EV71-infected Cells Research Articles

Anti-inflammatory and antiviral effects of minocycline in enterovirus 71 infections

Enterovirus 71 (EV71) brainstem encephalitis (BE) is divided into—uncomplicated BE, autonomic nervous system (ANS) dysregulation, and pulmonary edema (PE)—based on cytokine-mediated severe systemic and central nervous system (CNS) inflammatory responses. Minocycline has been found to have anti-inflammatory and immunomodulatory properties in infectious and inflammatory neurological disease models. The effects of minocycline on EV71 infection were studied in vitro and in vivo experiments. The minocycline treatment (100–300 μg/mL) on cytokine expressions and viral replications were investigated in rhabdomyosarcoma (RD), U-87MG, and THP-1 cells. The mouse-adapted-EV71 strain (MP4)-infected 7-day-old ICR mice model was used to explore the anti-inflammatory and antiviral effects of minocycline (1 and 5 μg/g) for the treatment of EV71 infection. In in vitro, minocycline reduced cytopathic effects (CPEs), viral protein expressions, viral titers, the levels of interleukin (IL)-6 and IL-8 and relative mRNA expressions of IL-12p40, IL-1β, and tumor necrosis factor (TNF) after EV71 infection. The levels of TNF, IL-1β, IL-6, and IL-8 decreased with a single dose of minocycline in EV71-infected THP-1 cells. Double-dose minocycline treatment demonstrated more effective reduction in cytokines. In the MP4-infected animal model, clinical scores, mortality rates and viral titers in various brain tissues were decreased evidently after double-dose minocycline treatment. Minocycline inhibited IL-6 and granulocyte colony-stimulating factor (G-CSF) in plasma and TNF in the cerebellum. Minocycline has properties that enable it to function both as an anti-inflammatory and antiviral agent in EV71 infection. These results evidence its potential usefulness in clinical treatment.

Chemical components from the twigs of Caesalpinia latisiliqua and their antiviral activity.

Three new compounds, (3S)-dihydrobonducellin 8-O-β-D-glucopyranoside (1), 3',5'-dimethoxy-jezonolid (2), and latisilinoid (3), along with 16 known compounds, were isolated from the twigs of Caesalpinia latisiliqua (Leguminosae). The known compounds were identified as flavonoids, stilbenes, and phenolics as determined by extensive spectroscopic methods, including 1D and 2D NMR. All the isolated compounds were evaluated for their antiviral activity in HRV1B-, CVB3-, and EV71-infected cells. Among the tested compounds, three flavonoids (4-6) and two stilbenes (12 and 14) exhibited significant antiviral activity. This is the first phytochemical investigation of C. latisiliqua twigs.

Human Vγ9Vδ2-T cells exhibit potent antiviral activity against EV71 infection

Abstract Enterovirus 71 (EV71) is a major pathogen of hand, foot and mouth disease (HFMD). It is highly contagious and usually causes mild disease. However, occasionlly HFMD caused by EV71 would develpoed into severe neurological complications or even deaths in young children. There is no effective anti-viral therapy against EV71 infection. Human Vγ9Vδ2-T cells, as the first line of human immune defense, exhibit antiviral activities against different viruses. However, whether human Vγ9Vδ2-T cells have antiviral activity against EV71 remains unkown. In this study, we used EV71-infected primary human monocyte-derived macrophages (MDMs) to examine the anti-EV71 activity of Vγ9Vδ2-T cells. We found that phosphoantigen pamidronate-expanded Vγ9Vδ2-T cells could kill EV71-infected MDMs, and thus reduce the viral load. The cytotoxicity of Vγ9Vδ2-T cells against EV71-infected MDMs required cell-cell contact and mediated by Fas/FasL pathway. Vγ9Vδ2-T cells also displayed potent capacity to produce IFN-γ and thus inhibit EV71 viral replication. CCR5 pathway was involved in the migration of Vγ9Vδ2 T cells towards EV71-infected cells. These results demonstrated that Vγ9Vδ2-T cells have both cytotoxic and non-cytolytic antiviral activities target EV71. Our data suggests a novel therapeutic approach against EV71 by boosting Vγ9Vδ2 T cell immunity. This work is supported in part by Health and Medical Research Fund, Hong Kong SAR government (13120972) and the General Research Fund (17115015 and 17126317), Research Grants Council of the Hong Kong SAR, China.

Characteristics of enterovirus 71-induced cell death and genome scanning to identify viral genes involved in virus-induced cell apoptosis

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease and severe neural complications in infants and young children. Viral pathogenesis is associated with virus-induced cell death and inflammatory cytokine production, which is usually correlated with the type of programmed cell death. EV71-infected cells were analyzed through microscopy, cell staining, and immunoblotting to determine the characteristics of EV71-induced cell death. Results demonstrated that EV71 infection induced cell shrinkage, nuclear condensation, decreased mitochondrial potential, and membrane phosphatidylserine translocation. Caspase-9 activation, poly (ADP-ribose) polymerase cleavage, and lactate dehydrogenase release were also observed during virus-induced cell death. The activated gasdermin D (GSDMD) and the phosphorylated mixed lineage kinase domain-like protein (p-MLKL) were not detected. These observations indicated that EV71-induced cell death was mainly executed by apoptosis through the intrinsic pathway rather than by GSDMD-mediated pyroptosis and p-MLKL-mediated necroptosis. Genome scanning analysis identified that EV71 2A, 2B, and 3C might be the determinant genes of virus-induced cell death. Further experiments showed that EV71 2A- and 3C-induced cell death exhibited dependence on their protease activities but involved different mechanisms. EV71 2A-induced cell death was correlated with the shut-off of host cap-dependent translation, whereas EV71 3C-induced cell death might not be ascribed to this mechanism. These findings would enhance our understanding of EV71 infection and viral pathogenesis, and help identify antiviral targets.

Pyroptosis induced by enterovirus 71 and coxsackievirus B3 infection affects viral replication and host response

Enterovirus 71 (EV71) is the primary causative pathogen of hand, foot, and mouth disease (HFMD), affecting children with severe neurological complications. Pyroptosis is a programmed cell death characterized by cell lysis and inflammatory response. Although proinflammatory response has been implicated to play important roles in EV71-caused diseases, the involvement of pyroptosis in the pathogenesis of EV71 is poorly defined. We show that EV71 infection induced caspase-1 activation. Responding to the activation of caspase-1, the expression and secretion of both IL-1β and IL-18 were increased in EV71-infected cells. The treatment of caspase-1 inhibitor markedly improved the systemic response of the EV71-infected mice. Importantly, caspase-1 inhibitor suppressed EV71 replication in mouse brains. Similarly, pyroptosis was activated by the infection of coxsackievirus B3 (CVB3), an important member of the Enterovirus genus. Caspase-1 activation and the increased expression of IL-18 and NLRP3 were demonstrated in HeLa cells infected with CVB3. Caspase-1 inhibitor also alleviated the overall conditions of virus-infected mice with markedly decreased replication of CVB3 and reduced expression of caspase-1. These results indicate that pyroptosis is involved in the pathogenesis of both EV71 and CVB3 infections, and the treatment of caspase-1 inhibitor is beneficial to the host response during enterovirus infection.

Inhibition of EV71 by curcumin in intestinal epithelial cells.

EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6), an active ingredient of turmeric (Curcuma longa Linn) with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES) activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK) signaling pathways is not involved. We found that protein kinase C delta (PKCδ) plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections.

Prohibitin plays a critical role in Enterovirus 71 neuropathogenesis.

A close relative of poliovirus, enterovirus 71 (EV71) is regarded as an important neurotropic virus of serious public health concern. EV71 causes Hand, Foot and Mouth Disease and has been associated with neurological complications in young children. Our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. Here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during EV71 infection of motor-neuron NSC-34 cells, which are found at the neuromuscular junctions where EV71 is believed to enter the central nervous system. Meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in NSC-34 cells. Among the candidate proteins, we focused our attention on prohibitin (PHB), a protein that is involved in multiple cellular functions and the target of anti-cancer drug Rocaglamide (Roc-A). We demonstrated that cell surface-expressed PHB is involved in EV71 entry into neuronal cells specifically, while membrane-bound mitochondrial PHB associates with the virus replication complex and facilitates viral replication. Furthermore, Roc-A treatment of EV71-infected neuronal cells reduced significantly virus yields. However, the inhibitory effect of Roc-A on PHB in NSC-34 cells was not through blocking the CRAF/MEK/ERK pathway as previously reported. Instead, Roc-A treated NSC-34 cells had lower mitochondria-associated PHB and lower ATP levels that correlated with impaired mitochondria integrity. In vivo, EV71-infected mice treated with Roc-A survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. Together, this study uncovers PHB as the first host factor that is specifically involved in EV71 neuropathogenesis and a potential drug target to limit neurological complications.

Anti-enterovirus 71 activities of Melissa officinalis extract and its biologically active constituent rosmarinic acid

Enterovirus 71 (EV71) infection is endemic in the Asia-Pacific region. No specific antiviral drug has been available to treat EV71 infection. Melissa officinalis (MO) is a medicinal plant with long history of usage in the European and Middle East. We investigated whether an aqueous solution of concentrated methanolic extract (MOM) possesses antiviral activity. MOM inhibited plaque formation, cytopathic effect, and viral protein synthesis in EV71-infected cells. Using spectral techniques, we identified rosmarinic acid (RA) as a biologically active constituent of MOM. RA reduced viral attachment and entry; cleavage of eukaryotic translation initiation factor 4 G (eIF4G); reactive oxygen species (ROS) generation; and translocation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) from nucleus to cytoplasm. It alleviated EV71-induced hyperphosphorylation of p38 kinase and EPS15. RA is likely to suppress ROS-mediated p38 kinase activation, and such downstream molecular events as hnRNP A1 translocation and EPS15-regulated membrane trafficking in EV71-infected cells. These findings suggest that MO and its constituent RA possess anti-EV71 activities, and may serve as a candidate drug for therapeutic and prophylactic uses against EV71 infection.

Exosome-mediated miR-146a transfer suppresses type I interferon response and facilitates EV71 infection.

Exosomes can transfer genetic materials between cells. Their roles in viral infections are beginning to be appreciated. Researches have shown that exosomes released from virus-infected cells contain a variety of viral and host cellular factors that are able to modulate recipient’s cellular response and result in productive infection of the recipient host. Here, we showed that EV71 infection resulted in upregulated exosome secretion and differential packaging of the viral genomic RNA and miR-146a into exosomes. We provided evidence showing that miR-146a was preferentially enriched in exosomes while the viral RNA was not in infected cells. Moreover, the exosomes contained replication-competent EV71 RNA in complex with miR-146a, Ago2, and GW182 and could mediate EV71 transmission independent of virus-specific receptor. The exosomal viral RNA could be transferred to and replicate in a new target cell while the exosomal miR-146a suppressed type I interferon response in the target cell, thus facilitating the viral replication. Additionally, we found that the IFN-stimulated gene factors (ISGs), BST-2/tetherin, were involved in regulating EV71-induced upregulation of exosome secretion. Importantly, in vivo study showed that exosomal viral RNA exhibited differential tissue accumulation as compared to the free virus particles. Together, our findings provide evidence that exosomes secreted by EV71-infected cells selectively packaged high level miR-146a that can be functionally transferred to and facilitate exosomal EV71 RNA to replicate in the recipient cells by suppressing type I interferon response.

Hsa-let-7c-5p augments enterovirus 71 replication through viral subversion of cell signaling in rhabdomyosarcoma cells

BackgroundHuman enterovirus 71 (EV71) causes severe hand, foot and mouse disease, accompanied by neurological complications. During the interaction between EV71 and the host, the virus subverts host cell machinery for its own replication. However, the roles of microRNAs (miRNAs) in this process remain obscure.ResultsIn this study, we found that the miRNA hsa-let-7c-5p was significantly upregulated in EV71-infected rhabdomyosarcoma cells. The overexpression of hsa-let-7c-5p promoted replication of the virus, and the hsa-let-7c-5p inhibitor suppressed viral replication. Furthermore, hsa-let-7c-5p targeted mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) and inhibited its expression. Interestingly, downregulation of MAP4K4 expression led to an increase in EV71 replication. In addition, MAP4K4 knockdown or transfection with the hsa-let-7c-5p mimic led to activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway, whereas the hsa-let-7c-5p inhibitor inhibited activation of this pathway. Moreover, EV71 infection promoted JNK pathway activation to facilitate viral replication.ConclusionsOur data suggested that hsa-let-7c-5p facilitated EV71 replication by inhibiting MAP4K4 expression, which might be related to subversion of the JNK pathway by the virus. These results may shed light on a novel mechanism underlying the defense of EV71 against cellular responses. In addition, these findings may facilitate the development of new antiviral strategies for use in future therapies.

Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway.

Enterovirus 71 (EV71) causing Hand, Foot and Mouth Disease, is regarded as the most important neurotropic virus worldwide. EV71 is believed to replicate in muscles and infect motor neurons to reach the central nervous system (CNS). To further investigate the mechanisms involved, we have employed the motor neuron cell line NSC-34. NSC-34 cells were permissive to EV71 and virus production yields were strain-dependent with differential efficacy at the entry, replication and egress steps. Furthermore, unlike all the other cell lines previously reported, EV71-infected NSC-34 cells neither displayed cytopathic effect nor underwent apoptosis. Instead, autophagy was markedly up-regulated and virus-containing autophagic vacuoles were isolated from the culture supernatant, providing the first experimental evidence that EV71 can adopt a non-lytic exit pathway. Finally, the ability of EV71 to infect productively NSC-34 cells correlated with its ability to invade the CNS in vivo, supporting the relevance of NSC-34 cells to study the intrinsic neurovirulence of EV71 strains.

Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication

Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets.

Differential Regulation of TLR Signaling on the Induction of Antiviral Interferons in Human Intestinal Epithelial Cells Infected with Enterovirus 71.

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease, which can lead to fatal neurological complications in young children and infants. Few gastrointestinal symptoms are observed clinically, suggesting the presence of a unique immunity to EV71 in the gut. We reported a robust induction of interferons (IFNs) in human intestinal epithelial cells (HT-29), which was suppressed in other types such as RD and HeLa cells. The underlying mechanism for the apparent difference remains obscure. In this study we report that in EV71-infected HT-29 cells, TLR/TRIF signaling was essential to IFN induction; viral replication increased and the induction of IFN-α, -β, -ω, -κ, and -ε decreased markedly in TRIF-silenced HT-29 cells. Importantly, TRIF was degraded by viral 3Cpro in RD cells, but resisted cleavage, and IRF3 was activated and translocated into the nucleus in HT-29 cells. Taken together, our data suggest that IFNs were induced differentially in human HT-29 cells through an intact TLR/TRIF signaling, which differs from other cell types and may be implicated in viral pathogenesis in EV71 infection.

Enterovirus 71 transmission by exosomes establishes a productive infection in human neuroblastoma cells.

Exosomes are small secreted cellular vesicles for intercellular communications which contain proteins, mRNAs, and microRNAs (miRNAs). Recent studies have shown that exosomes play an important role in the transmission of infectious agents including hepatitis C virus, human immunodeficiency virus, and so on. However, the role of exosomes in the transfer of enterovirus 71 (EV71) between host cells remains unknown. In this study, we show that the exosomes derived from EV71-infected rhabdomyosarcoma cells contain EV71 RNA and capsid protein VP1, determined by quantitative reverse transcription-PCR (QRT-PCR) and Western blot analysis. The shedding of exosomes containing virus can establish a productive infection in human neuroblastoma cell line (SK-N-SH). A comparative analysis of neutralization by EV71-specific immunoglobulins showed different levels of neutralization of exosomes-mediated infection compared with free virus. In conclusion, exosomes from EV71-infected cells may play an important role in virus dissemination and are partially resisted to antibody neutralization. Our results suggest that there is an exosomal route of EV71 transmission infection.

Systematic Identification and Bioinformatic Analysis of MicroRNAs in Response to Infections of Coxsackievirus A16 and Enterovirus 71.

Hand, foot, and mouth disease (HFMD), mainly caused by coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) infections, remains a serious public health issue with thousands of newly diagnostic cases each year since 2008 in China. The mechanisms underlying viral infection, however, are elusive to date. In the present study, we systematically investigated the host cellular microRNA (miRNA) expression patterns in response to CVA16 and EV71 infections. Through microarray examination, 27 miRNAs (15 upregulated and 12 downregulated) were found to be coassociated with the replication process of two viruses, while the expression levels of 15 and 5 miRNAs were significantly changed in CVA16- and EV71-infected cells, respectively. A great number of target genes of 27 common differentially expressed miRNAs were predicted by combined use of two computational target prediction algorithms, TargetScan and MiRanda. Comprehensive bioinformatic analysis of target genes in GO categories and KEGG pathways indicated the involvement of diverse biological functions and signaling pathways during viral infection. These results provide an overview of the roles of miRNAs in virus-host interaction, which will contribute to further understanding of HFMD pathological mechanisms.

Enterovirus 71 induces dsRNA/PKR-dependent cytoplasmic redistribution of GRP78/BiP to promote viral replication

Enterovirus 71 induces dsRNA/PKR-dependent cytoplasmic redistribution of GRP78/BiP to promote viral replicationGRP78/BiP is an endoplasmic reticulum (ER) chaperone protein with the important function of maintaining ER homeostasis, and the overexpression of GRP78/BiP alleviates ER stress. Our previous studies showed that infection with enterovirus 71 (EV71), a (+)RNA picornavirus, induced GRP78/BiP upregulation; however, ectopic GRP78/BiP overexpression in ER downregulates virus replication and viral particle formation. The fact that a virus infection increases GRP78/BiP expression, which is unfavorable for virus replication, is counterintuitive. In this study, we found that the GRP78/BiP protein level was elevated in the cytoplasm instead of in the ER in EV71-infected cells. Cells transfected with polyinosinic–polycytidylic acid, a synthetic analog of replicative double-stranded RNA (dsRNA), but not with viral proteins, also exhibited upregulation and elevation of GRP78/BiP in the cytosol. Our results further demonstrate that EV71 infections induce the dsRNA/protein kinase R-dependent cytosolic accumulation of GRP78/BiP. The overexpression of a GRP78/BiP mutant lacking a KDEL retention signal failed to inhibit both dithiothreitol-induced eIF2α phosphorylation and viral replication in the context of viral protein synthesis and viral titers. These data revealed that EV71 infection might cause upregulation and aberrant redistribution of GRP78/BiP to the cytosol, thereby facilitating virus replication.Emerging Microbes and Infections (2016) 5, e23; doi:10.1038/emi.2016.20; published online 23 March 2016

Global quantitative proteomic analysis of human glioma cells profiled host protein expression in response to enterovirus type 71 infection.

Enterovirus 71 (EV71) is one of the leading causes of hand, foot and mouth disease with neurological complications in some cases. To study the pathogenesis of EV71 infection, large-scale analyses of EV71 infected cells have been performed. However, most of these studies employed rhabdomyosarcoma (RD) cells or used transcriptomic strategy. Here, we performed SILAC-based quantitative proteomic analysis of EV71-infected U251 cells, a human glioma cell line. A total of 3125 host proteins were quantified, in which 451 were differentially regulated as a result of EV71 infection at 8 or 20 hpi or both. Gene Ontology analysis indicates the regulated proteins were enriched in "metabolic process", "biological regulation" and "cellular process", implying that these biological processes were affected by EV71 infection. Furthermore, functional study indicated that TRAF2 and TRAF6 among the up-regulated proteins could inhibit the replication of EV71 at the early phase post infection, and the anti-EV71 function of both proteins was independent of interferon β. Our study not only provided an overview of cellular response to EV71 infection in a human glioma cell line, but also found that TRAF2 and TRAF6 might be potential targets to inhibit the replication of EV71. All MS data have been deposited in the ProteomeXchange with identifier PXD002454 (http://proteomecentral.proteomexchange.org/dataset/PXD002454).

Norepinephrine and Epinephrine Enhanced the Infectivity of Enterovirus 71.

BackgroundEnterovirus 71 (EV71) infections may be associated with neurological complications, including brainstem encephalitis (BE). Severe EV71 BE may be complicated with autonomic nervous system (ANS) dysregulation and/or pulmonary edema (PE). ANS dysregulation is related to the overactivation of the sympathetic nervous system, which results from catecholamine release.ObjectiveThe aims of this study were to explore the effects of catecholamines on severe EV71 infection and to investigate the changes in the percentages of EV71-infected cells, virus titer, and cytokine production on the involvement of catecholamines.Study DesignPlasma levels of norepinephrine (NE) and epinephrine (EP) in EV71-infected patients were measured using an enzyme-linked immunoassay. The expression of adrenergic receptors (ADRs) on RD, A549, SK-N-SH, THP-1, Jurkat and human peripheral blood mononuclear cells (hPBMCs) were detected using flow cytometry. The percentages of EV71-infected cells, virus titer, and cytokine production were investigated after treatment with NE and EP.ResultsThe plasma levels of NE and EP were significantly higher in EV71-infected patients with ANS dysregulation and PE than in controls. Both α1A- and β2-ADRs were expressed on A549, RD, SK-N-SH, HL-60, THP-1, Jurkat cells and hPBMCs. NE treatment elevated the percentages of EV71-infected cells to 62.9% and 22.7% in THP-1 and Jurkat cells, respectively. Via treatment with EP, the percentages of EV71-infected cells were increased to 64.6% and 26.9% in THP-1 and Jurkat cells. The percentage of EV71-infected cells increased upon NE or EP treatment while the α- and β-blockers reduced the percentages of EV71-infected cells with NE or EP treatment. At least two-fold increase in virus titer was observed in EV71-infected A549, SK-N-SH and hPBMCs after treatment with NE or EP. IL-6 production was enhanced in EV71-infected hPBMCs at a concentration of 102 pg/mL NE.ConclusionThe plasma levels of NE and EP elevated in EV71-infected patients with ANS dysregulation and PE. Both NE and EP enhanced the percentages of infected cells and virus titers in EV71 infection in vitro. NE and EP may play a role in the pathogenesis of EV71 BE complicated with ANS dysregulation and PE.

Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation.

Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5' untranslated region (5'UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection.

Differential expression of miRNAs in enterovirus 71-infected cells.

BackgroundEnterovirus 71 (EV71) is one of the major etiological pathogens of hand, foot and mouth disease (HFMD) and can cause severe cerebral and pulmonary complications and even fatality. MicroRNAs (miRNAs), a class of small non-coding RNA molecules, play an important role in post-transcriptional regulation of gene expression and thereby influencing various physiological and pathological processes. Increasing evidence suggests that miRNAs act as key effector molecules in the complicated pathogen-host interactions. However, the roles of miRNAs in EV71 infection and pathogenesis are not well understood.MethodsTo identify special miRNAs involved in EV71 infection, a microarray assay was performed to study the expression pattern of miRNAs in EV71-infected human rhabdomyosarcoma cells (RD cells) and uninfected RD cells. We further predicted the putative target genes for the dysregulated miRNAs using the online bioinformatic algorithms (TargetScan, miRanda and PicTar) and carried out functional annotation including GO enrichment and KEGG pathway analysis for miRNA predicted targets. Then, the results of microarray were further confirmed by quantitative RT-PCR.ResultsTotally, 45 differentially expressed miRNAs ware identified by microarray, among which 36 miRNAs were up-regulated and 9 were down-regulated. 7166 predicted target genes for the dysregulated miRNAs were revealed by using TargetScan in conjunction with miRanda and PicTar. The GO annotation suggested that predicted targets of miRNAs were enriched into the category of signal transduction, regulation of transcription, metabolic process, protein phosphorylation, apoptotic process and immune response. KEGG pathway analysis suggested that these predicted target genes were involved in many important pathways, mainly including endocytosis and focal adhesion, MAPK signaling pathway, hypertrophic cardiomyopathy, melanogenesis and ErbB signaling pathway. The expression levels of 8 most differentially up-regulated miRNAs and 3 most differentially down-regulated miRNAs were confirmed by qRT-PCR. The expressions of hsa-miR-4530, hsa-miR-4492, hsa-miR-6125, hsa-miR-494-3p, hsa-miR-638, hsa-miR-6743-5p, hsa-miR-4459 and hsa-miR-4443 detected by qRT-PCR were consistent with the microarray data.ConclusionThese results might extend our understanding to the regulatory mechanism of miRNAs underlying the pathogenesis of EV71 infection, thus strengthening the preventative and therapeutic strategies of HFMD caused by EV71.