FIGURE 4 - uploaded by Hanzhong Wang
Content may be subject to copyright.
The effects of 2C on IKKb activation. A, 293T cells (5 3 10 5 ) were cotransfected with 0.5 mg pNF-kB-luc, 0.1 mg pRL-TK, and either 0.5 mg IKKb or IKKb SS/EE (a constitutively active mutant of IKKb) together with the indicated amount of 2C expression plasmid. Total amounts of
Contexts in source publication
Context 1
... (Fig. 3B), we further investigated whether IKKb is target by 2C protein. As shown in Fig. 4A, 2C signifi- cantly inhibited this activation in a dose-dependent manner, in- dicating that 2C protein acted at, or downstream of, the IKKb activation. The target site was further investigated using IKKb constitutively active mutant IKKb SS/EE (21) (Fig. 4A). There was only a small reduction (28%) of IKKb SS/EE-induced NF-kB activation in the presence of the highest amount of 2C (2.5 mg). These results imply that once IKKb is activated, 2C no longer inhibits NF-kB activation, supporting a notion that 2C inhibits NF-kB activation by preventing the phosphorylation of IKKb. To test this ...
Context 2
... in the presence of the highest amount of 2C (2.5 mg). These results imply that once IKKb is activated, 2C no longer inhibits NF-kB activation, supporting a notion that 2C inhibits NF-kB activation by preventing the phosphorylation of IKKb. To test this hypothesis, we used immunoblot assay to detect TNF-a- induced IKKb phosphorylation. As shown in Fig. 4B, the level of phosporylated IKKb was greatly reduced in samples from 2C-expressing cells when treated with TNF-a for 30 min, and to . Twenty-four hours posttransfection, cells were exposed to TNF-a (10 ng/ml) containing medium for 6 h, harvested, lysed, and assayed for luciferase activity. The resultant ratios were normalized to fold ...
Context 3
... inhibit TNF-a-induced IKKb phosphorylation. To elucidate whether 2C inhibits IKKb phosphorylation by targeting IKKb, IKKb-and 2C-expressing plasmids were cotransfected into 293T cells, and the phosphorylation of IKKb was detected by im- munoblot assay. When IKKb was overexpessed, the amount of phospho-IKKb decreased in the presence of 2C protein (Fig. 4C). Taken together, these data indicate that 2C inhibits NF-kB acti- vation by preventing IKKb ...
Citations
... Cell surface-specific receptors are recognized after the virus infected, then activate the downstream adaptor proteins and NF-κB signaling pathways to resist pathogen invasion by promoting the production of inflammatory cytokines [13]. For example, EV71 2C protein suppresses IκB kinase β phosphorylation, thereby inhibiting NF-κB activation and allowing the virus to escape [14]. CVA16 2C recruits protein phosphatase 1, inhibiting IKKβ phosphorylation to regulate the NF-κB pathway and viral replication [15]. ...
Coxsackievirus Group B type 5 (CVB5), an important pathogen of hand-foot-mouth disease, is also associated with neurological complications and poses a public health threat to young infants. Among the CVB5 proteins, the nonstructural protein 3D, known as the Enteroviral RNA-dependent RNA polymerase, is mainly involved in viral genome replication and transcription. In this study, we performed immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC–MS/MS) to identify host proteins that interacted with CVB5 3D polymerase. A total of 116 differentially expressed proteins were obtained. Gene Ontology analysis identified that the proteins were involved in cell development and cell adhesion, distributed in the desmosome and envelope, and participated in GTPase binding. Kyoto Encyclopedia of Genes and Genomes analysis further revealed they participated in nerve diseases, such as Parkinson disease. Among them, 35 proteins were significantly differentially expressed and the cellular protein TGF-BATA-activated kinase1 binding protein 1 (TAB1) was found to be specifically interacting with the 3D polymerase. 3D polymerase facilitated the entry of TAB1 into the nucleus and down-regulated TAB1 expression via the lysosomal pathway. In addition, TAB1 inhibited CVB5 replication via inducing inflammatory factors and activated the NF-κB pathway through IκBα phosphorylation. Moreover, the 90-96aa domain of TAB1 was an important structure for the function. Collectively, our findings demonstrate the mechanism by which cellular TAB1 inhibits the CVB5 replication via activation of the host innate immune response, providing a novel insight into the virus-host innate immunity.
... The nonstructural protein 2C has been widely reported to have ATPase activity and helicase activity, which plays an irreplaceable role in viral replication. In addition, 2C also helps EV-A71 escape from TNF-a-mediated NF-jB immune response by inhibiting IKKa/b phosphorylation [82][83][84][85]. As the most conserved protein in picornavirus, 2C protein has become a hot target for some broad-spectrum antiviral inhibitors in recent years. ...
Background: Enterovirus 71 (EV71) is capable of causing hand, foot and mouth disease (HFMD), which may lead to neurological sequelae and even death. As EV71 is resistant to environmental changes and mutates easily, there is still a lack of effective treatments or globally available vaccines.
Aim of Review: For more than 50 years since the HFMD epidemic, related drug research has been conducted. Progress in this area can promote the further application of existing potential drugs and develop more efficient and safe antiviral drugs, and provide useful reference for protecting the younger generation and maintaining public health security.
Key Scientific Concepts of Review: At present, researchers have identified hundreds of EV71 inhibitors based on screening repurposed drugs, targeted structural design, and rational modification of previously effective drugs as the main development strategies. This review systematically introduces the current potential drugs to inhibit EV71 infection, including viral inhibitors targeting key sites such as the viral capsid, RNA-dependent RNA polymerase (RdRp), internal ribosome entry site (IRES), 3C proteinase (3Cpro), and 2A proteinase (2Apro), starting from each stage of the viral life cycle. Meanwhile, the progress of host-targeting antiviral drugs and their development are summarized in terms of regulating host immunity, inhibiting autophagy or apoptosis, and regulating the cellular redox environment. In addition, the current clinical methods for the prevention and treatment of HFMD are summarized and discussed with the aim of providing support and recommendations for the treatment of enterovirus infections including EV71.
... 3C protease is shown to block the NF-κB pathway to proinflammatory cytokine production by targeting the TAK1/TAB1/TAB2/TAB3 complex [72]. EV-A71 2C targets IKKβ and p65 to suppress NF-κB activation [73][74][75]. In addition to viral factors, EV-A71 infection also induces the expression of host microRNA miR-146a, which in turn downregulates the expression of TRAF6 and IRAK1 involved in TLR signaling to type I IFN induction [76]. ...
Enterovirus A71 (EV-A71) is a growing threat to public health, particularly in the Asia-Pacific region. EV-A71 infection is most prevalent in infants and children and causes a wide spectrum of clinical complications, including hand-foot-and-mouth disease (HFMD), pulmonary and neurological disorders. The pathogenesis of EV-A71 infection is poorly understood at present. It is likely that viral factors and host immunity, and their interplay, affect the pathogenesis and outcome of EV-A71 infection. The mammalian innate immune system forms the first layer of defense against viral infections and triggers activation of adaptive immunity leading to full protection. In this review, we discuss recent advances in our understanding of the interaction between EV-A71 and the innate immune system. We discuss the role of pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and inflammasomes, in the detection of EV-A71 infection and induction of antiviral immunity. As a counteraction, EV-A71 viral proteins target multiple innate immune pathways to facilitate viral replication in host cells. These novel insights at the virus-host interphase may support the future development of vaccines and therapeutics against EV-A71 infection.
... The ATPase domain of the protein belongs to the superfamily of SF3 helicases of the AAA+ ATPases and contains Walker A and Walker B motifs and motif C. 7 Besides the ATPase domain, 2C harbors an Nterminal membrane-associated helical domain, a cysteine-rich motif, and putative RNA binding motifs. 2C has been implicated in pleiotropic functions such as uncoating, 8 cellular membrane rearrangement, 9−12 RNA binding, 13−15 RNA replication, 16−21 immune evasion, 22 and encapsidation. 23−26 Although 2C has a central role in the viral life cycle, the exact details of its involvement remain poorly understood. ...
Enteroviruses (family Picornaviridae) comprise a large group of human pathogens against which no licensed antiviral therapy exists. Drug-repurposing screens uncovered the FDA-approved drug fluoxetine as replication inhibitor of enterovirus B and D species. Fluoxetine likely targets the non-structural viral protein 2C, but detailed mode-of-actions studies are missing because structural information of 2C of fluoxetine-sensitive enteroviruses is lacking. We here show that broad-spectrum anti-enteroviral activity of fluoxetine is stereospecific concomitant with binding to recombinant 2C. (S)-fluoxetine inhibits with 5-fold lower EC50 than racemic fluoxetine. Using a homology model of 2C of the fluoxetine-sensitive enterovirus coxsackievirus B3 (CVB3) based upon a recently elucidated structure of a fluoxetine-insensitive enterovirus, we predicted stable binding of (S)-fluoxetine. Structure-guided mutations disrupted binding and rendered CVB3 resistant to fluoxetine. The study provides new insights into the anti-enteroviral mode-of-action of fluoxetine. Importantly, using only (S)-fluoxetine would allow for lower dosing in patients, thereby likely reducing side effects.
... Coatomer is required for EV-A71 replication and associates with 2C ( Wang et al., 2012). 2C binds IKKβ and protein phosphatase 1 to suppress IKKβ phosphorylation (Zheng et al., 2011;Li et al., 2016). By interacting with RelA, 2C inhibited the NF-kB pathway ( Du et al., 2015). ...
... We have previously performed yeast 2 hybrid unbiased screenings to identify 2C binding partners, and several important regulators including p65 have been identified ( Du et al., 2015). Others have identified more 2C-associated host proteins including reticulon3, coatomer, IKKβ and protein phosphatase 1 ( Tang et al., 2007;Zheng et al., 2011;Wang et al., 2012;Li et al., 2016). Three 2C-associated host proteins (hnRNPK, COPB2, and PKM) were on the list of our MS SPEC results, indicating the validity of our screen. ...
Viral protein 2C plays a critical role in EV-A71 replication. The discovery of 2C binding proteins will likely provide potential targets to treat EV-A71 infection. Here, we provide a global proteomic analysis of the human proteins that interact with the EV-A71 2C protein. TRIM4, exportin2, and ARFGAP1 were validated as 2C binding partners. Further functional studies revealed that TRIM4, exportin2, and ARFGAP1 were novel host dependency factors for EV-A71. Moreover, enteroviruses’ 2C family proteins interacted with exportin2 and ARFGAP1. In conclusion, our study provides a cellular interactome of the EV-A71 2C and identifies the proviral roles of TRIM4, exportin2, and ARFGAP1 in EV-A71 infection.
... NF-κB is a ubiquitous transcription factor that modulates not only cell death but also innate immunity and inflammatory responses. This pathway seems to play critical roles in the picornaviral life cycle (8,11,14,16,18,19,22,27,66). Activation of the NF-κB signaling pathway by viruses is mediated by pattern recognition receptors (PRRs) which detect pathogen associated molecular patterns (PAMPs; including double-stranded RNA [dsRNA] and/or viral proteins) and initiate the signaling cascade that leads to host gene transcription (67,68). ...
Senecavirus A (SVA), an oncolytic picornavirus used for cancer treatment in humans, has recently emerged as a vesicular disease (VD)-causing agent in swine worldwide. Notably, SVA-induced VD is indistinguishable from foot-and-mouth disease (FMD) and other high-consequence VDs of pigs. Here we investigated the role of apoptosis on infection and replication of SVA. Given the critical role of the nuclear factor-kappa B (NF-κB) signaling pathway on modulation of cell death, we first assessed activation of NF-κB during SVA infection. Results here show that while early during infection SVA induces activation of NF-κB, as evidenced by nuclear translocation of NF-κB-p65 and NF-κB-mediated transcription, late in infection a cleaved product corresponding to the C-terminus of NF-κB-p65 is detected in infected cells, resulting in lower NF-κB transcriptional activity. Additionally, we assessed the potential role of SVA 3C protease (3Cpro) in SVA-induced host-cell apoptosis and cleavage of NF-κB-p65. Transient expression of SVA 3Cpro was associated with cleavage of NF-κB-p65 and Poly (ADP-ribose) polymerase (PARP), suggesting its involvement in virus-induced apoptosis. Most importantly, we showed that while cleavage of NF-κB-p65 is secondary to caspase activation, the proteolytic activity of SVA 3Cpro is essential for induction of apoptosis. Experiments using the pan-caspase inhibitor Z-VAD-FMK confirmed the relevance of late apoptosis for SVA infection, indicating that SVA induces apoptosis, presumably, as a mechanism to facilitate virus release and/or spread from infected cells. Together, these results suggest an important role of apoptosis for SVA infection biology.
... The N-terminal of 2C (amino acids 1-125) interacts with all isoforms of the protein phosphatase 1 (PP1) catalytic subunit through PP1-docking motifs, which is efficient for EV71 2C-mediated inhibition of IKKb phosphorylation and NF-jB activation (Fig. 4). Moreover, 2C forms a complex with PP1 and IKKb to dephosphorylate IKKb activation Zheng et al. 2011). Coat protein complex I (COPI) may be directed to the viral replication complex through viral 2C protein to enhance EV71 infection, whereas the inhibition of COPI activity can weaken the replication of EV71 (Fig. 4) (Wang et al. 2012a). ...
Enterovirus 71 (EV71) is one of the main pathogens that causes hand-foot-and-mouth disease (HFMD). HFMD caused by EV71 infection is mostly self-limited; however, some infections can cause severe neurological diseases, such as aseptic meningitis, brain stem encephalitis, and even death. There are still no effective clinical drugs used for the prevention and treatment of HFMD. Studying EV71 protein function is essential for elucidating the EV71 replication process and developing anti-EV71 drugs and vaccines. In this review, we summarized the recent progress in the studies of EV71 non-coding regions (5′ UTR and 3′ UTR) and all structural and nonstructural proteins, especially the key motifs involving in viral infection, replication, and immune regulation. This review will promote our understanding of EV71 virus replication and pathogenesis, and will facilitate the development of novel drugs or vaccines to treat EV71.
... PP1 binding is crucial for EV71 2C-mediated inhibition of IKKβ phosphorylation. EV71 2C-mediated PP1 recruitment inhibits IKKβ phosphorylation, NF-κB activation and NF-κB signaling pathway-induced IFN production (Zheng et al., 2011;Li Q. et al., 2016). Other enteroviruses, such as PV, coxsackie A virus 16 (CVA16), and coxsackie B virus 3 (CVB3) also exploit this mechanism to inhibit the production of IFN ( Li Q. et al., 2016). ...
Viral infections trigger the innate immune system to produce interferons (IFNs), which play important role in host antiviral responses. Co-evolution of viruses with their hosts has favored development of various strategies to evade the effects of IFNs, enabling viruses to survive inside host cells. One such strategy involves inhibition of IFN signaling pathways by non-structural proteins. In this review, we provide a brief overview of host signaling pathways inducing IFN production and their suppression by picornavirus non-structural proteins. Using this strategy, picornaviruses can evade the host immune response and replicate inside host cells.
... In addition, EV71-induced generation of mitochondrial reactive oxygen species also participated in facilitating efficient replication of the virus [27]. ROS can induce or mediate the activation of the NF-κB pathway and the activation of NF-κB pathway was involved in EV71 replication [31,32]. NF-κB activation is usually emerging as the protective response in the host against the viral pathogens. ...
Enterovirus 71 (EV71) infection of young children can cause neurological manifestations, which is mainly responsible for the fatality. Although a vaccine is recently available for preventing enterovirus 71 infection, its efficacy remains to be seen. Therefore, there is a pressing need for anti-viral agents for the treatment of EV71 infection. By screening a natural compound library for inhibitory activity of EV71 replication, we identified a small molecule, harmine, that inhibited EV71 replication by targeting NF-κB signaling pathway. Harmine is a β-carboline alkaloid found in the medicinal plant Peganum harmala, which is used as a folk antitumor medicine in China and other parts of the Asia. The estimated EC50 value for harmine to block EV71 infection was 20 μM, while the CC50 was estimated at 500 μM in vitro. Harmine inhibited replication of EV71, as evidenced by its ability to diminish plague formation induced by EV71 and to reduce the level of viral RNA and protein. Mechanistic studies indicated that harmine suppressed EV71 replication through inhibition of NF-κB signaling pathway. Harmine treatment also reduced EV71-induced reactive oxygen species (ROS) formation, which was associated with a decline in EV71-associated NF-κB activation. In addition, the harmine treatment could protect AG129 mice against EV71 replication in vivo. These findings suggest that harmine may present as a candidate antiviral drug for the treatment of EV71 infection.
... Bioinformatics and mutagenesis studies predict that 2C harbors an N-terminal membrane-binding motif, an adenosine triphosphatase (ATPase) domain, a cysteine-rich motif, and RNA binding sites (7). The ATPase domain, which belongs to SF3 helicases of the AAA+ ATPase superfamily, contains Walker motifs and motif C (8). 2C has been shown to participate in many crucial events throughout the virus life cycle, such as uncoating (9), cellular membrane rearrangement (10), RNA replication, immune evasion (11), and encapsidation (12,13); however, the details of 2C involvement in these processes remain to be understood. Functional indispensability of 2C makes it an attractive target for anti-enterovirus drug development. ...
Enterovirus 71 (EV71) is the major pathogen responsible for outbreaks of hand, foot, and mouth disease. EV71 nonstructural protein 2C participates in many critical events throughout the virus life cycle; however, its precise role is not fully understood. Lack of a high-resolution structure made it difficult to elucidate 2C activity and prevented inhibitor development. We report the 2.5 Å–resolution crystal structure of the soluble part of EV71 2C, containing an adenosine triphosphatase (ATPase) domain, a cysteine-rich zinc finger with an unusual fold, and a carboxyl-terminal helical domain. Unlike other AAA+ ATPases, EV71 2C undergoes a carboxyl terminus–mediated self-oligomerization, which is dependent on a specific interaction between the carboxyl-terminal helix of one monomer and a deep pocket formed between the ATPase and the zinc finger domains of the neighboring monomer. The carboxyl terminus–mediated self-oligomerization is fundamental to 2C ATPase activity and EV71 replication. Our findings suggest a strategy for inhibition of enterovirus replication by disruption of the self-oligomerization interface of 2C.
