Decoding Enterovirus 71: Molecular Functions of Structural and Nonstructural Proteins in Viral Replication and Host Manipulation

1 Introduction

Enterovirus 71 (EV71), belonging to the family Picornaviridae and genus Enterovirus (species Enterovirus A), is a small, non-enveloped, positive-sense RNA virus and an emerging neurotropic pathogen that continues to drive seasonal outbreaks of hand, foot, and mouth disease (HFMD) with escalating severity and expanding geographic distribution [1,2,3]. Alongside Coxsackie virus A16 (CV-A16), EV71 remains a primary etiological agent of HFMD, yet it is uniquely associated with severe neurological and cardiopulmonary complications in a subset of pediatric patients [4,5].

HFMD predominantly affects infants and young children, typically presenting with low-grade fever, vesicular eruptions on the hands, feet, and buttocks, and oral ulcers as hallmark clinical manifestations of the disease [6]. Although most cases are self-limiting, EV71 infection can progress to serious neurological manifestations such as aseptic meningitis, brainstem encephalitis, poliomyelitis-like flaccid paralysis, and neurogenic pulmonary edema, occasionally leading to fatal outcomes [7]. EV71 infection is initiated primarily through fecal–oral and respiratory transmission, followed by viral replication in the gastrointestinal or respiratory epithelium and subsequent systemic dissemination via viremia. The virus may invade the central nervous system through several proposed mechanisms, including retrograde axonal transport from peripheral neuromuscular junctions, disruption of the blood–brain barrier that permits paracellular or transcellular viral passage across endothelial cells, and the so-called “Trojan horse” mechanism in which infected immune cells facilitate viral entry into neural tissues [8]. Since its first detection in New Zealand in 1956, EV71 has spread globally, with major epidemics reported across the Asia–Pacific region, including Australia, Cambodia, China, India, Japan, Malaysia, Singapore, Taiwan, Thailand, and Vietnam [9,10]. In China, HFMD was classified as a noticeable Class C infectious disease in 2008 [11]. The temporary decline in HFMD incidence during the COVID-19 pandemic, attributed to non-pharmaceutical interventions (NPIs), was followed by a rapid resurgence of EV71 cases after restrictions were lifted, accompanied by shifts in genotype prevalence and an increase in neurological complications among younger age groups [7,12,13].

Genotypically, EV71 is classified into three main genogroups (A, B, and C) and multiple subgenotypes (e.g., B1–B5, C1–C5), with C4 having predominated in China over the past two decades [14,15]. Recent surveillance has revealed the emergence of novel recombinant lineages and co-circulation with other enteroviruses such as CV-A6 and CV-A16, raising concerns about limited vaccine cross-protection and the potential for immune-escape variants [16].

Although over 280 human Enterovirus (EV) serotypes have been documented, only four currently have approved vaccines worldwide, and no antiviral agents are available for the treatment of EV infections [17,18]. In addition, global monitoring of EV circulation remains limited and lacks coordinated standardization, making it difficult to rapidly detect and respond to new variants. Because Enterovirus A71 (EV-A71) has emerged as a major public health threat in China, three alum-adjuvanted and inactivated EV-A71 vaccines were launched between 2015 and 2016. However, these vaccines predominantly exhibit genotype-restricted protection and do not provide broad immunity against diverse circulating strains [17,19]. Recent studies have identified that EV71 structural (e.g., VP1) and nonstructural proteins (2A, 3C) play pivotal roles in modulating viral replication, host immune evasion, and neuropathogenesis. For instance, the host E3 ligase SPOP was found to degrade the viral 2A protease to restrict replication, while VP1 induces endoplasmic reticulum stress via PMP22 accumulation in neuronal cells, contributing to virus-induced neuropathology [20]. Similarly, the ubiquitination of VP1 by ZYG11B (Zyg-11 family member B), an E3 ubiquitin ligase adaptor protein, suppresses viral propagation, identifying VP1 as both a virulence factor and a potential drug target [21].

Despite these advances, the molecular understanding of how EV71 proteins collectively coordinate viral replication, immune evasion, and host responses remains fragmented. Previous reviews have largely focused on individual proteins or limited mechanistic aspects, lacking a holistic synthesis of structural and nonstructural protein functions. The present review aims to fill this gap by providing a comprehensive, mechanistic, and integrative analysis of all EV71-encoded proteins, summarizing their structural characteristics, host interactions, and immunomodulatory roles. By critically evaluating recent findings and highlighting unresolved questions, this review seeks to provide a unified conceptual framework that may guide future antiviral and vaccine development strategies.

2 Genome Structure and Replication Process of EV71

EV71 is a non-enveloped, positive-sense single-stranded RNA (+ssRNA) virus belonging to the family Picornaviridae, genus Enterovirus. The genome is approximately 7.4 kb in length and contains a 5′ untranslated region (5′UTR), a single open reading frame (ORF), and a 3′UTR followed by a poly(A) tail (Fig. 1) [22]. The ORF is divided into three regions, P1, P2, and P3, where the P1 region encodes the four structural capsid proteins (VP1–VP4), while P2 and P3 encode seven nonstructural proteins (2A–3D) that regulate viral replication, host interaction, and immune evasion [3].

Recent structural analyzes have refined the topology of the EV71 genome, revealing conserved cis-acting replication elements within both the 5′ and 3′ UTRs, including a cloverleaf-like structure essential for polymerase recruitment and genome circularization [23]. Together, the 11 encoded proteins form a coordinated molecular network that drives viral replication, immune evasion, and host inflammatory responses [7]. A concise overview of VP1–VP4, including their structural features, functions, host interactions, and therapeutic potential, is provided in Table 1 to enable systematic comparison and facilitate translational interpretation.

In particular, VP1 not only contributes to receptor recognition but also triggers host stress responses and is implicated in endoplasmic reticulum dysfunction and neurotoxicity [20]. Similarly, nonstructural proteins such as 2A protease and 3C protease are multifunctional enzymes involved in viral polyprotein cleavage, host shutoff, and immune modulation. New findings indicate that 2A is tightly regulated by host ubiquitination machinery, with E3 ligase SPOP promoting its degradation and thereby limiting viral replication [19].

Figure 1: Structure of the enterovirus 71 (EV71) genome.

EV71 entry begins with the receptor-mediated attachment, followed by capsid rearrangement and VP4 release, which drive endocytosis and genome uncoating [24,25]. The interaction between EV-A71 capsid proteins and host receptors, followed by conformational changes leading to VP4 release, is depicted in Fig. 1, offering a visual framework that complements the mechanistic description provided in this section.

Within the host cell, the viral capsid is removed, and genomic RNA is released into the cytoplasm. The viral proteases 2A and 3C rapidly process the translated polymer proteins into structural capsid proteins (VP1–VP4) and nonstructural proteins (2A−2C, 3A−3D) of daughter viruses by directly using the parent viral RNA as mRNA. Notably, 3C has been shown to target and degrade host RNA-binding proteins and nuclear pore components, contributing to immune evasion and facilitating efficient replication [23,26].

Genome replication occurs within membrane-associated replication organelles, where the viral RNA-dependent RNA polymerase (3D) synthesizes a negative-strand RNA template, subsequently used to generate multiple positive-strand RNAs for progeny virion assembly [27]. Viral replication organelle formation depends on VPS34 (class III phosphatidylinositol 3-kinase) and its effector DFCP1, which remodels host lipid droplets and endoplasmic reticulum membranes. Inhibition of VPS34 has therefore emerged as a potential antiviral strategy [23].

3 Function of EV71’s Structural Proteins

The EV71 capsid is formed by four structural proteins (VP1-VP4) that assemble into an icosahedral shell encapsulating the viral genome [28]. While VP1–VP3 are surface-exposed and involved in host cell receptor engagement and immune recognition, VP4 is located internally and contributes to capsid stabilization and uncoating [24]. Together, these proteins play mechanistic roles beyond structural support, participating in viral entry, host modulation, and immune evasion. A comparative summary of VP1–VP4, including their molecular functions, host interactions, and translational relevance, is provided in Table 1.

3.1 VP1 Protein

3.1.1 Antigenicity and Receptor Recognition

VP1 is the most exposed capsid protein (297 aa) and serves as the principal determinant for receptor usage, serotype, and immune recognition [3]. Its canyon and pocket-factor domains are critical for receptor-mediated viral entry, consistent with high-resolution structural analyses (Fig. 2). SCARB2 is the primary entry receptor, interacting with VP1 GH and VP2 EF loops in a pH-dependent manner to facilitate uncoating [29]. Mutations such as K98E, E145A, and L169F enhance VP1–SCARB2 binding and increase infectivity, particularly in murine models [30].

P-selectin glycoprotein ligand-1 (PSGL-1) binds explicitly to VP1 variants containing G or Q at residue 145, whereas the 145E variant abolishes PSGL1 binding [31,32]. VP1-145 acts as a molecular switch that controls both PSGL-1 usage and neutralisation sensitivity, indicating strong immune selection pressure [23]. When heparan sulfate (HS) is used as an attachment receptor, residues VP1-98 and VP1-145 regulate viral affinity and tropism, and VP1-E98K variants show reduced pathogenicity in mice [33,34].

Additional host factors, including annexin II, vimentin, fibronectin, and Galectin-1, interact with distinct VP1 regions to enhance viral entry and thermal stability, supporting EV71 survival in vivo [24,35]. Thermostable VP1 mutants (e.g., K215A) have shown promise for recombinant vaccine design, especially in high-temperature regions [27,36].

VP1 contains multiple conformational B-cell epitopes located in surface loops such as BC, GH, and HI, which are targets for potent monoclonal antibodies, including mAb 2G8, mAb 51, and mAb 22A12 [5,37]. Recombinant VP1 vaccines have demonstrated cross-genotype neutralisation responses, supporting its use as a universal vaccine antigen [37]. Fig. 2 illustrates the key domains and receptor-binding motifs of VP1.

3.1.2 Neurovirulence

VP1 is a major determinant of neurotropism, acting through endoplasmic reticulum (ER) stress, autophagy regulation, and mitochondrial dysfunction (Fig. 2). The VP1/ER stress/PMP22 axis modulates autophagy in Schwann cells, promoting viral replication and neuronal damage [38]. Downregulation of mTOR signaling further supports autophagic flux, facilitating viral spread and increased disease severity [39].

Mammalian target protein (mTOR) is an important signalling molecule in the phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (Akt)/mTOR autophagy pathway, and the inhibition of mTOR activity can lead to the initiation of autophagy and the formation of autophagosomes [40]. The EV71 VP1 protein regulates viral replication in nerve cells via the mTOR autophagy signalling pathway. Experiments have shown that autophagy promotes the release and spread of the virus, thereby increasing disease severity and the viral titer [39].

Recent works reveal that VP1 induces mitochondrial fragmentation and reactive oxygen species (ROS) accumulation, impairing axonal integrity [41]. In infected neurons, VP1 sustains autophagic flux through the PI3K/Akt/mTOR pathway, thereby suppressing apoptosis and enabling prolonged viral persistence [42]. VP1 also upregulates serotonin-related regulators ERICH3 and ZC3H13, disrupting neurotransmission via the VP1–ERICH3–ZC3H13 axis, which has been linked to brainstem encephalitis in mouse models [43,44]. Collectively, these findings implicate VP1 as a multifunctional virulence factor with roles beyond its structural role.

Figure 2: Molecular functions and host interactions of viral protein 1 (VP1) during EV71 infection. VP1 engages multiple host receptors—including SCARB2, PSGL-1, heparan sulfate (HS), annexin A2 (Anx2), vimentin, and fibronectin (FN)—to mediate viral attachment, entry, and uncoating. Binding of cell-surface receptors triggers the expulsion of VP4 and the exposure of the VP1 N-terminus, facilitating membrane penetration and genome release. VP1 also induces endoplasmic reticulum (ER) stress by upregulating peripheral myelin protein 22 (PMP22), thereby activating PI3K/Akt/mTOR-dependent autophagy and promoting viral replication. Additionally, VP1 modulates serotonergic signaling by regulating ERICH3–ZC3H13 interactions, contributing to neurovirulence. MIR2911, a plant-derived miRNA, directly targets VP1 and inhibits viral replication. Abbreviation: VP1, viral protein 1; SCARB2, scavenger receptor class B member 2; PSGL-1, P-selectin glycoprotein ligand-1; HS, heparan sulfate; Anx2, annexin A2; FN, fibronectin; PM P22, peripheral myelin protein 22; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; mTOR, mechanistic target of rapamycin; ER, endoplasmic reticulum; dsRNA, double-stranded RNA; 5-HT, 5-hydroxytryptamine (serotonin); ERICH3, glutamate-rich protein 3; ZC3H13, zinc finger CCCH-type containing 13; MIR2911, microRNA-2911.

3.1.3 Virus Neutralisation

Due to its surface exposure, VP1 is the primary target of neutralising antibodies, especially within the GH loop. Mutations at VP1-145 modulate both receptor usage and antibody sensitivity, enabling immune escape [29,45,46]. VP2 and VP3 also contribute to neutralising epitopes, owing to shared β-sandwich “jelly-roll” folds [47]. Conserved VP2 regions (141–150 amino acids) remain stable after formalin inactivation—making them highly suitable for vaccine potency assays [48]. Synergistic mutations at VP1-145 and VP2-149 increase cell apoptosis and enhance viral fitness, suggesting coordinated modulation of receptor binding and replication [49].

VP3 harbors a conserved “knob” region, targeted by diagnostic mAbs such as 10D3 and 5H7 [50]. MicroRNAs (e.g., miR-18a and miR-452) further regulate EV71 replication by targeting VP3 transcripts, reinforcing VP3’s role beyond capsid support [51,52].

VP4 (69 aa) is internal and is expelled during uncoating, inserting into host membranes to form a transient pore for genome release [53]. Its N-terminal VP4N20 peptide fused to HBcAg successfully produced virus-like particles (VLPs) that neutralized viruses across genotypes, making VP4 a promising next-generation vaccine platform [54].

VP1–VP4 function as a coordinated molecular system that drives viral attachment, entry, genome delivery, immunogenicity, and capsid dynamics. VP1 is the best-characterized component; however, mechanistic insights into VP2–VP4 remain comparatively limited. The cooperative roles of these proteins during uncoating, endosomal escape, and capsid destabilization remain incompletely resolved, particularly in physiologically relevant in vivo models.

Future studies should prioritize high-resolution structural imaging, protein–protein interaction mapping, and host interaction profiling to define the temporal sequence of events during early infection. Such work will be essential for rational vaccine engineering and for identifying structural determinants amenable to capsid-targeting antiviral strategies. Importantly, most available evidence regarding VP1–VP4 function derives from in vitro systems, with limited validation in neuronal models or animal studies. Establishing the physiological relevance of these mechanisms will require in vivo evaluation using organoids, transgenic mouse models, or humanized infection systems.

4 Functions of EV71’s Nonstructural Proteins

EV71 nonstructural proteins (2A–3D) are translated as part of a single polyprotein and subsequently processed into enzymatic and regulatory components that coordinate viral RNA replication, polyprotein processing, intracellular membrane remodeling, and evasion of host innate and adaptive immunity [28,55]. In addition to their canonical roles in replication, accumulating evidence indicates that these proteins extensively rewire host signaling pathways, stress responses, and cell death programs, thereby shaping tissue tropism and disease severity. To synthesize the multifaceted roles of EV71 nonstructural proteins, we provide a structured overview (Table 2) highlighting their functions, host interactions, system-level relevance, and current therapeutic opportunities. The integration of replication machinery and innate immune evasion suggests an emergent model of coordinated viral control over cellular decision-making pathways.

4.1 Protein 2A

Protein 2A is a cysteine protease of approximately 150 amino acids composed of an N-terminal four-stranded β-sheet domain and a C-terminal six-stranded β-barrel domain that coordinates a tightly bound zinc ion [56]. According to the crystal structure, 2A adopts a chymotrypsin-like fold, and the catalytic triad (H21, D39, C110) is positioned within the cleft between the N- and C-terminal domains. The cI–eI2 loop at the N terminus forms a highly conserved, surface-exposed conformation that increases hydrophilicity, whereas two C-terminal motifs, 144DEE146 and LLWL, are critical for EV71 replication [57]. Structural analysis indicates that 144DEE146 forms an acidic surface patch, with E145 and E146 more solvent-exposed than D144. Functional studies showed that a D144A substitution does not impair viral replication, whereas E145A markedly reduces replication, and the D144A/E145A double mutant is lethal despite preserving correct folding and protease activity [57,58]. Deletion of residues 146–149 (EAME) similarly abrogates viral transcriptional activity without disrupting protease function, and removal of the adjacent LLWL motif abolishes polyprotein processing and replication, indicating that this hydrophobic motif is essential for positioning the acidic motif and maintaining an active protease conformation [57]. Notably, 2A also exhibits transcriptional activity in yeast, contributing to replication and virulence independently of its protease function, as demonstrated in 2A-substituted chimeric strains [57,59].

As a protease, 2A catalyses cleavage of the capsid precursor and 3CD polyprotein, thereby generating 3C and 3D and initiating the maturation of viral proteins. Beyond viral substrates, 2A cleaves host far upstream element-binding protein 1 (FBP1) at Gly-371 to produce FBP1_1–371, which synergizes with full-length FBP1 to enhance internal ribosome entry site (IRES)–driven translation from the 5′UTR of the viral genome [60,61,62]. ERK1/2-dependent signaling positively regulates IRES activity, and 2A mediates both cis-cleavage of viral polyproteins and trans-cleavage of the translation initiation factor eIF4G, thereby shutting off cap-dependent host translation while favoring cap-independent viral mRNA translation [63,64]. By cleaving eIF4GI, 2A disrupts the formation of canonical stress granules and promotes atypical stress particle assembly, further biasing translation toward viral RNAs and contributing to virus-induced apoptosis [63,64,65]. 2A also interferes with RNA processing bodies (P-bodies) by preventing their assembly and recruiting P-body components to viral RNA, generating a microenvironment that supports efficient viral RNA synthesis [66].

2A is a central antagonist of innate antiviral pathways. It inhibits IRF3-dependent signaling by cleaving MAVS and the RIG-I-like receptor MDA5, thereby blocking type I IFN induction (Fig. 3) [67,68,69]. In addition, 2A reduces surface IFNAR1 expression and dampens JAK1, Tyk2, and STAT phosphorylation, attenuating IFN-stimulated gene induction and enabling escape from IFN-α/β responses [67,68,69]. 2A also targets the DEAD-box helicase DDX6, reducing the DDX6–viral RNA complex and impairing its positive regulatory role in RIG-I–mediated IFN-I signaling, thereby further favouring replication [70].

At the level of cellular immunity, 2A downregulates NKG2D ligands and upregulates PD-L1 on intestinal epithelial cells, reducing recognition and killing by innate lymphoid cells and CD3+ intraepithelial lymphocytes [71]. Furthermore, 2A suppresses NLRP3 inflammasome activity by cleaving NLRP3 at the Q225–G226 and G493–L494 junctions, thereby preventing IL-1β maturation and permitting enhanced viral replication [72,73]. Taken together, these findings position 2A as a multifunctional enzyme that links polyprotein processing with broad suppression of interferon, inflammasome, and RNA quality-control pathways.

4.2 Protein 2B

Protein 2B is a small, ~100-residue ion channel–like protein that integrates into intracellular membranes and perturbs ion homeostasis. It contains two predicted transmembrane domains (TM1 and TM2), and 2B-mediated chloride-dependent currents elevate cytosolic Ca2+ by disturbing anion balance in the Golgi complex, thereby promoting EV71 replication; these currents can be inhibited by the anion exchange blocker DIDS [28]. The C-terminal segment (amino acids 63–80) mediates mitochondrial targeting during infection. Within this compartment, protein 2B interacts with the mitochondrial voltage-dependent anion channel 3 (VDAC3), thereby elevating mitochondrial ROS production and perturbing cellular redox balance—conditions conducive to efficient viral replication [74]. The N-terminal hydrophilic segment (the first 14 residues) interacts with the proapoptotic protein Bax, inducing its conformational activation and redistribution, and triggering mitochondrial apoptosis (Fig. 4).

2B also modulates host antiviral factors and transcriptional regulators. It binds interleukin enhancer-binding factor 2 (ILF2), impairs its nuclear translocation, and reduces ILF2 mRNA and protein levels, thereby antagonizing ILF2-mediated antiviral effects [75]. The ER-resident acetyltransferase NAT8 interacts with 2B, as well as 3AB and 3C, stabilizing these viral proteins in an acetyltransferase-dependent manner and enhancing replication [76]. Collectively, 2B integrates ion dysregulation, mitochondrial stress, apoptosis, and antagonism of antiviral host factors to create a cellular environment conducive to EV71 replication.

Figure 3: Mechanisms of EV71 nonstructural proteins in host immune suppression and cell death regulation. EV71 nonstructural proteins 2A and 3C employ multiple mechanisms to evade host immunity, modulate inflammatory signalling, and regulate apoptosis and pyroptosis. Both proteases inhibit type I and type II interferon responses by suppressing JAK/STAT signalling, cleaving MAVS, MDA5, TRIF, and IRF7, and disrupting the formation of antiviral transcriptional complexes. They further inhibit NF-κB activation by blocking IKK phosphorylation and cleaving key scaffold proteins (TAK1, TAB1, TAB2, TAB3). Moreover, 2A and 3C regulate programmed cell death via targeted cleavage of caspase-8, caspase-9, and gasdermin-D, thereby modulating apoptosis and pyroptosis to enhance viral replication and dissemination. Collectively, these interactions establish EV71 proteases as central determinants of virulence and immune evasion. Abbreviation: IFN-γ, interferon-gamma; IFN-I/IFN-β, type I interferon/interferon-beta; IFNAR1, interferon-alpha/beta receptor subunit 1; JAK1/JAK2, Janus kinase 1/2; TYK2, tyrosine kinase 2; STAT1/STAT2/STAT3, signal transducer and activator of transcription 1/2/3; IRF1/IRF3/IRF7, interferon regulatory factors 1/3/7; OAS3, 2′–5′ oligoadenylate synthetase 3; RNase L, ribonuclease L; GAS, gamma-activated sequence; ISRE, interferon-stimulated response element; RIG-I, retinoic acid-inducible gene I; MDA5, melanoma differentiation–associated protein 5; MAVS, mitochondrial antiviral signaling protein; TLR3, Toll-like receptor 3; TRIF, TIR-domain-containing adaptor inducing interferon-β; TRAF3/TRAF6, TNF receptor-associated factors 3/6; TRIM25, tripartite motif-containing protein 25; DDX6, DEAD-box helicase 6; TAK1, transforming growth factor-beta–activated kinase 1; TAB1/TAB2/TAB3, TAK1-binding proteins; IKKα/IKKβ, IκB kinases α/β; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; TBK1, TANK-binding kinase 1; ASC, apoptosis-associated speck-like protein containing a CARD; NLRP3, NOD-like receptor family pyrin domain containing 3; GSDMD/GSDME, gasdermin D/E; hnRNP A1, heterogeneous nuclear ribonucleoprotein A1; Apaf-1, apoptotic peptidase activating factor 1; PinX1, PIN2/TERF1-interacting telomeric protein 1; Caspase-3/-8/-9/-11, cysteine-aspartic proteases involved in apoptosis and pyroptosis.

4.3 Protein 2C

2C, comprising 329 amino acid residues, is one of the most conserved nonstructural proteins of Protein 2C (329 aa) is one of the most conserved EV71 nonstructural proteins and is essential for RNA replication. It comprises an NTPase/RNA helicase domain, a zinc-binding motif, and a C-terminal helical region that supports self-oligomerisation [77,78]. 2C functions as an ATP-dependent RNA helicase and ATP-independent RNA chaperone, mediating RNA unwinding and remodeling of viral RNA structures required for replication; these two RNA-remodeling activities are mechanistically distinct yet functionally interdependent within the same viral RNA substrate [77,78]. The C-terminal oligomerisation domain is critical for both ATPase activity and formation of the replication complex.

2C is a key organizer of replication organelles. It binds the ER-associated protein reticulon-3 (RTN3) and coatomer protein complex I (COPI), recruiting these host factors to intracellular membranes to assemble viral replication complexes (Fig. 4) [79,80]. Interference with COPI function markedly reduces EV71 replication, underscoring the importance of 2C-dependent membrane remodeling [80].

In innate immunity, 2C suppresses NF-κB signaling by targeting components of the IKK complex. 2C interacts with the IPT domain of RelA(p65), recruits protein phosphatase 1 (PP1) together with IKKβ, and inhibits IKKβ phosphorylation, thereby preventing TNF-α–induced NF-κB activation [81,82,83]. 2C also forms inclusion bodies that sequester IKKβ and IKKα, further limiting their phosphorylation and NF-κB–dependent transcription [81,82,83]. Moreover, 2C promotes degradation of the cytidine deaminase APOBEC3G (A3G) via the autophagy–lysosome pathway, counteracting A3G-mediated suppression of 5′UTR activity and enabling sustained viral replication [84]. These data identify 2C as a multifunctional hub linking replication complex formation to antagonism of NF-κB signaling and intrinsic antiviral restriction factors.

Figure 4: Functional roles of EV71 nonstructural proteins 2b and 2c in apoptosis, nf-κb suppression, and viral replication. EV71 proteins 2B and 2C orchestrate multiple host-modulating mechanisms to promote viral replication and disrupt immune defence. Protein 2B localises to mitochondria and directly interacts with VDAC3 to elevate reactive oxygen species (ROS), triggering Bax-mediated mitochondrial apoptosis and enhancing virus propagation. Additionally, 2B inhibits the transcription factor ILF2, thereby downregulating IL-2 expression and facilitating immune evasion. Protein 2C interferes with NF-κB signalling by binding to RelA/p65 and recruiting protein phosphatase 1 (PP1), which inhibits phosphorylation of IKKα/IKKβ and leads to cytosolic sequestration within inclusion bodies (IBs). Furthermore, 2C interacts with COPI and RTN3 to assemble the viral replication complex on intracellular membranes, enabling efficient genome replication and viral egress. Collectively, these mechanisms highlight 2B and 2C as central regulators of host cell death, inflammatory signalling, and replication complex formation during EV71 infection. Abbreviation: ROS, reactive oxygen species; VDAC3, voltage-dependent anion channel 3; Bax, Bcl-2-associated X protein; Caspase, cysteine-aspartic protease; ILF2, interleukin enhancer-binding factor 2; IL-2, interleukin-2; PP1, protein phosphatase 1; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; RelA/p65, NF-κB subunit p65; IKKα/IKKβ, IκB kinase α/β; IBs, inclusion bodies; COPI, coat protein complex I; RTN3, reticulon-3.

4.4 Protein 3A

Protein 3A (86 aa) is a membrane-associated protein that coordinates intracellular trafficking and the formation of replication organelles. EV71 infection increases the expression of Na+/K+-transporting ATPase subunit β3 (ATP1β3), which exerts antiviral activity by promoting type I IFN production; 3A interacts with ATP1β3, potentially modulating this antiviral response, although the mechanistic details remain to be clarified [84]. 3A also antagonizes innate immune sensors by targeting Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1). Through upregulation of the autophagy-related protein LRRC25, 3A promotes G3BP1 degradation and suppresses RIG-I-like helicase (RLH) signaling, thereby dampening IFN-β induction and enhancing EV71 replication (Fig. 5) [85].

A central function of 3A is to remodel Golgi-derived membranes for viral RNA synthesis. 3A interacts with the Golgi resident protein acyl-CoA–binding domain containing 3 (ACBD3), which is required to recruit phosphatidylinositol 4-kinase IIIβ (PI4KB) to sites of genomic replication [86,87]. The 3A–ACBD3–PI4KB axis generates PI4P-enriched membranes that serve as scaffolds for the replication complex; mutations at residues I44 or H54 in 3A disrupt these interactions and strongly inhibit RNA replication [86,87]. More recently, 3A has been shown to bind vacuolar protein sorting 25 (VPS25) on exosomes, enhancing exosome biogenesis and promoting the release of EV71-containing vesicles (Fig. 5) [88]. This exosome-mediated dissemination pathway may facilitate immune evasion and long-range viral spread.

4.5 Protein 3B

Protein 3B, also known as viral protein genome-linked (VPg), is a 22-residue nucleic acid chaperone covalently linked to the 5′ end of the viral genome. VPg binds 3Dl and anchors into a site near residue 311 at the base of the polymerase, adopting a V-shaped extended conformation that positions its hydroxylated Tyr3 in the active site [28,89,90]. Uridylation of VPg by 3D generates VPg-pUpU, which primes RNA synthesis; mutations that impair VPg uridylation severely compromise viral replication [90]. A bimolecular model proposes that one 3D position VPg, while a second 3D catalyses uridylation, underscoring the complexity of this priming reaction [91]. Although 3B is small, its essential role in replication initiation and its high conservation make it an attractive candidate for antiviral targeting.

4.6 Protein 3C

Protein 3C (3C, 183 aa) is a chymotrypsin-like cysteine protease that is indispensable for polyprotein processing and also functions as an RNA-binding protein and potent immune antagonist. Structural studies revealed that 3C comprises two β-barrel domains forming a catalytic cleft, and a flexible β-loop containing hinge residues Gly123 and His133 that regulate conformational dynamics and hydrolytic activity [92]. 3C cleaves multiple sites within the viral precursor polyprotein, generating both structural and nonstructural proteins required for virion maturation.

3C harbors two principal RNA-binding motifs, KFRDI (residues 82–86) and VGK (154–156), that mediate interaction with viral RNA; mutations in these motifs impair both RNA binding and protease activity, whereas catalytic-site mutations do not abolish RNA binding [93]. A single amino acid substitution at position 69 significantly reduces replication and virulence in recombinant viruses, highlighting this residue as a virulence determinant [94,95].

Beyond its proteolytic role, 3C extensively remodels innate immune pathways. It cleaves the cytosolic sensor RIG-I and reduces expression of the E3 ligase TRIM25, thereby blocking RIG-I ubiquitination and activation and suppressing type I IFN induction. 3C also cleaves IRF7 and modulates miR-526a, further dampening IFN-I signaling. By targeting the TAK1–TAB1/TAB2/TAB3 complex, 3C prevents NF-κB activation, and by cleaving TRIF, it disrupts TLR3-mediated antiviral signaling (Fig. 3) [96]. 3C additionally cleaves the antiviral effector OAS3 at Q982–G983 and the NLRP3 inflammasome component at Q225–G226, weakening OAS3-dependent RNA degradation and inhibiting inflammasome activation [97,98]. TRAF3-interacting protein 3 (TRAF3IP3) and promyelocytic leukemia protein IV (PMLIV), both antiviral factors, are similarly targeted and cleaved by 3C, leading to disruption of PML nuclear bodies and diminished antiviral autophagy [99,100,101]. NAT8-mediated stabilization of 3C further amplifies these immune evasion effects [76].

3C also orchestrates multiple forms of programmed cell death. It cleaves telomere-binding protein PinX1 at Q51–G52, promotes caspase-8 and caspase-9 activation, and indirectly induces caspase-3–dependent apoptosis (Fig. 3) [102,103,104]. By cleaving hnRNP A1, 3C relieves repression of apaf-1 IRES translation, further enhancing caspase-3 activation and apoptotic cell death [103,104]. In pyroptosis, 3C directly cleaves gasdermin D (GSDMD) at Q193–G194, inactivating its pore-forming N-terminal fragment and suppressing pyroptotic cell death, while 3C-driven caspase-3 activation contributes indirectly to gasdermin E (GSDME) cleavage [71,105]. These combined actions ensure efficient viral release while limiting inflammatory cell death pathways that could restrict viral spread.

4.7 Protein 3D

3D polymerase (3D, 462 aa) is the viral RNA-dependent RNA polymerase responsible for synthesizing negative- and positive-strand RNA during replication and for catalyzing VPg uridylation. Post-translational modification by SUMO-1 stabilizes 3D, and SUMO-1–dependent ubiquitination further enhances polymerase abundance and activity, leading to increased EV71 replication [106]. 3D also interacts with the methyltransferase METTL3, promoting N6-methyladenosine (m6A) modification of EV71 RNA and facilitating viral RNA translation and stability [106]. Similarly, N4-acetylcytidine (ac4C) modification of viral RNA augments translation and RNA–3D binding, with ac4C-deficient mutants showing reduced pathogenicity in mice [107,108].

3D can manipulate cell cycle progression and innate immunity. It induces S-phase arrest, creating a cellular state favorable to viral replication [109,110]. 3D directly binds NLRP3 to promote assembly of the 3D–NLRP3–ASC inflammasome complex and IL-1β release (Fig. 5), yet it concurrently suppresses antiviral signaling by inhibiting IFN-γ–STAT1 signaling and interacting with the CARD domain of MDA5 to block IFN-β promoter activation [68,111]. 3D also engages the autophagy machinery via Beclin1, whose ECD and CCD domains bind 3D to support the formation of autophagic structures that are exploited as replication sites [112]. Furthermore, 3D interacts with acyl-CoA oxidase 1 (ACOX1), reduces peroxisome biogenesis, and enhances ROS production by inhibiting the DJ-1/Nrf2/HO-1 antioxidant pathway, thereby inducing autophagy and apoptosis in neural cells and contributing to neuropathogenesis [112,113].

A3G, discussed above, can associate with 3D and viral RNA and be packaged into progeny virions, thereby restricting infectivity; however, EV71 counteracts this through 2C- and 3D-mediated modulation of A3G stability and function [114,115]. Overall, 3D integrates RNA synthesis with extensive post-transcriptional RNA modification and modulation of cell stress pathways, positioning it as a prime target for broad-spectrum antiviral intervention.

Figure 5: Multifunctional roles of EV71 proteins 3A and 3D in viral replication, innate immune evasion, and host cell fate regulation. EV71 nonstructural proteins 3A and 3D coordinate distinct yet complementary mechanisms to enhance viral replication and suppress host antiviral responses. Protein 3A disrupts innate immune signalling by interacting with ATP1B3 to suppress IFN-I production, and by promoting degradation of G3BP1 via LRRC25 to block RIG-I/MDA5-mediated RLH signalling. It further recruits ACBD3 to engage PI4KB at viral replication sites, facilitating membrane remodeling and genome synthesis. Additionally, 3A binds VPS25 on exosomes, enabling exosome-mediated viral dissemination. Protein 3D functions as an RNA-dependent RNA polymerase and modulates host cell fate by promoting NLRP3 inflammasome activation, inducing S-phase arrest, driving autophagy via Beclin1, and impairing peroxisomal function through ACOX1-mediated oxidative stress. Furthermore, 3D suppresses IFN-γ signaling and MDA5-mediated IFN-β production, serving as a dual regulator of viral immune evasion and host cell survival. Together, 3A and 3D represent key molecular determinants of EV71 pathogenesis and therapeutic intervention targets. Abbreviation: IFN-I, type I interferon; IFN-γ, interferon-gamma; ATP1B3, sodium/potassium-transporting ATPase subunit beta-3; G3BP1, Ras-GTPase–activating protein SH3 domain–binding protein 1; LRRC25, leucine-rich repeat-containing protein 25; RIG-I, retinoic acid-inducible gene I; MDA5, melanoma differentiation-associated protein 5; RLH, RIG-I-like helicase pathway; ACBD3, acyl-CoA-binding domain–containing protein 3; PI4KB, phosphatidylinositol-4-kinase IIIβ; VPS25, vacuolar protein sorting–associated protein 25; 3Dpol, EV71 RNA-dependent RNA polymerase; NLRP3, NOD-like receptor family pyrin domain containing 3; ASC, apoptosis-associated speck-like protein containing a CARD; Beclin1, autophagy regulator; ACOX1, acyl-CoA oxidase 1.

Nonstructural proteins 2A–3D serve as the functional core of the EV71 replication machinery and orchestrate immune evasion, intracellular remodeling, and regulated cell death. However, most mechanistic insights originate from in vitro overexpression systems or limited animal models, and the temporal hierarchy of these proteins during natural human infection remains unresolved. Deciphering how these proteins interact as a coordinated network—rather than as isolated effectors—will be crucial to understanding EV71 pathogenesis.

Future research should prioritize in vivo validation of protein–host interactions, temporal mapping of immune evasion events, and evaluation of 2A, 3C, and 3D as conserved enzymatic targets for small-molecule inhibition. The integration of stem cell–derived neural organoids, CRISPR-based functional screening, and multi-omics profiling may provide a physiologically relevant platform to dissect viral strategies and identify actionable host dependencies. Ultimately, systematic dissection of the nonstructural protein network—through structure-guided drug design and advanced infection models—will be essential to convert current mechanistic insights into clinically viable antiviral strategies.

5 Conclusions and Future Perspectives

EV71 remains a persistent threat to child health due to its neurotropic potential, genetic diversity, and absence of broadly protective antiviral or vaccine strategies. The coordinated actions of its structural proteins (VP1–VP4) and nonstructural proteins (2A–3D) extend beyond viral assembly and replication, orchestrating receptor binding, immune evasion, intracellular remodeling, and host cell death. This expanding mechanistic landscape suggests that EV71 operates as a systems-level pathogen rather than through isolated linear pathways.

Key host factors—including ACBD3, RTN3, VPS25, NAT8, METTL3, and SPOP—have emerged as central nodes in virus–host interplay, indicating that targeting viral–host interfaces may offer broader therapeutic potential than virus-directed approaches alone. However, much of the current evidence stems from in vitro studies, and mechanistic validation in physiologically relevant models—such as human organoids, hSCARB2 knock-in mice, and primary neuronal cultures—remains limited.

To accelerate translational progress, several priority questions warrant systematic investigation. A central objective is to determine whether cross-genotype vaccines can be rationally engineered by exploring conserved structural motifs or epitope-scaffolding strategies to achieve broad and durable protection. Equally important is the identification of viral proteins and post-translational modifications that constitute the most tractable druggable nodes within the EV71 interactome, thereby enabling the development of targeted antiviral interventions. Advances in computational biology further invite exploration into whether artificial intelligence–assisted protein design platforms, including RFdiffusion and AlphaFold2-multimer, can generate synthetic antibodies or receptor mimetics with broad-spectrum neutralizing capacity. In parallel, the therapeutic feasibility of host-directed strategies—such as inhibition of METTL3 or VPS34—should be rigorously evaluated to determine whether viral replication can be suppressed without perturbing essential cellular homeostasis. Finally, a deeper mechanistic understanding is needed to clarify how EV71 remodels neuroimmune crosstalk during the earliest stages of infection, particularly within brainstem neurons, microglia, and gut-resident immune compartments.

A unified model of EV71 pathogenesis will require combining structural virology with systems immunology and translational biology. By bridging these disciplines, the field can advance toward broad-spectrum antivirals and next-generation vaccine platforms capable of mitigating the global burden of EV71 and related enteroviruses.