Enterovirus 71 Represses Interleukin Enhancer-Binding Factor 2 Production and Nucleus Translocation to Antagonize ILF2 Antiviral Effects

Enterovirus 71 (EV71) infection causes hand-foot-mouth disease (HFMD), meningoencephalitis, neonatal sepsis, and even fatal encephalitis in children, thereby presenting a serious risk to public health. It is important to determine the mechanisms underlying the regulation of EV71 infection. In this study, we initially show that the interleukin enhancer-binding factor 2 (ILF2) reduces EV71 50% tissue culture infective dose (TCID50) and attenuates EV71 plaque-formation unit (PFU), thereby repressing EV71 infection. Microarray data analyses show that ILF2 mRNA is reduced upon EV71 infection. Cellular studies indicate that EV71 infection represses ILF2 mRNA expression and protein production in human leukemic monocytes (THP-1) -differentiated macrophages and human rhabdomyosarcoma (RD) cells. In addition, EV71 nonstructural protein 2B interacts with ILF2 in human embryonic kidney (HEK293T) cells. Interestingly, in the presence of EV71 2B, ILF2 is translocated from the nucleus to the cytoplasm, and it colocalizes with 2B in the cytoplasm. Therefore, we present a distinct mechanism by which EV71 antagonizes ILF2-mediated antiviral effects by inhibiting ILF2 expression and promoting ILF2 translocation from the nucleus to the cytoplasm through its 2B protein.

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Enterovirus 71 Represses Interleukin Enhancer Binding Factor 2 Production and Nucleus Translocation to Antagonize ILF2 Antiviral Effects

10.20944/preprints201910.0161.v1 ◽

2019 ◽

Author(s):

Jing Jin ◽

Wenbiao Wang ◽

Sha Ai ◽

Qi Zhang ◽

Kailang Wu ◽

...

Keyword(s):

Health Hazard ◽

Enterovirus 71 ◽

Ev71 Infection ◽

Structural Protein ◽

Antiviral Effects ◽

Distinct Mechanism ◽

Binding Factor ◽

2B Protein ◽

Rd Cells ◽

Hand Foot Mouth Disease

Enterovirus 71 (EV71) infection hand-foot-mouth disease (HFMD), meningoencephalitis, neonatal sepsis, and even fatal encephalitis in children, thereby representing a serious public health hazard. It is important to determine the mechanisms underlying the regulation of EV71 infection. In this study, we initially reveal that the interleukin enhancer binding factor 2 (ILF2) down-regulates EV71 50% tissue culture infective dose (TCID50), attenuates EV71 plaque formation unit (PFU), thereby repressing EV71 infection. Moreover, we reveal a distinct mechanism by which EV71 antagonizes ILF2-mediated antiviral effects. Chip data analyses show that ILF2 mRNA is reduced upon EV71 infection. Cellular studies indicate that EV71 infection represses ILF2 mRNA expression and protein production in human leukemic monocytes (THP-1) differentiated macrophages and in human rhabdomyosarcoma (RD) cells. Additionally, EV71 non-structural protein 2B interacts with ILF2 in human embryonic kidney (HEK293T) cells. Interestingly, in the presence of EV71 2B, ILF2 is translocated from the nucleus to the cytoplasm and co-localizes with 2B in the cytoplasm. Therefore, we reveal a distinct mechanism by which EV71 antagonizes ILF2-mediated antiviral effects by inhibiting ILF2 expression and promoting ILF2 translocation from the nucleus to cytoplasm through its 2B protein.

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Characterization of Critical Functions of Long Non-Coding RNAs and mRNAs in Rhabdomyosarcoma Cells and Mouse Skeletal Muscle Infected by Enterovirus 71 Using RNA-Seq

Viruses ◽

10.3390/v10100556 ◽

2018 ◽

Vol 10 (10) ◽

pp. 556 ◽

Cited By ~ 8

Author(s):

Ying Li ◽

Chao Zhang ◽

Luwei Qin ◽

Dong Li ◽

Guangyuan Zhou ◽

...

Keyword(s):

Skeletal Muscle ◽

Gene Prediction ◽

Expression Profiles ◽

Enterovirus 71 ◽

Differentially Expressed ◽

Ev71 Infection ◽

Rd Cells ◽

Hand Foot Mouth Disease ◽

Second Generation Sequencing ◽

Non Coding Rnas

Enterovirus 71 (EV71) is the main pathogen of severe hand-foot-mouth disease (HFMD). Long non-coding RNAs (lncRNAs) are recognized as pivotal factors during the pathogenesis of viral infection. However, the critical functions of lncRNAs in EV71–host interactions have not been characterized. Here, for the first time, we performed global transcriptome analysis of lncRNA and mRNA expression profiles in EV71-infected human rhabdomyosarcoma (RD) cells and skeletal muscle of mice using second-generation sequencing. In our study, a total of 3801 novel lncRNAs were identified. In addition, 23 lncRNAs and 372 mRNAs exhibited remarkable differences in expression levels between infected and uninfected RD cells, while 104 lncRNAs and 2647 mRNAs were differentially expressed in infected skeletal muscle from neonatal mice. Comprehensive bioinformatics analysis included target gene prediction, lncRNA‑mRNA co-expression network construction, as well as gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis mainly focused on differentially-expressed genes (DEGs). Our results suggest that lncRNAs may participate in EV71 infection-induced pathogenesis through regulating immune responses, protein binding, cellular component biogenesis and metabolism. The present study provides novel insights into the functions of lncRNAs and the possible pathogenic mechanism following EV71 infection.

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Plant-Produced Anti-Enterovirus 71 (EV71) Monoclonal Antibody Efficiently Protects Mice Against EV71 Infection

Plants ◽

10.3390/plants8120560 ◽

2019 ◽

Vol 8 (12) ◽

pp. 560 ◽

Cited By ~ 5

Author(s):

Kaewta Rattanapisit ◽

Zhang Chao ◽

Konlavat Siriwattananon ◽

Zhong Huang ◽

Waranyoo Phoolcharoen

Keyword(s):

Monoclonal Antibody ◽

Therapeutic Option ◽

Enterovirus 71 ◽

Cost Effective ◽

Ev71 Infection ◽

Asia Pacific ◽

Variable Regions ◽

Hand Foot Mouth Disease ◽

Protection Against Infection

Enterovirus 71 (EV71) is the main causative agent of severe hand-foot-mouth disease. EV71 affects countries mainly in the Asia-Pacific region, which makes it unattractive for pharmaceutical companies to develop drugs or vaccine to combat EV71 infection. However, development of these drugs and vaccines is vital to protect younger generations. This study aims to develop a specific monoclonal antibody (mAb) to EV71 using a plant platform, which is a cost-effective and scalable production technology. A previous report showed that D5, a murine anti-EV71 mAb, binds to VP1 protein of EV71, potently neutralizes EV71 in vitro, and effectively protects mice against EV71 infection. Herein, plant-produced chimeric D5 (cD5) mAb, variable regions of murine D5 antibody linked with constant regions of human IgG1, was transiently expressed in Nicotiana benthamiana using geminiviral vectors. The antibody was expressed at high levels within six days of infiltration. Plant-produced cD5 retained its in vitro high-affinity binding and neutralizing activity against EV71. Furthermore, a single dose (10 µg/g body weight) of plant-produced cD5 mAb offered 100% protection against infection in mice after a lethal EV71 challenge. Therefore, our results showed that plant-produced anti-EV71 mAb is an effective, safe, and affordable therapeutic option against EV71 infection.

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Enterovirus 71 2B Induces Cell Apoptosis by Directly Inducing the Conformational Activation of the Proapoptotic Protein Bax

Journal of Virology ◽

10.1128/jvi.01499-16 ◽

2016 ◽

Vol 90 (21) ◽

pp. 9862-9877 ◽

Cited By ~ 22

Author(s):

Haolong Cong ◽

Ning Du ◽

Yang Yang ◽

Lei Song ◽

Wenliang Zhang ◽

...

Keyword(s):

Cell Apoptosis ◽

Enterovirus 71 ◽

Terminal Region ◽

Ev71 Infection ◽

Apoptotic Pathway ◽

Hydrophobic Region ◽

Mitochondrial Apoptotic Pathway ◽

Proapoptotic Protein ◽

2B Protein ◽

Induced Apoptosis

ABSTRACTTo survive and replicate within a host, many viruses have evolved strategies that target crucial components within the apoptotic cascade, leading to either inhibition or induction of cell apoptosis. Enterovirus 71 (EV71) infections have been demonstrated to impact the mitochondrial apoptotic pathway and induce apoptosis in many cell lines. However, the detailed mechanism of EV71-induced apoptosis remains to be elucidated. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. 2B recruited Bax to the mitochondria and induced Bax conformational activation. In addition, mitochondria isolated from 2B-expressing cells that were treated with a recombinant Bax showed increased Bax interaction and cytochromec(Cytc) release. Importantly, apoptosis in cells with either EV71 infection or 2B expression was dramatically reduced in Bax knockdown cells but not in Bak knockdown cells, suggesting that Bax played a pivotal role in EV71- or 2B-induced apoptosis. Further studies indicate that a hydrophobic region of 18 amino acids (aa) in the C-terminal region of 2B (aa 63 to 80) was responsible for the location of 2B in the mitochondria. A hydrophilic region of 14 aa in the N-terminal region of 2B was functional in Bax interaction and its subsequent activation. Moreover, overexpression of the antiapoptotic protein Bcl-XLabrogates 2B-induced release of Cytcand caspase activation. Therefore, this study provides direct evidence that EV71 2B induces cell apoptosis and impacts the mitochondrial apoptotic pathway by directly modulating the redistribution and activation of proapoptotic protein Bax.IMPORTANCEEV71 infections are usually accompanied by severe neurological complications. It has also been postulated that the induction of cell apoptosis resulting from tissue damage is a possible process of EV71-related pathogenesis. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. This study provides evidence that EV71 induces cell apoptosis by modulating Bax activation and reveals important clues regarding the mechanism of Cytcrelease and mitochondrial permeabilization during EV71 infection.

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3C protease of enterovirus 71 cleaves promyelocytic leukemia protein and impairs PML-NBs production

Virology Journal ◽

10.1186/s12985-021-01725-7 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Zhuoran Li ◽

Ya’ni Wu ◽

Hui Li ◽

Wenqian Li ◽

Juan Tan ◽

...

Keyword(s):

Enterovirus 71 ◽

Promyelocytic Leukemia ◽

Nuclear Bodies ◽

Ev71 Infection ◽

3C Protease ◽

Pml Nuclear Bodies ◽

Antiviral Function ◽

Hand Foot Mouth Disease ◽

Antiviral Activities ◽

Novel Target

Abstract Background Enterovirus 71 (EV71) usually infects infants causing hand-foot-mouth disease (HFMD), even fatal neurological disease like aseptic meningitis. Effective drug for preventing and treating EV71 infection is unavailable currently. EV71 3C mediated the cleavage of many proteins and played an important role in viral inhibiting host innate immunity. Promyelocytic leukemia (PML) protein, the primary organizer of PML nuclear bodies (PML-NBs), can be induced by interferon and is involved in antiviral activity. PML inhibits EV71 replication, and EV71 infection reduces PML expression, but the molecular mechanism is unclear. Methods The cleavage of PMLIII and IV was confirmed by co-transfection of EV71 3C protease and PML. The detailed cleavage sites were evaluated further by constructing the Q to A mutant of PML. PML knockout cells were infected with EV71 to identify the effect of cleavage on EV71 replication. Immunofluorescence analysis to examine the interference of EV71 3C on the formation of PML-NBs. Results EV71 3C directly cleaved PMLIII and IV. Furthermore, 3C cleaved PMLIV at the sites of Q430–A431 and Q444–S445 through its protease activity. Overexpression of PMLIV Q430A/Q444A variant exhibited stronger antiviral potential than the wild type. PMLIV Q430A/Q444A formed normal nuclear bodies that were not affected by 3C, suggesting that 3C may impair PML-NBs production via PMLIV cleavage and counter its antiviral activities. PML, especially PMLIV, which sequesters viral proteins in PML-NBs and inhibits viral production, is a novel target of EV71 3C cleavage. Conclusions EV71 3C cleaves PMLIV at Q430–A431 and Q444–S445. Cleavage reduces the antiviral function of PML and decomposes the formation of PML-NBs, which is conducive to virus replication.

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Beclin1 Binds to Enterovirus 71 3D Protein to Promote the Virus Replication

Viruses ◽

10.3390/v12070756 ◽

2020 ◽

Vol 12 (7) ◽

pp. 756 ◽

Cited By ~ 1

Author(s):

Qi Xiang ◽

Pin Wan ◽

Ge Yang ◽

Siyu Huang ◽

Mengying Qin ◽

...

Keyword(s):

Prevention And Control ◽

Core Protein ◽

Neurological Diseases ◽

Mrna Level ◽

Enterovirus 71 ◽

Structural Protein ◽

Innate Immune Signaling ◽

Rd Cells ◽

Hand Foot Mouth Disease ◽

And Control

Enterovirus 71 (EV71) is the main pathogen causing hand-foot-mouth disease (HFMD) in infants and children, which can also lead to severe neurological diseases and even death. Therefore, understanding the replication mechanism of EV71 is of great significance for the prevention and control of EV71-induced diseases. Beclin1 (BECN1, a mammalian homologue of ATG6 in yeast) is an important core protein for the initiation and the normal process of autophagy in cells. In addition to its involvement in autophagy, Beclin1 has also been reported to play an important role in cancer and innate immune signaling pathways. However, the role of Beclin1 in EV71 replication remains elusive. Here, we primarily found that Beclin1 facilitates EV71 replication in human rhabdomyosarcoma (RD) cells and the autophagy was actually induced, but Beclin1 was not significantly affected at either mRNA level or protein level during early EV71 infection. Further studies discovered that Beclin1 could interacts with EV71 non-structural protein 3D mainly through its evolutionary conserved domain (ECD) and coiled-coiled domain (CCD), thus promoting the replication of EV71 in human rhabdomyosarcoma (RD) cells and human astroglioma (U251) cells. Collectively, we reveal a novel regulatory mechanism associated with Beclin1 to promote EV71 replication, thus providing a potential therapeutic target for the prevention and control of EV71-associated diseases.

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Pathogenesis Study of Enterovirus 71 Using a Novel Human SCARB2 Knock-In Mouse Model

mSphere ◽

10.1128/msphere.01048-20 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Yuefei Jin ◽

Tiantian Sun ◽

Guangyuan Zhou ◽

Dong Li ◽

Shuaiyin Chen ◽

...

Keyword(s):

Animal Models ◽

Mouse Model ◽

Scavenger Receptor ◽

Enterovirus 71 ◽

Antiviral Drugs ◽

Ev71 Infection ◽

Cellular Receptor ◽

Scavenger Receptor Class ◽

Class B ◽

Hand Foot Mouth Disease

ABSTRACT Enterovirus 71 (EV71) can cause a severe hand-foot-mouth disease in children. However, the precise mechanism of EV71-associated disease, particularly the neuropathogenesis and pulmonary disorder, is still not fully understood because no suitable animal models are available. The human scavenger receptor class B, member 2 (hSCARB2), is a cellular receptor for EV71. Here, we generated a novel knock-in (KI) mouse model using the CRISPR/Cas9 system to insert the hSCARB2 gene into the mouse Rosa26 locus to study the pathogenesis of EV71. The hSCARB2 KI mice infected with clinical isolates of EV71 showed neurological symptoms, such as ataxia, paralysis, and death. Viral replication was detected in mainly astrocytes and a limited number of neurons and microglia, accompanied by gliosis. Vascular leakage and alveoli filled with erythrocytes were detected, suggesting that edema and hemorrhage, which are observed in human patients, also occurred in EV71-infected KI mice. In addition, proinflammatory cytokines and chemokines were significantly increased in the serum of infected KI mice. These pathological features of the KI mice after infection resembled those of EV71 encephalomyelitis in humans. Therefore, our KI mouse model is suitable to study the pathogenesis of EV71 and is of great significance for development of antiviral drugs and vaccines to treat or prevent EV71 infection. IMPORTANCE Enterovirus 71 (EV71) is associated with severe hand-foot-mouth disease. Recently, outbreaks of EV71 infection with high mortality have been reported in the Asia-Pacific region, posing a great challenge for global public health. To date, the precise mechanism of EV71-induced disease, particularly the neuropathogenesis and respiratory disorders, is still not fully understood because no suitable animal models are available. Human scavenger receptor class B, member 2 (hSCARB2), has been identified as a cellular receptor for EV71. Here, we introduce a novel CRISPR/Cas9-mediated hSCARB2 knock-in (KI) mouse model for the study of EV71 pathogenesis, which is of great significance for the development of antiviral drugs and vaccines.

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Inhibition of EV71 replication by L3HYPDH, a newly identified interferon-stimulated gene product

10.1101/304345 ◽

2018 ◽

Author(s):

Jian Liu ◽

Luogen Liu ◽

Shinuan Zeng ◽

Xiaobin Meng ◽

Nanfeng Lei ◽

...

Keyword(s):

Enterovirus 71 ◽

Protein Translation ◽

Amino Acid Position ◽

Terminal Region ◽

Entry Site ◽

Interferon Stimulated Genes ◽

Antiviral Effects ◽

Hand Foot Mouth Disease ◽

Underlying Mechanisms ◽

Hcv Ires

ABSTRACTUp-regulation of interferon-stimulated genes (ISGs) is key to antiviral states mediated by interferon (IFN) but little is known about activity and underlying mechanisms of most ISGs against Enterovirus 71 (EV71). EV71 causes hand-foot-mouth disease in infants and occasionally severe neurological symptoms. Here we report that the product ofL3HYPDH, a newly identified ISG, inhibits the replication of EV71. This anti-EV71 activity was mapped to the C-terminal 60 amino acids region as well as the N-terminal region spanning from amino acid position 61 to 120 of L3HYPDH protein. L3HYPDH was shown to interfere with EV71 propagation at the RNA replication and protein translation levels. Specifically, L3HYPDH impairs translation mediated by the EV71 international ribosome entry site (IRES) but not by the HCV IRES, and this activity is conferred by the C-terminal region of L3HYPDH. Thus, L3HYPDH has antiviral activity against EV71, suggesting a potential mechanism for broad-spectrum antiviral effects of IFN.

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VGLL4 Promotes EV71 Virus Proliferation and Accelerates the Apoptosis of Infected RD Cells

10.21203/rs.3.rs-272535/v1 ◽

2021 ◽

Author(s):

Shengyu Wang ◽

Xin Sun ◽

Junhua Qiao ◽

Cancan He ◽

Langfei Tian ◽

...

Keyword(s):

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Eukaryotic Expression ◽

Host Cells ◽

Ev71 Infection ◽

Western Blot Assay ◽

Multifunctional Protein ◽

Rd Cells ◽

Infected Cells ◽

Induced Apoptosis

Abstract Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot and mouth disease (HFMD) which affects public health increasingly. Apoptosis plays an important role in EV71 infection, but the molecular mechanism involved in EV71 induced apoptosis is not completely clear. VGLL4 is a multifunctional protein in host cells, which has been studied in tumor and cell apoptosis, but has not been reported in pathogen. In this study, the mammalian eukaryotic expression plasmid of VGLL4 fused with HA tag (HA-VGLL4) and the model of overexpression VGLL4 RD cells were successfully constructed. The effect of VGLL4 on the proliferation of EV71 was detected by western-blot assay, fluorescence quantitative PCR and cytotoxicity assay (CCK assay), and the mechanism of its effect on the proliferation of the virus was researched. The experimental results showed that VGLL4 may promote the replication of EV71 by promoting the apoptosis of infected cells. VGLL4 can be an important target for prevention and treatment of EV71 infection.

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Enterovirus 71 Induces INF2 Cleavage via Activated Caspase-2 in Infected RD Cells

Frontiers in Microbiology ◽

10.3389/fmicb.2021.684953 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bei Wang ◽

Chongyang Zhang ◽

Congci Yu ◽

Yue Zhu ◽

Qing Tang ◽

...

Keyword(s):

Regulatory Mechanism ◽

Mitochondrial Fission ◽

Enterovirus 71 ◽

Foot And Mouth Disease ◽

Ev71 Infection ◽

Causative Pathogen ◽

Rd Cells ◽

Infected Cells ◽

Caspase 2 ◽

Feedback Regulatory Mechanism

Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease. The lack of understanding of the virus’s pathogenesis hinders the development of anti-virus drugs and the control of EV71 infection. Our previous studies have demonstrated that both mitochondria and endoplasmic reticulum (ER) were altered significantly in EV71 infected cells, but the mechanism is still unclear. In this study, we investigated the effects of EV71 infection on the expression of INF2, a key regulator factor in ER-Mitochondria communication and mitochondrial fission. We found that INF2 was cleaved in EV71 infected RD cells. The INF2 cleavage occurred at Aspartic 1,051 of INF2 and is mediated by activated caspases, predominantly by activated caspase-2. The subcellular localization of INF2 and caspase-2 was significantly altered in infected cells. We speculate that caspase-2-mediated INF2 cleavage is involved in forming viral replication organelles (ROs) and is a positive feedback regulatory mechanism of mitochondrial disorders caused by EV71 infection.

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