scholarly journals Curcumin Assists Anti-EV71 Activity of IFN-α by Inhibiting IFNAR1 Reduction in SH-SY5Y Cells

2021 ◽  
Author(s):  
Yanfang Wang ◽  
Kena Dan ◽  
Xiaoling Xue ◽  
Bangtao Chen ◽  
Cheng Chen
Keyword(s):  
Internal Ribosomal Entry Site ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Luciferase Assay ◽  
Interferon Α ◽  
Entry Site ◽  
Human Neuroblastoma ◽  
Virus Attachment ◽  
Synergistic Inhibition ◽  
Ubiquitin Proteasome

Abstract Background and aim Enterovirus 71(EV71) can cause severe hand, foot, and mouth disease (HFMD) with brain tissue involvement. Few effective anti-EV71 drugs are presently available in clinical practice. Interferon-α (IFN-α) was ineffective while Curcumin was effective in restricting EV71 replication in non-neuronal cells. Ubiquitin-proteasome-mediated degradation of interferon-alpha receptor 1 (IFNAR1) protein contributes to IFN-α resistance. Current study aimed to determine synergistic inhibition of EV71 by Curcumin and IFN-α in human neuroblastoma SH-SY5Y cells. Methods SH-SY5Y cells were infected with mock-/Curcumin-pre-incubated EV71 or transfected with plasmid containing interferon-stimulated response element (ISRE) or mRNA containing viral internal ribosomal entry site (IRES) following by post-treatment with Curcumin with or without IFN-α. Supernatant IFN-α/β was detected by ELISA. ISRE, IRSE, proteasome and deubiquitinating activity were measured by luciferase assay. EV71 RNA and viral protein or IFNAR1 were determined by qPCR and western blot, respectively. Results EV71 flailed to completely block IFN-α/β production but inhibited IFN-α signal. Curcumin only slightly inhibited EV71 proliferation without modulating virus attachment and internalization. However, Curcumin addition restored IFN-α-mediated ISRE activity thus significantly inhibiting EV71 replication. Furthermore, EV71 also reduced IFNAR1 protein with proteasome-dependence in SH-SY5Y cells, which can be reversed by Curcumin addition with the evidence that it lowered proteasome activity. Conclusion These data demonstrate that Curcumin assists anti-EV71 activity of IFN-α by inhibiting IFNAR1 reduction via ubiquitin-proteasome disruption in SH-SY5Y cells

scholarly journals Enterovirus 71 Infection Cleaves a Negative Regulator for Viral Internal Ribosomal Entry Site-Driven Translation

2013 ◽  
Vol 87 (7) ◽  
pp. 3828-3838 ◽  
Author(s):  
L.-L. Chen ◽  
Y.-A. Kung ◽  
K.-F. Weng ◽  
J.-Y. Lin ◽  
J.-T. Horng ◽  
...  
Keyword(s):  
Internal Ribosomal Entry Site ◽  
Enterovirus 71 ◽  
Negative Regulator ◽  
Entry Site ◽  
Ribosomal Entry ◽  
Enterovirus 71 Infection

scholarly journals hnRNP K Is a Novel Internal Ribosomal Entry Site-Transacting Factor That Negatively Regulates Foot-and-Mouth Disease Virus Translation and Replication and Is Antagonized by Viral 3C Protease

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Wenming Liu ◽  
Decheng Yang ◽  
Chao Sun ◽  
Haiwei Wang ◽  
Bo Zhao ◽  
...  
Keyword(s):  
Translational Control ◽  
Translational Regulation ◽  
Internal Ribosomal Entry Site ◽  
Foot And Mouth Disease ◽  
Cleavage Product ◽  
Entry Site ◽  
Hnrnp K ◽  
Mouth Disease Virus ◽  
Ribosomal Entry ◽  
Fmdv Replication

ABSTRACT Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5′ untranslated region. The regulation of internal initiation requires the interaction of IRES-transacting factors (ITAFs) with the IRES. In this study, we identified a novel ITAF, heterogeneous nuclear ribonucleoprotein K (hnRNP K), which negatively regulates foot-and-mouth disease virus (FMDV) translation and viral replication. Further investigation revealed that the KH2 and KH3 domains of hnRNP K directly bind to domains II, III, and IV of the FMDV IRES, resulting in the inhibition of IRES-mediated translation by interfering with the recognition of another positive ITAF, polypyrimidine tract-binding protein (PTB). Conversely, hnRNP K-mediated inhibition was antagonized by the viral 3C protease through the cleavage of hnRNP K at the Glu-364 residue during FMDV infection. Interestingly, the N-terminal cleavage product, hnRNP K1–364, retained partial inhibitory effects on IRES activity, whereas the C-terminal cleavage product, hnRNP K364–465, became a positive regulator of FMDV replication. Our findings expand the current understanding of virus-host interactions concerning viral recruitment and the modulation of ITAFs, providing new insights into translational control during viral infection. IMPORTANCE The translation of picornaviral genome RNA mediated by the internal ribosomal entry site (IRES) is a crucial step for virus infections. Virus-host interactions play a critical role in the regulation of IRES-dependent translation, but the regulatory mechanism remains largely unknown. In this study, we identified an ITAF, hnRNP K, that negatively regulates FMDV replication by inhibiting viral IRES-mediated translation. In addition, we describe a novel translational regulation mechanism involving the proteolytic cleavage of hnRNP K by FMDV protease 3C. The cleavage of hnRNP K yields two cleavage products with opposite functions: the cleavage product hnRNP K1–364 retains a partial inhibitory effect on IRES activity, and the cleavage product hnRNP K364–465 becomes a positive regulator of FMDV replication. Our findings shed light on the effect of a novel ITAF on the translational regulation of picornavirus and provide new insights into translational control during viral infection.

scholarly journals Hsp27 Responds to and Facilitates Enterovirus A71 Replication by Enhancing Viral Internal Ribosome Entry Site-Mediated Translation

2019 ◽  
Vol 93 (9) ◽  
Author(s):  
Xuelian Dan ◽  
Qianya Wan ◽  
Lina Yi ◽  
Jing Lu ◽  
Yang Jiao ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Chinese Herb ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Initiation Factor ◽  
Hnrnp A1 ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Foot And Mouth

ABSTRACTEnterovirus 71 (EV-A71) is a human pathogen that causes hand, foot, and mouth disease (HFMD) and fatal neurological diseases, and no effective treatment is available. Characterization of key host factors is important for understanding its pathogenesis and developing antiviral drugs. Here we report that Hsp27 is one of the most upregulated proteins in response to EV-A71 infection, as revealed by two-dimensional gel electrophoresis-based proteomics studies. Depletion of Hsp27 by small interfering RNA or CRISPR/Cas9-mediated knockout significantly inhibited viral replication, protein expression, and reproduction, while restoration of Hsp27 restored such virus activities. Furthermore, we show that Hsp27 plays a crucial role in regulating viral internal ribosome entry site (IRES) activities by two different mechanisms. Hsp27 markedly promoted 2Apro-mediated eukaryotic initiation factor 4G cleavage, an important process for selecting and initiating IRES-mediated translation. hnRNP A1 is a key IREStrans-acting factor (ITAF) for enhancing IRES-mediated translation. Surprisingly, knockout of Hsp27 differentially blocked hnRNP A1 but not FBP1 translocation from the nucleus to the cytoplasm and therefore abolished the hnRNP A1 interaction with IRES. Most importantly, the Hsp27 inhibitor 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone (TDP), a compound isolated from a traditional Chinese herb, significantly protected against cytopathic effects and inhibited EV-A71 infection. Collectively, our results demonstrate new functions of Hsp27 in facilitating virus infection and provide novel options for combating EV-A71 infection by targeting Hsp27.IMPORTANCEOutbreaks of infections with EV-A71, which causes hand, foot, and mouth disease, severe neurological disorders, and even death, have been repeatedly reported worldwide in recent decades and are a great public health problem for which no approved treatments are available. We show that Hsp27, a heat shock protein, supports EV-A71 infection in two distinct ways to promote viral IRES-dependent translation. A small-molecule Hsp27 inhibitor isolated from a traditional Chinese medicinal herb effectively reduces virus yields. Together, our findings demonstrate that Hsp27 plays an important role in EV-A71 infection and may serve as an antiviral target.

scholarly journals Translocating lipopolysaccharide correlates with the severity of enterovirus A71-induced HFMD by promoting pro-inflammation and viral IRES activity

2021 ◽  
Author(s):  
Yuya Wang ◽  
Xiaoling Xue ◽  
Kena Dan ◽  
Xiongbo Yang ◽  
Qingqing Yang ◽  
...  
Keyword(s):  
Inflammatory Mediators ◽  
Internal Ribosomal Entry Site ◽  
Foot And Mouth Disease ◽  
Serum Levels ◽  
Interferon Γ ◽  
Healthy Children ◽  
Entry Site ◽  
Fatty Acid Binding ◽  
Enterovirus A71

Abstract BackgroundThe increase of inflammation-inducing enterobacteria was recently observed in severe hand, foot, and mouth disease (HFMD) caused by Enterovirus A71 (EV-A71). This study aimed to verify the occurrence of bacterial translocation (BT) and further explore the contributory role of BT to severity of EV-A71-mediated HFMD cases. MethodsSerum specimens from 65 mild and 65 severe EV-A71-associated HFMD cases and 65 healthy children were collected. EV-A71 VP1 in serum, inflammatory mediators including C-reactive protein, IL-1β, IL-6, interferon-γ and tumor necrosis factor-α, BT related biomarkers including Claudin-3, intestinal fatty acid binding protein, lipopolysaccharide (LPS), soluble CD14(sCD14) and endotoxin core antibody were measured by ELISA. Bacterial DNA (BactDNA) fragments were quantified by quantified PCR (qPCR). Rhabdomyosarcoma (RD) cells, infected with LPS-pre-incubated EV-A71 or transfected with plasmid containing viral 2A pro or mRNA containing viral internal ribosomal entry site (IRES), were post-treated with or without LPS in vitro . EV-A71 RNA and viral or cellular proteins were determined by qPCR and western blot, respectively. ResultsCompared to mild HFMD patients, remarkably higher inflammatory mediators as well as BT-related biomarkers except BactDNA were observed in severe HFMD cases (all P <0.05). In severe HFMD group, circulating concentrations of LPS and sCD14 showed statistical correlations with inflammation indices (all P <0.05), serum levels of EV-A71 VP1 were found to be positively correlated with serum LPS ( r =0.341, P =0.005) and serum sCD14( r =0.458, P <0.001). In vitro , EV-A71 attachment and internalization were only slightly promoted by LPS pre-incubation; however, EV-A71 proliferation and viral 2A pro -mediated IRES activity were significantly accelerated by LPS post-treatment. ConclusionsOur results collectively indicate that gut-derived translocating LPS contributes to the severity of EV-A71-induced HFMD by driving inflammatory response and viral proliferation via viral 2A pro -mediated IRES.

scholarly journals Single Neutralizing Monoclonal Antibodies Targeting the VP1 GH Loop of Enterovirus 71 Inhibit both Virus Attachment and Internalization during Viral Entry

2015 ◽  
Vol 89 (23) ◽  
pp. 12084-12095 ◽  
Author(s):  
Zhiqiang Ku ◽  
Xiaohua Ye ◽  
Jinping Shi ◽  
Xiaoli Wang ◽  
Qingwei Liu ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Heparan Sulfate ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Critical Role ◽  
Antiviral Drug ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Independent Manner ◽  
Virus Attachment

ABSTRACTAntibodies play a critical role in immunity against enterovirus 71 (EV71). However, how EV71-specific antibodies neutralize infections remains poorly understood. Here we report the working mechanism for a group of three monoclonal antibodies (MAbs) that potently neutralize EV71. We found that these three MAbs (termed D5, H7, and C4, respectively) recognize the same conserved neutralizing epitope within the VP1 GH loop of EV71. Single MAbs in this group, exemplified by D5, could inhibit EV71 infection in cell cultures at both the pre- and postattachment stages in a cell type-independent manner. Specifically, MAb treatment resulted in the blockade of multiple steps of EV71 entry, including virus attachment, internalization, and subsequent uncoating and RNA release. Furthermore, we show that the D5 and C4 antibodies can interfere with EV71 binding to its key receptors, including heparan sulfate, SCARB2, and PSGL-1, thus providing a possible explanation for the observed multi-inhibitory function of the MAbs. Collectively, our study unravels the mechanism of neutralization by a unique group of anti-EV71 MAbs targeting the conserved VP1 GH loop. The findings should enhance our understanding of MAb-mediated immunity against enterovirus infections and accelerate the development of MAb-based anti-EV71 therapeutic drugs.IMPORTANCEEnterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), which has caused significant morbidities and mortalities in young children. Neither a vaccine nor an antiviral drug is available. Neutralizing antibodies are major protective components in EV71 immunity. Here, we unraveled an unusual mechanism of EV71 neutralization by a group of three neutralizing monoclonal antibodies (MAbs). All of these MAbs bound the same conserved epitope located at the VP1 GH loop of EV71. Interestingly, mechanistic studies showed that single antibodies in this MAb group could block EV71 attachment and internalization during the viral entry process and interfere with EV71 binding to heparan sulfate, SCARB2, and PSGL-1 molecules, which are key receptors involved in different steps of EV71 entry. Our findings greatly enhance the understanding of the interplays among EV71, neutralizing antibodies, and host receptors, which in turn should facilitate the development of an MAb-based anti-EV71 therapy.

scholarly journals First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Sotaro Chiba ◽  
Atif Jamal ◽  
Nobuhiro Suzuki
Keyword(s):  
Filamentous Fungi ◽  
Rna Viruses ◽  
Internal Ribosomal Entry Site ◽  
Cryphonectria Parasitica ◽  
Luciferase Assay ◽  
Untranslated Regions ◽  
Entry Site ◽  
Translational Initiation ◽  
Dsrna Viruses ◽  
Ribosomal Entry

ABSTRACTIn contrast to well-established internal ribosomal entry site (IRES)-mediated translational initiation in animals and plants, no IRESs were established in fungal viral or cellular RNAs. To identify IRES elements in mycoviruses, we developed a luciferase-based dual-reporter detection system inCryphonectria parasitica, a model filamentous fungus for virus-host interactions. A bicistronic construct entails a codon-optimizedRenillaand firefly luciferase (ORlucandOFluc, respectively) gene, between which potential IRES sequences can be inserted. In this system, ORluc serves as an internal control, while OFluc represents IRES activity. Virus sequences in the 5′ untranslated regions (UTRs) of the genomes of diverse positive-sense single-stranded RNA and double-stranded RNA (dsRNA) viruses were analyzed. The results show relatively high IRES activities for Cryphonectria hypovirus 1 (CHV1) and CHV2 and faint but measurable activity for CHV3. The weak IRES signal of CHV3 may be explained by its monocistronic nature, differing from the bicistronic nature of CHV1 and CHV2. This would allow these three hypoviruses to have similar rates of translation of replication-associated protein per viral mRNA molecule. The importance of 24 5′-proximal codons of CHV1 as well as the 5′ UTR for IRES function was confirmed. Furthermore, victoriviruses and chrysoviruses tested IRES positive, whereas mycoreoviruses, partitiviruses, and quadriviruses showed similar Fluc activities as the negative controls. Overall, this study represents the first development of an IRES identification system in filamentous fungi based on the codon-optimized dual-luciferase assay and provides evidence for IRESs in filamentous fungi.IMPORTANCECap-independent, internal ribosomal entry site (IRES)-mediated translational initiation is often used by virus mRNAs and infrequently by cellular mRNAs in animals and plants. However, no IRESs have been established in fungal virus RNAs or cellular RNAs in filamentous fungi. Here, we report the development of a dual-luciferase assay system and measurement of the IRES activities of fungal RNA viruses in a model filamentous fungal host,Cryphonectria parasitica. Viruses identified as IRES positive include hypoviruses (positive-sense RNA viruses, members of the expandedPicornavirussupergroup), totiviruses (nonsegmented dsRNA viruses), and chrysoviruses (tetrasegmented dsRNA viruses). No IRES activities were observed in the 5′ untranslated regions of mycoreoviruses (11-segmented dsRNA viruses), quadriviruses (tetrasegmented dsRNA viruses), or partitiviruses (bisegmented dsRNA viruses). This study provides the first evidence for IRES activities in diverse RNA viruses in filamentous fungi and is a first step toward identifyingtrans-acting host factors andcis-regulatory viral RNA elements.

scholarly journals Translocating lipopolysaccharide correlates with the severity of enterovirus A71-induced HFMD by promoting pro-inflammation and viral IRES activity

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Yuya Wang ◽  
Kena Dan ◽  
Xiaoling Xue ◽  
Xiongbo Yang ◽  
Xujiao Feng ◽  
...  
Keyword(s):  
Inflammatory Mediators ◽  
Internal Ribosomal Entry Site ◽  
Foot And Mouth Disease ◽  
Serum Levels ◽  
Interferon Γ ◽  
Healthy Children ◽  
Entry Site ◽  
Fatty Acid Binding ◽  
Statistical Correlations

Abstract Background The increase of inflammation-inducing enterobacteria was recently observed in severe hand, foot, and mouth disease (HFMD) caused by Enterovirus A71 (EV-A71). This study aimed to verify the occurrence of bacterial translocation (BT) and further explore the contributory role of BT to severity of EV-A71-mediated HFMD cases. Methods Serum specimens from 65 mild and 65 severe EV-A71-associated HFMD cases and 65 healthy children were collected. EV-A71 VP1 in serum, inflammatory mediators including C-reactive protein, IL-1β, IL-6, interferon-γ and tumor necrosis factor-α, BT related biomarkers including Claudin-3, intestinal fatty acid binding protein, lipopolysaccharide (LPS), soluble CD14 (sCD14) and endotoxin core antibody were measured by ELISA. Bacterial DNA (BactDNA) fragments were quantified by quantified PCR (qPCR). Rhabdomyosarcoma (RD) or SH-SY5Y cells, infected with LPS-pre-incubated EV-A71 or transfected with plasmid containing viral 2Apro or mRNA containing viral internal ribosomal entry site (IRES), were post-treated with or without LPS in vitro. EV-A71 RNA and viral or cellular proteins were determined by qPCR and western blot, respectively. Results Compared to mild HFMD patients, remarkably higher inflammatory mediators as well as BT-related biomarkers except BactDNA were observed in severe HFMD cases (all P < 0.05). In severe HFMD group, circulating concentrations of LPS and sCD14 showed statistical correlations with inflammation indices (all P < 0.05), serum levels of EV-A71 VP1 were found to be positively correlated with serum LPS (r = 0.341, P = 0.005) and serum sCD14 (r = 0.458, P < 0.001). In vitro, EV-A71 attachment and internalization were only slightly promoted by LPS pre-incubation; however, EV-A71 proliferation and viral 2Apro-mediated IRES activity were significantly accelerated by LPS post-treatment. Conclusions Our results collectively indicate that gut-derived translocating LPS contributes to the severity of EV-A71-induced HFMD by driving inflammatory response and viral proliferation via viral 2Apro-mediated IRES.

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