Abstract
Enterovirus 71 (EV71) is the main pathogen of the hand, foot, and mouth disease. It was firstly isolated from sputum specimens of infants with central nervous system diseases in California in 1969, and has been repeatedly reported in various parts of the world, especially in the Asia-Pacific region. EV71 3C protein is a 183 amino acid cysteine protease that can cleave most structural and non-structural proteins of EV71. Based on the analysis and understanding of EV71 3C protease, it is helpful to study and treat diseases caused by EV71 virus infection. The EV71 3C protease promotes virus replication by cleaving EV71 synthesis or host proteins. Moreover, EV71 3C protease inhibits the innate immune system and causes apoptosis. At present, in order to deal with the damage caused by the EV71, it is urgent to develop antiviral drugs targeting 3C protease. This review will focus on the structure, function, and mechanism of EV71 3C protease.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
EV71 is a member of the picornaviridae family and is highly infectious in the central nervous system, which causes serious clinical symptoms including encephalitis, poliomyelitis-like paralysis, and even death. However, the pathogenesis of EV71 remains unclear and there is no effective vaccines or drugs to prevent it [1]. The genome of EV71 is a single positive-stranded RNA of approximately 7.4 kb in length and contains an open reading frame (ORF), encoding a polypeptide with 2194 amino acids. The polyprotein is further proteolyzed into P1, P2, and P3 precursor proteins. The P1 precursor protein is proteolyzed into four structural proteins (VP1, VP2, VP3, and VP4). The P2 and P3 precursor proteins are proteolyzed into seven non-structural proteins (2A–2C and 3A–3D) [2]. Among them, the viral protease 3C has been proved to be involved in multiple pathological processes of EV71. EV71 3C protease can cleave the connection site Gln–Gly of P2–P3 [3]. EV71 3C protease can degrade DNA repair enzymes, thereby activating caspase and inducing apoptosis of host cells [4]. EV71 3C protease can inhibit innate immunity by inhibiting type I interferon response [5]. In this article, we will summarize the structure, function, and mechanism of EV71 3C protease.
The Structure of EV71 3C Protease
The crystal structure of EV71 3C protease consists of two similar β-ribbon folded. The long, shallow groove region between the β-ribbon regions is the substrate binding site [6]. The β-ribbon exists in the cleft of the picornaviral 3C protease binding sites to the substrate, and the tip of β-ribbon faces the protease active site. We call this β-ribbon conformation "closed conformation". Unlike picornaviral 3C protease, EV71 3C protease is an open conformation of β-ribbon between βB2 and βC2 (123–133 aa). The β-ribbon flips and the apical end are away from the active site of the EV71 3C protease and are located over the substrate binding cleft [7]. Gly123 and His133 in the β-ribbon form a hinge structure, which plays a key role in the catalytic activity and conformational change of β-ribbon [8]. Previous studies have found that most picornaviral 3C proteases cleave Gln–Gly proteins. Similarly, the catalytic center of EV71 3C protease is Cys–His–Glu [6]. EV71 3C protease contains KFRDI motif (positions 82–86) and VGK motif (positions 154–156) RNA binding domain [3].
The Role of 3C Protease in the Pathway of Induction of Interferon
Most virus-infected cells produce type I interferons (IFN-α/β) and type III interferons (IFN-λ) or interleukin-28/29 (IL-28/29) [9]. Type II interferons (IFN-γ) are produced only by T cells or NK cells [10, 11]. Picornavirals are mainly recognized by three classical pattern recognition receptors: Toll-like receptors (TLRs), Retinoic acid-induced gene I-like receptors (RLRs), and Nucleotide oligomerization domain-like receptors (NLRs) [12]. We will introduce the role of EV71 3C protease in the three receptor pathways.
The Role of 3C Protease in TLRs Pathway
Studies have shown that TLR1,2,4,5,6 exist on the cell membrane and TLR3,7,8,9,10,11,12,13 exist in the endosomal compartments. TLR4/MD2 (myeloid differentiation) complex, TLR1/6, TLR2 and TLR5 recognize LPS, lipoproteins, and flagellin to activate NF-κB, and induce type I interferon production [13]. TLR7/8 recognizes single-stranded RNA of RNA virus. TLR9 recognizes unmethylated cytosine-phosphate-guanosine (CpG) DNA in bacteria. TLR3 recognizes viral double-stranded RNA and recruits TRIF to induce TRAF3 and activate the TBK1/IKKε complex. Moreover, TRIF also induces TRAF6 to activate the TAK1/TAB2/TAB3/TAB1 complex. After activating TBK1/IKKε, it can phosphorylate IRF7 or IRF3 to induce IFN-I production [14]. After activating TAK1/TAB2/TAB3/TAB1 complex, NF-κB is dimerized and enters into the nucleus, thereby producing proinflammatory cytokines [15] (Fig. 1).
EV71 3C protease can cleave the Q312–S313 site of toll-like ligand TRIF and affect the IFN-β production and NF-κB activation. When the catalytic site H40D of EV71 3C protease is mutated, it cannot inhibit the activation of NF-κB and IFN-β promoters [16]. EV71 3C protease also inhibits NF-κB promoter activation by cleavage of the TAK1 complex by its catalytic activity. EV71 3C protease can cut TAK1 complex, including TAK1Q360-S361, TAB1Q414-G415, Q451-S452, TAB2Q113-S114 and TAB3Q173-G174, and Q343-G344. Overexpression of TAB2 inhibits EV71 replication. However, fragmentation of TAB2 has no impact on EV71 replication [17]. EV71 3C protease inhibits IRF3 entry into the nucleus and prevents RIG-I pathway from inducing IFN-β production in HT-29 cells [18]. However, another study showed that EV71 3C protease cleaves IRF7 in site of Q189-S190, but had no effect on IRF3 [5]. Furthermore, the 3C protease can cleave purified IRF7, but not IRF3. These inconsistencies have not been clarified [19]. In summary, these studies have shown that EV71 3C protease affects IFN-I production and proinflammatory factor production in part through the TLRs signaling pathway.
The Role of 3C Protease in RLRs Pathway
Members of the RLRs family include RIG-I, MDA5, and LGP2.LGP2 cannot induce type I IFN because it has no CARD structure [20]. RIG-I recognizes double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) virus containing 5′-triphosphate structure.MDA5 mainly recognizes double strand (dsRNA) and positive strand RNA [(+) ssRNA] viruses [21]. RGI-1 and MDA5 have two N-terminal caspase recruitment domains and a C-terminal repressor domain (RD).RIG-I and MDA5 recognize RNA viruses, cause conformational changes, and expose their N-terminal caspase recruitment domains. Subsequently, activated RIG-1 and MDA5 interact with adaptor IFN promoter-stimulating factor 1(MAVS). MAVS binds to IKKα-IKKβ-IKKγ and activates the NF-κB pathway (Fig. 1). In addition, MAVS also activates the TRADD/TANK/TRAF3 complex, thereby activating the TBK1 and IKKε complexes. Activated TBK1 and IKKε complexes can phosphorylate IRF3/IRF7 and induce IFN-I production [22].
EV71 3C protease blocks the binding of RIG-I to IPS-1 and TBK1 by binding to the caspase recruitment domain of RIG-I. EV71 3C protease inhibits IRF3 entry into the nucleus and affected IFN-β production. EV71 3C protease also binds to MDA5, but does not inhibit MDA5-regulated IFN-β production. H40, KFRDI and VDK regions of EV71 3C protease may bind to RIG-I, resulting in inhibition of IFN-β production [5]. EV71-infected cells reduced Lys 63-linked polyubiquitin chains in the N-terminal CARDs region of the RIG-I and inhibit the IFN-I signaling pathway. Increasing the level of ubiquitination of RIG-I can promote the expression of IFN-β and ISGs [23].
Cylindromatosis (CYLD) is a deubiquitinating enzyme that removes Lys 63-linked polyubiquitin chains from RIG-I. CYLD can inhibit the production of IRF3 pathway and IFN-β production [24]. MiR-526a can downregulate the mRNA and protein of CYLD, which increases the expression levels of IFN-I. However, EV71 3C protease downregulated the expression of miR-526a, thereby inhibiting RIG-I regulated interferon type I production [25]. In addition, EV71 encoded-2A protease can cleave MDA5 and MAVS, and then inhibit the IRF3 pathway to mediate IFN-β production [26, 27]. Therefore, EV71 2A protease and 3C protease regulate the activity of MDA5 and RIG-I, thereby affecting the innate immune response.
The Role of 3C Protease in NLRP3 Pathway
In human, there are 22 Nucleotide-binding and oligomerization domains (NOD)-like receptors (NLRs).NLRs recognize pathogens such as bacteria and viruses, and produce activated inflammatory factors such as interleukin-1β (IL-1β) and interleukins-18 (IL-18).Therefore, NLRs can kill or eliminate invading pathogens to maintain the body's immune system balance [28]. The NLRP3 inflammasome is an important component of the NLR inflammatory pathway. Classical NLRP3 inflammasome activation is stimulated by two signals. The first signal activates TLR4 signal pathway, promotes nuclear transcription factor κB (NF-κB) activation and activates the production of precursors such as IL-1β and IL-18. The second signal promotes the assembly of the NLRP3/ASC/pro-caspase-1 protein complex. Pro-caspase-1 self-cuts into an activated form. Activated Caspase-1 also helps pro-IL-1β, pro-IL-18 matures into IL-1β, IL-18 [30] (Fig. 1). The activation of Non-classical NLRP3 inflammasome does not depend on the activation of TLR4 signaling pathway. The latest research shows that caspase-11 directly recognizes intracellular LPS, initiates the activation of NLRP3 inflammasome, promotes the activation and release of gasdermin D, and mediates cell death [29, 31].
After EV71 infection, human primary monocyte-derived macrophages (MDMs) induce proinflammatory cytokines, such as IL-1, IL-6 and tumor necrosis factor a (TNF-a) [32]. Several studies revealed that children with EV71 infection have increased levels of cytokines such as IL-6, IL-10 and IL-13 [33]. Sendai virus and influenza A virus can activate NLRP3 inflammasome [34]. EV71 2A protease specifically cleaves NLRP3 G493-L494. EV71 3C protease specifically cleaves Q225-G226 of NLRP3, but has no cleavage effect on ANL2, ASC, caspase-1, and IL-1β of NLRP3 signaling pathway. Thus, NLRP3 pathway loses regulation of IL-1β and IL-18 production, and does not inhibit EV71 replication. In this review, it is proposed that the patient infection period is divided into 2 stages. Initially, EV71 protein and RNA activates NLRP3 to regulate interleukin production against infection. However, in the later stage, EV71 replicate and produce a large amount of 3C protease and 2A protease. 3C protease and 2A protease specifically cleaves NLRP3, which hinders immune activity [35]. (As shown in Fig. 1, we summarized that EV71 3C protease is involved in the mechanism of interferon-inducing pathways).
The Role of 3C Protease in Interferon Signal Transduction Pathway
Type I IFNs (IFN-α/β) and type II IFN (IFN-γ) are widely expressed in the cell as the first line of defense. These cytokines are capable of combating viral infection and inhibiting virus replication [36]. There are 14 types of type I interferon (IFN-α) in mice, 13 in humans, and one type of β (IFN-β) [37]. Most cells can produce IFNβ. But IFNβ is mainly produced by hematopoietic cells, especially plasma cell-like dendritic cells [38]. The type I interferon (IFN-α/β/ω) receptor is mainly composed of two subunits, IFNAR1 and IFNAR-2. The intracellular domains of IFNAR-1 and IFNAR-2 can bind to Jak1 and Jak2 and activates Jak signaling pathway, resulting in phosphorylation of STAT1 and STAT2. Activated STAT1/STAT2 and IRF9 (p48) form the transcription factor complex interferon-stimulated gene factor 3 (ISGF3). ISGF3 and IFN-stimulated regulatory elements (ISREs) responsible for inducing transcription of related target genes (Fig. 2).
IFN-γ receptor also contains two subunits, IFNGR-1 and IFNGR-2. IFN-γ activates Jak1 and Jak2 pathway and subsequently activates the STAT1/STAT1 complex. STAT1/STAT1 and IRF1 form a homodimer GAF (IFN-γ activating factor) into the nucleus. IFN-γ activating factor combines with GAS elements to induce ISGs transcription [39]. ISGs induce transcription products: including oligoadenylate synthase (OAS), protein kinase R (PKR), and interferon-induced GTP-binding protein Mx (MX) [40, 41]. OAS and PKR can regulate virus replication. RNAase L and MX can inhibit viral transcription [42] (Fig. 2).
3C Protease Affects Type I Interferon-Induced Signaling Pathway
There are a lot of evidence that IFNs have a strong antiviral effect and can be used to treat patients [43, 44]. However, only high concentration of type I IFNs can control EV71 infection and replication. In mice, IFNs pretreatment can avoid infection of EV71. If mice are injected with IFNs after infection with EV71, the antiviral effect of IFNs is small [45]. Studies showed that EV71 reduced the expression of type I interferon receptor IFNAR1, which affects the production of ISGs [46]. However, Liu et al. revealed that EV71 did not significantly downregulate the expression of IFNAR1, but downregulate the expression of JAK1 [47]. Wang et al. indicated that EV71 did not downregulate the expression of JAK1 and IFNAR1 [48].
In conclusion, EV71 blocks IFN signaling probably not by downregulating IFNAR1 and JAK1 expression. Hung et al. revealed that the 3C protease of EV71 had a cleaving effect on IRF9, and the C147S point mutation of 3C protease had no cleaving effect on IRF9 [49]. In general, it may be that EV71 3C protease affects the effect of type I interferon by cleaving IRF9, which promotes EV71 replication in the host cells. Thus, it is necessary to further explore the mechanism of EV71 blocking interferon signal transduction.
Effect of 3C Protease on Type II Interferon-Induced Signaling Pathway
Several studies have shown that pulmonary edema and encephalitis resulting from EV71 infection are associated with gene polymorphism and expression of IFN-γ [50, 51]. The mice lacking A129 (IFN-α/β receptor deficient) has a higher mortality rate than AG129(IFN-α/β, γ receptor deficient) [52]. Moreover, the knockdown of the interferon type II receptor and STAT1 in mice increased the EV71 lethality [53]. It indicates that type II interferon may also play an important role in antiviral activity.
Wang et al. showed that EV71 2A protease and 3D protease prevented IFN-γ induction of IRF1 activation, resulting in failure of STAT1 phosphate nucleation [54]. Therefore, EV71 attenuates IRF1 activation in the IFN-γ signaling pathway and promotes replication in the host cells (as shown in Fig. 2, we summarized that EV71 3C protease is involved in the interferon signal transduction pathway).
The Role of EV71 3C Protease in Host Cell Apoptosis
Picornaviral 3C protease can activate caspase and induce apoptosis of host cells. EV71 3C protease triggers cell DNA degradation and apoptotic bodies [55]. Li et al. reveals that the 3C protease can degrade poly (ADP-ribose) polymerase, a DNA repair enzyme. This study showed that 3C protease can activate caspase and induce apoptosis in SF268 cells. Caspase inhibitors (DEVD-fmk and VAD-fmk) can block apoptosis caused by 3C protease [4]. It has been reported that EV71 3C protease affects the polyadenylation of host mRNA by cleaving CstF64 [56]. EV71 3C protease relies on its protease activity to cleave GSDMD, a significant component of pyroptosis, resulting in cell pyroptosis [57]. Li et al. demonstrated that EV71 3C protease promotes apoptosis through cleaving pinx1, a telomere binding protein. What's more, 3C protease cleaves the pinx1at the site of Q50-G51 pair, and accelerates EV71 release [58]. Chen et al. recently confirmed that SUMO E2-conjugating enzyme Ubc9 promotes ubiquitination of EV71 3C protease, which helps to reduce EV71 replication and cell apoptosis [59]. The ubiquitination of EV-A71 3C protease inhibits viral replication and host cell apoptosis.
In addition, endoplasmic reticulum-associated degradation (ERAD) component p97 can participate in EV71 replication. EV71 3C protease induced Ubc6e cleavage may be a key mechanism for EV71 to inhibit ERAD [60]. In conclusion, EV71 3C protease facilitates EV71 replication by affecting endoplasmic reticulum molecules.
EV71 3C Protease Inhibitors and Potential Treatment for EV71 Infection
The 3C protease play important roles in EV71 replication and apoptosis of host cells. Thus, the development of antiviral drugs targeting 3C protease has become a hot spot. EV71 3C protease has no homology with mammalian proteases, so 3C proteases can be used as antiviral targets. According to the specificity of 3C protease structure, anti-EV71 drugs were designed, such as inhibitors of 3–5 amino acids and aldehyde peptides, which can form irreversible covalent bonds with the 3C protease active site.
Broad-Spectrum Inhibitors of 3C Protease
Rupintrivir (AG7088) has extensive antiviral activity against HRV and a variety of enteroviruses. EV71 3C protease catalytic structural amino acid residues (His40, Glu71 and Cys147) are strictly conserved. Rupintrivir can covalently bind to the active site of EV71 3C protease. Studies have shown that this binding mode is relatively stable. Yeast two-hybrid cell experiments also demonstrated that Rupintrivir effectively binds to EV71 3C protease. Besides, Ruprintrivir does not reduce the frequency of the common cold, but has a significant antiviral effect in the common cold [61].
Modified Peptide Inhibitor of 3C Protease
Based on the covalent combination of Rupintrivir and 3C protease, a series of highly inhibitory drugs have been developed, such as substance-based peptidomimetics, aldehydes and derivatives. Kuo et al. found that Rupintrivir enhanced antiviral activity by replacing the unsaturated ester with the aldehyde at the P1’ position [7].The EV71 3C protease inhibitor, pseudopeptide aldehyde 5×, has strong inhibitory activity and low cytotoxicity [62]. Wang et al. reported that Peptide NK-1.8k inhibits EV71 3C protease activity and EV71 proliferation [63]. α-Keto amide exhibits good inhibition of 3C protease activity and relatively low cytotoxicity, so it can be used as an inhibitor of EV71 3C protease [64]. And the experimental results show that if the inhibitor has a rotatable ester bond, it can enhance the interaction of 3C protease at the substrate binding site of S4 and increase the antiviral activity [65].
Natural Medicine and Other Inhibitors
Hung et al. found that Rupintrivir and interferon have a synergistic effect on the inhibition of EV71. Fisetin and rutin have been reported to inhibit EV71 3C protease with an IC50 value of 85 μM and 110 μM, respectively [66]. CPI (Diisopropyl chrysin-7-yl phosphate) can bind toEV71 3C protease and inhibits its activity. The main component of the CPI, chrysin, can be extracted from natural flavonoids in many plants [67]. Due to the excellent inhibitory activity of Cyanohydrin on 3C protease, Cyanohydrin derivatives can be used as EV71 3C protease inhibitors [68]. Adenosine analog (NITD008) inhibits EV71 replication. Its EC50 value is 0.67 mM, but it has relatively high cytotoxicity (CC50 1/4 119.97 mM) [69].It is reported that EV71 3C protease inhibitor DC07090 exhibited the inhibition potency with an IC50 value of 21.72 ± 0.95 mM without apparent toxicity (CC50 > 200 mM) [70].
Conclusion
EV71 is the leading cause of severe hand, foot and mouth disease, which is a serious threat to the world, especially in the Asia-Pacific region. Studies have shown that EV71 3C protease plays important roles in its pathogenesis. The research on EV71 3C protease has made great progress. The crystal structure of 3C protease has been determined, the catalytic active site and RNA binding site have been found. The innate immune system, as the body's first line of defense, plays key roles in the fight against microbial infections, especially the type I interferon response. EV71 attenuates interferon signaling, allowing EV71 to evade immune mechanisms. However, the EV71 escaping mechanism has not been fully elucidated. The 2A and 3C protease of EV71 have cleavage inhibition on some important linker molecules. Research on anti-EV71 3C protease drugs has made some progress. The role of EV71 in the pathogenic mechanism still needs further study. The targeting medicines against 3C protease are still needed to be further explored.
References
Yuan J, Shen L, Wu J, Zou X, Gu J, Chen J, Mao L (2018) Enterovirus A71 proteins: structure and function. Front Microbiol 9:286. https://doi.org/10.3389/fmicb.2018.00286
Lin JY, Chen TC, Weng KF, Chang SC, Chen LL, Shih SR (2009) Viral and host proteins involved in picornavirus life cycle. J Biomed Sci 16:103. https://doi.org/10.1186/1423-0127-16-103
Shih SR, Chiang C, Chen TC, Wu CN, Hsu JT, Lee JC, Hwang MJ, Li ML, Chen GW, Ho MS (2004) Mutations at KFRDI and VGK domains of enterovirus 71 3C protease affect its RNA binding and proteolytic activities. J Biomed Sci 11(2):239–248. https://doi.org/10.1007/bf02256567
Li ML, Hsu TA, Chen TC, Chang SC, Lee JC, Chen CC, Stollar V, Shih SR (2002) The 3C protease activity of enterovirus 71 induces human neural cell apoptosis. Virology 293(2):386–395. https://doi.org/10.1006/viro.2001.1310
Lei X, Liu X, Ma Y, Sun Z, Yang Y, Jin Q, He B, Wang J (2010) The 3C protein of enterovirus 71 inhibits retinoid acid-inducible gene I-mediated interferon regulatory factor 3 activation and type I interferon responses. J Virol 84(16):8051–8061. https://doi.org/10.1128/JVI.02491-09
Sweeney TR, Roque-Rosell N, Birtley JR, Leatherbarrow RJ, Curry S (2007) Structural and mutagenic analysis of foot-and-mouth disease virus 3C protease reveals the role of the beta-ribbon in proteolysis. J Virol 81(1):115–124. https://doi.org/10.1128/jvi.01587-06
Kuo CJ, Shie JJ, Fang JM, Yen GR, Hsu JT, Liu HG, Tseng SN, Chang SC, Lee CY, Shih SR, Liang PH (2008) Design, synthesis, and evaluation of 3C protease inhibitors as anti-enterovirus 71 agents. Bioorg Med Chem 16(15):7388–7398. https://doi.org/10.1016/j.bmc.2008.06.015
Sun D, Chen S, Cheng A, Wang M (2016) Roles of the picornaviral 3C proteinase in the viral life cycle and host cells. Viruses 8(3):82. https://doi.org/10.3390/v8030082
Ank N, West H, Bartholdy C, Eriksson K, Thomsen AR, Paludan SR (2006) Lambda interferon (IFN-lambda), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo. J Virol 80(9):4501–4509. https://doi.org/10.1128/jvi.80.9.4501-4509.2006
Schroder K, Hertzog PJ, Ravasi T, Hume DA (2004) Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol 75(2):163–189. https://doi.org/10.1189/jlb.0603252
Schoenborn JR, Wilson CB (2007) Regulation of interferon-gamma during innate and adaptive immune responses. Adv Immunol 96:41–101. https://doi.org/10.1016/s0065-2776(07)96002-2
Iwasaki A, Pillai PS (2014) Innate immunity to influenza virus infection. Nat Rev Immunol 14(5):315–328. https://doi.org/10.1038/nri3665
Tartey S, Takeuchi O (2017) Pathogen recognition and Toll-like receptor targeted therapeutics in innate immune cells. Int Rev Immunol 36(2):57–73. https://doi.org/10.1080/08830185.2016.1261318
Honda K, Taniguchi T (2006) IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol 6(9):644–658. https://doi.org/10.1038/nri1900
Mishra S, Kumar H (2018) Balancing anti-viral innate immunity and immune homeostasis. Cell Mol Immunol 15(4):408–410. https://doi.org/10.1038/cmi.2017.98
Lei X, Sun Z, Liu X, Jin Q, He B, Wang J (2011) Cleavage of the adaptor protein TRIF by enterovirus 71 3C inhibits antiviral responses mediated by Toll-like receptor 3. J Virol 85(17):8811–8818. https://doi.org/10.1128/jvi.00447-11
Lei X, Han N, Xiao X, Jin Q, He B, Wang J (2014) Enterovirus 71 3C inhibits cytokine expression through cleavage of the TAK1/TAB1/TAB2/TAB3 complex. J Virol 88(17):9830–9841. https://doi.org/10.1128/jvi.01425-14
Wang C, Ji L, Yuan X, Jin Y, Cardona CJ, Xing Z (2016) Differential regulation of TLR signaling on the induction of antiviral interferons in human intestinal epithelial cells infected with enterovirus 71. PLoS ONE 11(3):e0152177. https://doi.org/10.1371/journal.pone.0152177
Lei X, Xiao X, Xue Q, Jin Q, He B, Wang J (2013) Cleavage of interferon regulatory factor 7 by enterovirus 71 3C suppresses cellular responses. J Virol 87(3):1690–1698. https://doi.org/10.1128/jvi.01855-12
Rodriguez KR, Bruns AM, Horvath CM (2014) MDA5 and LGP2: accomplices and antagonists of antiviral signal transduction. J Virol 88(15):8194–8200. https://doi.org/10.1128/jvi.00640-14
Schlee M (2013) Master sensors of pathogenic RNA-RIG-I like receptors. Immunobiology 218(11):1322–1335. https://doi.org/10.1016/j.imbio.2013.06.007
Chiang JJ, Davis ME, Gack MU (2014) Regulation of RIG-I-like receptor signaling by host and viral proteins. Cytokine Growth Factor Rev 25(5):491–505. https://doi.org/10.1016/j.cytogfr.2014.06.005
Chen N, Li X, Li P, Pan Z, Ding Y, Zou D, Zheng L, Zhang Y, Li L, Xiao L, Song B, Cui Y, Cao H, Zhang H (2016) Enterovirus 71 inhibits cellular type I interferon signaling by inhibiting host RIG-I ubiquitination. Microb Pathog 100:84–89. https://doi.org/10.1016/j.micpath.2016.09.001
Friedman CS, O'Donnell MA, Legarda-Addison D, Ng A, Cardenas WB, Yount JS, Moran TM, Basler CF, Komuro A, Horvath CM, Xavier R, Ting AT (2008) The tumour suppressor CYLD is a negative regulator of RIG-I-mediated antiviral response. EMBO Rep 9(9):930–936. https://doi.org/10.1038/embor.2008.136
Xu C, He X, Zheng Z, Zhang Z, Wei C, Guan K, Hou L, Zhang B, Zhu L, Cao Y, Zhang Y, Cao Y, Ma S, Wang P, Zhang P, Xu Q, Ling Y, Yang X, Zhong H (2014) Downregulation of microRNA miR-526a by enterovirus inhibits RIG-I-dependent innate immune response. J Virol 88(19):11356–11368. https://doi.org/10.1128/jvi.01400-14
Wang B, Xi X, Lei X, Zhang X, Cui S, Wang J, Jin Q, Zhao Z (2013) Enterovirus 71 protease 2Apro targets MAVS to inhibit anti-viral type I interferon responses. PLoS Pathog 9(3):e1003231. https://doi.org/10.1371/journal.ppat.1003231
Feng Q, Langereis MA, Lork M, Nguyen M, Hato SV, Lanke K, Emdad L, Bhoopathi P, Fisher PB, Lloyd RE, van Kuppeveld FJ (2014) Enterovirus 2Apro targets MDA5 and MAVS in infected cells. J Virol 88(6):3369–3378. https://doi.org/10.1128/jvi.02712-13
Kim YK, Shin JS, Nahm MH (2016) NOD-like receptors in infection, immunity, and diseases. Yonsei Med J 57(1):5–14. https://doi.org/10.3349/ymj.2016.57.1.5
Amin J, Boche D, Rakic S (2017) What do we know about the inflammasome in humans? Brain Pathol 27(2):192–204. https://doi.org/10.1111/bpa.12479
Zhou W, Chen C, Chen Z, Liu L, Jiang J, Wu Z, Zhao M, Chen Y (2018) NLRP3: a novel mediator in cardiovascular disease. J Immunol Res 2018:5702103. https://doi.org/10.1155/2018/5702103
Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H, Zhuang Y, Cai T, Wang F, Shao F (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526(7575):660–665. https://doi.org/10.1038/nature15514
Gong X, Zhou J, Zhu W, Liu N, Li J, Li L, Jin Y, Duan Z (2012) Excessive proinflammatory cytokine and chemokine responses of human monocyte-derived macrophages to enterovirus 71 infection. BMC Infect Dis 12:224. https://doi.org/10.1186/1471-2334-12-224
Chen Z, Li R, Xie Z, Huang G, Yuan Q, Zeng J (2014) IL-6, IL-10 and IL-13 are associated with pathogenesis in children with enterovirus 71 infection. Int J Clin Exp Med 7(9):2718–2723
Kanneganti TD, Body-Malapel M, Amer A, Park JH, Whitfield J, Franchi L, Taraporewala ZF, Miller D, Patton JT, Inohara N, Nunez G (2006) Critical role for Cryopyrin/Nalp3 in activation of caspase-1 in response to viral infection and double-stranded RNA. J Biol Chem 281(48):36560–36568. https://doi.org/10.1074/jbc.M607594200
Wang H, Lei X, Xiao X, Yang C, Lu W, Huang Z, Leng Q, Jin Q, He B, Meng G, Wang J (2015) Reciprocal regulation between enterovirus 71 and the NLRP3 inflammasome. Cell Rep 12(1):42–48. https://doi.org/10.1016/j.celrep.2015.05.047
Platanias LC (2005) Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol 5(5):375–386. https://doi.org/10.1038/nri1604
Ng CT, Mendoza JL, Garcia KC, Oldstone MB (2016) Alpha and beta type 1 interferon signaling: passage for diverse biologic outcomes. Cell 164(3):349–352. https://doi.org/10.1016/j.cell.2015.12.027
Ivashkiv LB, Donlin LT (2014) Regulation of type I interferon responses. Nat Rev Immunol 14(1):36–49. https://doi.org/10.1038/nri3581
Michalska A, Blaszczyk K, Wesoly J, Bluyssen HAR (2018) A positive feedback amplifier circuit that regulates interferon (IFN)-stimulated gene expression and controls type I and type II IFN responses. Front Immunol 9:1135. https://doi.org/10.3389/fimmu.2018.01135
Garcia MA, Gil J, Ventoso I, Guerra S, Domingo E, Rivas C, Esteban M (2006) Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. Microbiol Mol Biol Rev 70(4):1032–1060. https://doi.org/10.1128/mmbr.00027-06
Silverman RH (1994) Fascination with 2–5A-dependent RNase: a unique enzyme that functions in interferon action. J Interferon Res 14(3):101–104
Goodbourn S, Didcock L, Randall RE (2000) Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. J Gen Virol 81(Pt 10):2341–2364. https://doi.org/10.1099/0022-1317-81-10-2341
Katze MG, He Y, Gale M Jr (2002) Viruses and interferon: a fight for supremacy. Nat Rev Immunol 2(9):675–687. https://doi.org/10.1038/nri888
Weber F, Kochs G, Haller O (2004) Inverse interference: how viruses fight the interferon system. Viral Immunol 17(4):498–515. https://doi.org/10.1089/vim.2004.17.498
Liu ML, Lee YP, Wang YF, Lei HY, Liu CC, Wang SM, Su IJ, Wang JR, Yeh TM, Chen SH, Yu CK (2005) Type I interferons protect mice against enterovirus 71 infection. J Gen Virol 86(Pt 12):3263–3269. https://doi.org/10.1099/vir.0.81195-0
Lu J, Yi L, Zhao J, Yu J, Chen Y, Lin MC, Kung HF, He ML (2012) Enterovirus 71 disrupts interferon signaling by reducing the level of interferon receptor 1. J Virol 86(7):3767–3776. https://doi.org/10.1128/jvi.06687-11
Liu Y, Zhang Z, Zhao X, Yu R, Zhang X, Wu S, Liu J, Chi X, Song X, Fu L, Yu Y, Hou L, Chen W (2014) Enterovirus 71 inhibits cellular type I interferon signaling by downregulating JAK1 protein expression. Viral Immunol 27(6):267–276. https://doi.org/10.1089/vim.2013.0127
Wang C, Sun M, Yuan X, Ji L, Jin Y, Cardona CJ, Xing Z (2017) Enterovirus 71 suppresses interferon responses by blocking Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling through inducing karyopherin-alpha1 degradation. J Biol Chem 292(24):10262–10274. https://doi.org/10.1074/jbc.M116.745729
Hung HC, Wang HC, Shih SR, Teng IF, Tseng CP, Hsu JT (2011) Synergistic inhibition of enterovirus 71 replication by interferon and rupintrivir. J Infect Dis 203(12):1784–1790. https://doi.org/10.1093/infdis/jir174
Wang SM, Lei HY, Huang KJ, Wu JM, Wang JR, Yu CK, Su IJ, Liu CC (2003) Pathogenesis of enterovirus 71 brainstem encephalitis in pediatric patients: roles of cytokines and cellular immune activation in patients with pulmonary edema. J Infect Dis 188(4):564–570. https://doi.org/10.1086/376998
Yang J, Zhao N, Su NL, Sun JL, Lv TG, Chen ZB (2012) Association of interleukin 10 and interferon gamma gene polymorphisms with enterovirus 71 encephalitis in patients with hand, foot and mouth disease. Scand J Infect Dis 44(6):465–469. https://doi.org/10.3109/00365548.2011.649490
Caine EA, Partidos CD, Santangelo JD, Osorio JE (2013) Adaptation of enterovirus 71 to adult interferon deficient mice. PLoS ONE 8(3):e59501. https://doi.org/10.1371/journal.pone.0059501
Liao CC, Liou AT, Chang YS, Wu SY, Chang CS, Lee CK, Kung JT, Tu PH, Yu YY, Lin CY, Lin JS, Shih C (2014) Immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of enterovirus 71. J Virol 88(21):12485–12499. https://doi.org/10.1128/jvi.00692-14
Wang LC, Chen SO, Chang SP, Lee YP, Yu CK, Chen CL, Tseng PC, Hsieh CY, Chen SH, Lin CF (2015) Enterovirus 71 proteins 2A and 3D antagonize the antiviral activity of gamma interferon via signaling attenuation. J Virol 89(14):7028–7037. https://doi.org/10.1128/jvi.00205-15
Barco A, Feduchi E, Carrasco L (2000) Poliovirus protease 3C(pro) kills cells by apoptosis. Virology 266(2):352–360. https://doi.org/10.1006/viro.1999.0043
Weng KF, Li ML, Hung CT, Shih SR (2009) Enterovirus 71 3C protease cleaves a novel target CstF-64 and inhibits cellular polyadenylation. PLoS Pathog 5(9):e1000593. https://doi.org/10.1371/journal.ppat.1000593
Lei X, Zhang Z, Xiao X, Qi J, He B, Wang J (2017) Enterovirus 71 inhibits pyroptosis through cleavage of gasdermin D. J Virol. https://doi.org/10.1128/jvi.01069-17
Li J, Yao Y, Chen Y, Xu X, Lin Y, Yang Z, Qiao W, Tan J (2017) Enterovirus 71 3C promotes apoptosis through cleavage of PinX1, a telomere binding protein. J Virol. https://doi.org/10.1128/jvi.02016-16
Chen SC, Chang LY, Wang YW, Chen YC, Weng KF, Shih SR, Shih HM (2011) Sumoylation-promoted enterovirus 71 3C degradation correlates with a reduction in viral replication and cell apoptosis. J Biol Chem 286(36):31373–31384. https://doi.org/10.1074/jbc.M111.254896
Wang T, Wang B, Huang H, Zhang C, Zhu Y, Pei B, Cheng C, Sun L, Wang J, Jin Q, Zhao Z (2017) Enterovirus 71 protease 2Apro and 3Cpro differentially inhibit the cellular endoplasmic reticulum-associated degradation (ERAD) pathway via distinct mechanisms, and enterovirus 71 hijacks ERAD component p97 to promote its replication. PLoS Pathog 13(10):e1006674. https://doi.org/10.1371/journal.ppat.1006674
Hayden FG, Turner RB, Gwaltney JM, Chi-Burris K, Gersten M, Hsyu P, Patick AK, Smith GJ 3rd, Zalman LS (2003) Phase II, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers. Antimicrob Agents Chemother 47(12):3907–3916
Zhai Y, Ma Y, Ma F, Nie Q, Ren X, Wang Y, Shang L, Yin Z (2016) Structure-activity relationship study of peptidomimetic aldehydes as enterovirus 71 3C protease inhibitors. Eur J Med Chem 124:559–573. https://doi.org/10.1016/j.ejmech.2016.08.064
Wang Y, Yang B, Zhai Y, Yin Z, Sun Y, Rao Z (2015) Peptidyl aldehyde NK-1.8k suppresses enterovirus 71 and enterovirus 68 infection by targeting protease 3C. Antimicrob Agents Chemother 59(5):2636–2646. https://doi.org/10.1128/aac.00049-15
Zeng D, Ma Y, Zhang R, Nie Q, Cui Z, Wang Y, Shang L, Yin Z (2016) Synthesis and structure-activity relationship of alpha-keto amides as enterovirus 71 3C protease inhibitors. Bioorg Med Chem Lett 26(7):1762–1766. https://doi.org/10.1016/j.bmcl.2016.02.039
Zhang L, Huang G, Cai Q, Zhao C, Tang L, Ren H, Li P, Li N, Huang J, Chen X, Guan Y, You H, Chen S, Li J, Lin T (2016) Optimize the interactions at S4 with efficient inhibitors targeting 3C proteinase from enterovirus 71. J Mol Recogn 29(11):520–527. https://doi.org/10.1002/jmr.2551
Lin YJ, Chang YC, Hsiao NW, Hsieh JL, Wang CY, Kung SH, Tsai FJ, Lan YC, Lin CW (2012) Fisetin and rutin as 3C protease inhibitors of enterovirus A71. J Virol Methods 182(1–2):93–98. https://doi.org/10.1016/j.jviromet.2012.03.020
Wang J, Zhang T, Du J, Cui S, Yang F, Jin Q (2014) Anti-enterovirus 71 effects of chrysin and its phosphate ester. PLoS ONE 9(3):e89668. https://doi.org/10.1371/journal.pone.0089668
Zhai Y, Zhao X, Cui Z, Wang M, Wang Y, Li L, Sun Q, Yang X, Zeng D, Liu Y, Sun Y, Lou Z, Shang L, Yin Z (2015) Cyanohydrin as an anchoring group for potent and selective inhibitors of enterovirus 71 3C protease. J Med Chem 58(23):9414–9420. https://doi.org/10.1021/acs.jmedchem.5b01013
Shang L, Wang Y, Qing J, Shu B, Cao L, Lou Z, Gong P, Sun Y, Yin Z (2014) An adenosine nucleoside analogue NITD008 inhibits EV71 proliferation. Antiviral Res 112:47–58. https://doi.org/10.1016/j.antiviral.2014.10.009
Ma GH, Ye Y, Zhang D, Xu X, Si P, Peng JL, Xiao YL, Cao RY, Yin YL, Chen J, Zhao LX, Zhou Y, Zhong W, Liu H, Luo XM, Chen LL, Shen X (2016) Identification and biochemical characterization of DC07090 as a novel potent small molecule inhibitor against human enterovirus 71 3C protease by structure-based virtual screening. Eur J Med Chem 124:981–991. https://doi.org/10.1016/j.ejmech.2016.10.019
Acknowledgements
This work was supported by National Natural Science Foundation of China Grants 81660332, Natural Science Foundation of Jiangxi province Grants 20151BAB205057 and Health and family planning project of Jiangxi province Grants 20155634.
Author information
Authors and Affiliations
Contributions
Weihui Wen and Zixuan Qi drafted the manuscript together and Jing Wang was responsible for conception and design.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Wen, W., Qi, Z. & Wang, J. The Function and Mechanism of Enterovirus 71 (EV71) 3C Protease. Curr Microbiol 77, 1968–1975 (2020). https://doi.org/10.1007/s00284-020-02082-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00284-020-02082-4