (Figure 5C,D). To examine its behaviour during EV71 infection, immunofluorescence analysis was first performed to assess the intracellular localization of NLRC5. The result indicated that in uninfected cells, NLRC5 was predominantly localized in the cytoplasm; however, EV71 infection markedly promoted its nuclear accumulation (Figure 5E). These results suggest that NLRC5 translocates into the nucleus upon viral infection, where it may activate its transcriptional regulatory programme. As a transcription factor, the mechanism by which its nuclear import facilitates the activation of MHC-I molecules requires further investigation.

To further determine whether EV71-induced MHC-I upregulation depends on IFN-β and NLRC5, we detected MHC-I expression levels under viral infection conditions after IFN-β treatment. As expected, IFN-β pretreatment upregulated the expression of MHC-I, and the magnitude of MHC-I upregulation was higher after additional EV71 infection than that with IFN-β pretreatment alone (Figure 5F). Then we transfected Hela cells with Flag-NLRC5 or an empty vector for 24 h, followed by EV71 infection for 12 h. The results demonstrated a marked increase in MHC-I expression in the NLRC5-transfected cells relative to the vector control (Figure 5G).

Our previous studies showed that EV71 infection of interferon-deficient Vero cells did not alter the expression of NLRC5 or MHC-I. In contrast, treatment of Vero cells with IFN-β significantly enhanced NLRC5 expression, which in turn promoted MHC-I activation, with both molecules showing a positively correlated expression pattern (Figure 4I,J). These results indicate that EV71-induced MHC-I expression is mediated by NLRC5, and this process depends on the IFN-β–NLRC5 signal axis.

3.6. The Negative Feedback Effect of NLRC5 on IFN-β Expression

Since NLRC5 can activate cytotoxic effects to inhibit viral replication, uncontrolled and persistent inflammatory responses may lead to tissue damage. Therefore, under physiological conditions, the body maintains precise control over inflammatory reactions. In this study, we observed that IFN-β expression decreased during the late stage of EV71 infection in both RD and Hela cells. Based on this finding, we further investigated whether NLRC5 is involved in the negative regulation of IFN-β expression. Our results showed that NLRC5 suppressed the expression of its upstream signal molecules, RIG-I and IRF3, in a dose-dependent manner (Figure 6A). Consistent with this observation, transfection with NLRC5 also led to a significant reduction in IFN-β transcription [26], as confirmed by both mRNA analysis (Figure 6B) and luciferase reporter assays (Figure 6C). Collectively, these data suggest that NLRC5 modulates inflammatory signals through a self-limiting negative feedback mechanism, which may help prevent excessive immune activation and protect the host from cytokine storm-mediated damage.

4. Discussion

The NLR family member NLRC5 has been recognized as a critical regulator of innate immunity; however, its dual functions in antiviral defence and inflammatory control remain incompletely defined. In this study, we reveal a previously uncharacterized mechanism through which NLRC5 coordinates antiviral and immunomodulatory responses during EV71 infection. We show that EV71 triggers NLRC5 expression via the RIG-I–IRF3–IFN-β axis, leading to the suppression of viral replication and simultaneous negative feedback on IFN-β production to maintain immune balance.

Our study presents a key advance by delineating the specific pathway through which EV71 infection upregulates NLRC5 expression. While prior studies have linked NLRC5 induction to IFN-γ or TLR ligands in certain contexts [26,27,28], we identified IFN-β as the principal cytokine responsible for driving NLRC5 expression during EV71 infection (Figure 4). This specificity was further supported by the finding that IRF3-but not IRF7-mediated the transcriptional upregulation of NLRC5 (Figure 4E–H), and its expression dynamics were positively correlated with those of NLRC5. As a signal molecule, IFN-β binds to the type I interferon receptor on the cell surface, thereby activating the JAK-STAT signal pathway. Activated STAT1 forms a complex with IRF3, known as the ISGF complex. The ISGF complex translocates into the nucleus and binds to the interferon-sensitive response elements (ISREs) located within the promoter regions of specific target genes [29]. Since the promoter region of the NLRC5 gene contains ISRE sequences, it can be directly and potently induced by IFN-β signals, leading to a significant upregulation of NLRC5 expression. This ensures that during the early stages of viral infection, once IFN-β is produced, NLRC5 is rapidly induced to carry out its downstream functions. These results underscore a virus-specific sensing mechanism that differs from those reported for other RNA viruses, such as RSV or PDCoV [15,16]. Furthermore, a key observation in our study was that the mechanism by which EV71 infection activates NLRC5 expression via IFN-β is conserved across different cell lines (Figure 1F–H). The RD and HeLa cells used here differ in their susceptibility to EV71, leading to distinct viral replication kinetics and consequently divergent dynamic changes in NLRC5 expression after infection. Despite these variations in the magnitude and timing of NLRC5 induction, the overall expression pattern triggered by EV71 remained conserved between the two cell types. This consistency suggests that the functional interaction between NLRC5 and EV71 is not cell-type-specific, but rather reflects a conserved host–virus regulatory mechanism. Together, our data demonstrate that while cell-intrinsic factors shape the extent and pace of NLRC5 modulation during EV71 infection, the core interplay between NLRC5 and the virus is maintained. This provides a foundation for further investigation into the broader role of NLRC5 in restricting EV71 replication.

Furthermore, our study demonstrates that NLRC5 directly restricts EV71 replication, as supported by the dose-dependent decrease in VP1 expression and viral titres following NLRC5 overexpression (Figure 3A–D). This finding extends the functional spectrum of NLRC5 beyond its classical function in immune recognition and is consistent with recent reports on other viral systems. For example, NLRC5 has been implicated in promoting viral protein degradation during dengue virus infection, while for influenza virus, it can modulate the inflammatory landscape, affecting disease outcomes [22,30]. Our work thus positions NLRC5 as a multifunctional player in antiviral defence, capable of both inhibiting viral replication and enhancing immune recognition.

Our study further identifies a novel negative feedback mechanism through which NLRC5 constrains IFN-β production (Figure 6A–C). While IFN-β is critical for early antiviral defence, its sustained overproduction can trigger cytokine storms, as observed in severe COVID-19 [31]. We demonstrate that NLRC5 attenuates IFN-β signal through two distinct mechanisms. First, it directly downregulates key upstream mediators of the IFN-β induction pathway, including RIG-I and IRF3. Second, as indicated in Figure 3E, we found that NLRC5 inhibit EV71 replication through targeting the 5′UTR of viral RNA thereby attenuates IFN-β signal. However, as the direct physical interaction between NLRC5 and the viral RNA replication require further investigation. In a word, this dual regulation helps to limit excessive inflammation while preserving the initial antiviral immune response. This dual functionality–enhancing MHC-I-mediated adaptive immunity while tempering innate immune hyperactivation–suggests that NLRC5 acts as an immunological rheostat that maintains immune equilibrium during infection. Such a regulatory mechanism is particularly relevant for severe EV71, where uncontrolled inflammation underlies neuropathology. Notably, unlike its reported role in stabilizing RIG-I during influenza infection [32], NLRC5 selectively suppresses the RIG-I–IRF3 axis during EV71 infection. This context-dependent action expands the emerging paradigm of NLR-mediated immunoregulation by revealing a previously unrecognized mechanism specific to enteroviral pathogenesis. It also parallels other feedback systems that fine-tune interferon responses, such as the IFN-induced Tetherin pathway, which recruits MARCH8 to promote K27-linked ubiquitination and NDP52-dependent autophagic degradation of MAVS, thereby attenuating RLR-mediated type I IFN signals [33]. This context-dependent function extends the emerging paradigm of NLR-mediated immunoregulation by revealing a previously uncharacterized mechanism specific to enteroviral pathogenesis.

The translational implications of our findings are twofold. First, enhancing NLRC5 activity may represent a viable strategy to strengthen antiviral immunity in EV71 infection, for which targeted therapies remain limited. The development of small-molecule agonists or gene-based approaches to potentiate NLRC5 function could improve MHC-I-mediated antigen presentation and promote viral clearance. Second, leveraging the immunoregulatory function of NLRC5 may help control excessive inflammation in severe cases. For example, NLRC5-based biologics or mimetics could be evaluated in cytokine storm models, similar to strategies targeting IL-6 in severe COVID-19 [34,35].

Taken together, our findings provide important insights into EV71 pathogenesis and contribute to the broader understanding of NLRC5′s function in host–virus interactions. Future studies should determine whether the NLRC5-mediated feedback mechanism operates in other enteroviruses or RNA viruses and whether modulating its activity improves clinical outcomes in HFMD animal models.

5. Conclusions

We delineate a coherent pathway in which EV71 triggers NLRC5 expression through the RIG-I–IRF3–IFN-β axis, enabling NLRC5 to concurrently restrict viral replication and temper excessive inflammation. These results provide a conceptual framework for understanding immune homeostasis during infection and not only advance the mechanistic knowledge of EV71 pathogenesis but also highlight NLRC5 as a promising target for immunomodulatory strategies against viral and inflammatory disorders.

In summary, our study establishes NLRC5 as a central coordinator of the host response to EV71, integrating both antiviral defence and inflammatory control. We delineate a coherent pathway in which EV71 triggers NLRC5 expression through the RIG-I–IRF3–IFN-β axis, enabling NLRC5 to concurrently restrict viral replication and temper excessive inflammation. These results provide a conceptual framework for understanding immune homeostasis during infection and not only advance the mechanistic knowledge of EV71 pathogenesis but also highlight NLRC5 as a promising target for immunomodulatory strategies against viral and inflammatory disorders.