EV71 infection induces neurodegeneration via activating TLR7 signaling and IL-6 production.

As a neurotropic virus, human Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease (HFMD) and may develop severe neurological disorders in infants. Toll-like receptor 7 (TLR7) acts as an innate immune receptor and is also a death receptor in the central nervous system (CNS). However, the mechanisms underlying the regulation of TLR7-mediated brain pathogenesis upon EV71 infection remain largely elusive. Here we reveal a novel mechanism by which EV71 infects astrocytes in the brain and induces neural pathogenesis via TLR7 and interleukin-6 (IL-6) in C57BL/6 mice and in human astroglioma U251 cells. Upon EV71 infection, wild-type (WT) mice displayed more significant body weight loss, higher clinical scores, and lower survival rates as compared with TLR7-/- mice. In the cerebral cortex of EV71-infected mice, neurofilament integrity was disrupted, and inflammatory cell infiltration and neurodegeneration were induced in WT mice, whereas these were largely absent in TLR7-/- mice. Similarly, IL-6 production, Caspase-3 cleavage, and cell apoptosis were significantly higher in EV71-infected WT mice as compared with TLR7-/- mice. Moreover, EV71 preferentially infected and induced IL-6 in astrocytes of mice brain. In U251 cells, EV71-induced IL-6 production and cell apoptosis were suppressed by shRNA-mediated knockdown of TLR7 (shTLR7). Moreover, in the cerebral cortex of EV71-infected mice, the blockade of IL-6 with anti-IL-6 antibody (IL-6-Ab) restored the body weight loss, attenuated clinical scores, improved survival rates, reduced the disruption of neurofilament integrity, decreased cell apoptotic induction, and lowered levels of Caspase-3 cleavage. Similarly, in EV71-infected U251 cells, IL-6-Ab blocked EV71-induced IL-6 production and cell apoptosis in response to viral infection. Collectively, it's exhibited TLR7 upregulation, IL-6 induction and astrocytic cell apoptosis in EV71-infected human brain. Taken together, we propose that EV71 infects astrocytes of the cerebral cortex in mice and human and triggers TLR7 signaling and IL-6 release, subsequently inducing neural pathogenesis in the brain.

HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection.

Enterovirus 71 (EV71) is an RNA virus that causes hand-foot-mouth disease (HFMD), and even fatal encephalitis in children. Although EV71 pathogenesis remains largely obscure, host immune responses may play important roles in the development of diseases. Recognition of pathogens mediated by Toll-like receptors (TLRs) induces host immune and inflammatory responses. Intracellular TLRs must traffic from the endoplasmic reticulum (ER) to the endolysosomal network from where they initiate complete signaling, leading to inflammatory response. This study reveals a novel mechanism underlying the regulation of TLR7 signaling during EV71 infection. Initially, we show that multiple cytokines are differentially expressed during viral infection and demonstrate that EV71 infection induces the production of proinflammatory cytokines through regulating TLR7-mediated p38 MAPK, and NF-κB signaling pathways. Further studies reveal that the expression of the endosome-associated protein hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is upregulated and highly correlated with the expression of TLR7 in EV71 infected patients, mice, and cultured cells. Virus-induced HRS subsequently enhances TLR7 complex formation in early- and late-endosome by interacting with TLR7 and TAB1. Moreover, HRS is involved in the regulation of the TLR7/NF-κB/p38 MAPK and the TLR7/NF-κB/IRF3 signaling pathways to induce proinflammatory cytokines and interferons, respectively, resulting in the orchestration of inflammatory and immune responses to the EV71 infection. Therefore, this study demonstrates that HRS acts as a key component of TLR7 signaling to orchestrate immune and inflammatory responses during EV71 infection, and provides new insights into the mechanisms underlying the regulation of host inflammation and innate immunity during EV71 infection.

HERP Binds TBK1 To Activate Innate Immunity and Repress Virus Replication in Response to Endoplasmic Reticulum Stress.

Host innate immunity is crucial for cellular responses against viral infection sensed by distinct pattern recognition receptors and endoplasmic reticulum (ER) stress. Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and neurological diseases. However, the exact mechanism underlying the link between ER stress induced by EV71 infection and host innate immunity is largely unknown. In this study, we demonstrated that EV71 infection induces the homocysteine-induced ER protein (HERP), a modulator of the ER stress response which is dependent on the participation of MAVS. Virus-induced HERP subsequently stimulates host innate immunity to repress viral replication by promoting type-I IFNs (IFN-α and IFN-ß) and type-III IFN (IFN-λ1) expression. Through interacting with TANK-binding kinase 1, HERP amplifies the MAVS signaling and facilitates the phosphorylation and nuclear translocation of IFN regulatory factor 3 and NF-κB to enhance the expression of IFNs, which leads to a broad inhibition of the replication of RNA viruses, including EV71, Sendai virus, influenza A virus, and vesicular stomatitis virus. Therefore, we demonstrated that HERP plays an important role in the regulation of host innate immunity in response to ER stress during the infection of RNA viruses. These findings provide new insights into the mechanism underlying the replication of RNA viruses and the production of IFNs, and also demonstrate a new role of HERP in the regulation of host innate immunity in response to viral infection.

Antiviral effects of two Ganoderma lucidum triterpenoids against enterovirus 71 infection.

Enterovirus 71 (EV71) is a major causative agent for hand, foot and mouth disease (HFMD), and fatal neurological and systemic complications in children. However, there is currently no clinical approved antiviral drug available for the prevention and treatment of the viral infection. Here, we evaluated the antiviral activities of two Ganoderma lucidum triterpenoids (GLTs), Lanosta-7,9(11),24-trien-3-one,15;26-dihydroxy (GLTA) and Ganoderic acid Y (GLTB), against EV71 infection. The results showed that the two natural compounds display significant anti-EV71 activities without cytotoxicity in human rhabdomyosarcoma (RD) cells as evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. The mechanisms by which the two compounds affect EV71 infection were further elucidated by three action modes using Ribavirin, a common antiviral drug, as a positive control. The results suggested that GLTA and GLTB prevent EV71 infection through interacting with the viral particle to block the adsorption of virus to the cells. In addition, the interactions between EV71 virion and the compounds were predicated by computer molecular docking, which illustrated that GLTA and GLTB may bind to the viral capsid protein at a hydrophobic pocket (F site), and thus may block uncoating of EV71. Moreover, we demonstrated that GLTA and GLTB significantly inhibit the replication of the viral RNA (vRNA) of EV71 replication through blocking EV71 uncoating. Thus, GLTA and GLTB may represent two potential therapeutic agents to control and treat EV71 infection.

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

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.

Enterovirus 71 induces neural cell apoptosis and autophagy through promoting ACOX1 downregulation and ROS generation.

Enterovirus 71 (EV71) infection causes hand, foot, and mouth disease (HFMD), and even fatal neurological complications. However, the mechanisms underlying EV71 neurological pathogeneses are largely unknown. This study reveals a distinct mechanism by which EV71 induces apoptosis and autophagy in neural cells. EV71 non-structure protein 3D (also known as RNA-dependent RNA polymerase, RdRp) interacts with the peroxisomal protein acyl-CoA oxidase 1 (ACOX1), and contributes to ACOX1 downregulation. Further studies demonstrate that EV71 reduces peroxisome numbers. Additionally, knockdown of ACOX1 or peroxin 19 (PEX19) induces apoptosis and autophagy in neural cells including human neuroblastoma (SK-N-SH) cells and human astrocytoma (U251) cells, and EV71 infection induces neural cell death through attenuating ACOX1 production. Moreover, EV71 infection and ACOX1 knockdown facilitate reactive oxygen species (ROS) production and attenuate the cytoprotective protein deglycase (DJ-1)/Nuclear factor erythroid 2-related factor 2 (NRF2)/Heme oxygenase 1 (HO-1) pathway (DJ-1/NRF2/HO-1), which collectively result in ROS accumulation in neural cells. In conclusion, EV71 downregulates ACOX1 protein expression, reduces peroxisome numbers, enhances ROS generation, and attenuates the DJ-1/NRF2/HO-1 pathway, thereby inducing apoptosis and autophagy in neural cells. These findings provide new insights into the mechanism underlying EV71-induced neural pathogenesis, and suggest potential treatments for EV71-associated diseases.

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.

Early growth response-1 facilitates enterovirus 71 replication by direct binding to the viral genome RNA.

Enterovirus 71 (EV71) infections can cause hand, foot and mouth disease (HFMD), meningoencephalitis, neonatal sepsis, and even fatal encephalitis in children. Unfortunately, there is currently no effective treatment for EV71 infection due to the lack of understanding of viral replication and infection; and viral infections have emerged as an imperative global hazard. Thus, it is extremely important to understand the mechanism of EV71 replication in order to prevent and control the diseases associated with EV71 infections. Early growth response-1 (EGR1) is a multifunctional transcription factor that regulates diverse biological functions, including inflammation, apoptosis, differentiation, tumorigenesis, and even viral infection. Here, we provide new insight into the role of EV71 infection in regulating EGR1 production; and reveal a novel mechanism by which EGR1 facilitates EV71 replication. We demonstrate that EV71 activates EGR1 expression during infection by stimulating the protein kinase A/protein kinase Cɛ/phosphoinositide 3-kinase/Akt (PKA/PKCɛ/PI3K/Akt) cascade. We further reveal that EV71-activated EGR1, in turn, regulates the internal ribosomal entry site (IRES) of EV71 to enhance viral replication. In addition, EGR1 facilitates EV71 replication by binding directly to stem-loops I and IV of EV71 5'-untranslated region (5'UTR) with its first two zinc fingers. Moreover, EGR1 protein co-localizes with EV71 RNA in the cytoplasm of infected cells to facilitate viral replication. Our results reveal an important new role of EGR1 in viral infection, provide new insight into the novel mechanism underlying the regulation of EV71 replication, and suggest a potential application of EGR1 in the control of EV71 infection.

PolyC-binding protein 1 interacts with 5'-untranslated region of enterovirus 71 RNA in membrane-associated complex to facilitate viral replication.

Enterovirus 71 (EV71) is one causative agent of hand, foot, and mouth disease (HFMD), which may lead to severe neurological disorders and mortality in children. EV71 genome is a positive single-stranded RNA containing a single open reading frame (ORF) flanked by 5'-untranslated region (5'UTR) and 3'UTR. The 5'UTR is fundamentally important for virus replication by interacting with cellular proteins. Here, we revealed that poly(C)-binding protein 1 (PCBP1) specifically binds to the 5'UTR of EV71. Detailed studies indicated that the RNA-binding K-homologous 1 (KH1) domain of PCBP1 is responsible for its binding to the stem-loop I and IV of EV71 5'UTR. Interestingly, we revealed that PCBP1 is distributed in the nucleus and cytoplasm of uninfected cells, but mainly localized in the cytoplasm of EV71-infected cells due to interaction and co-localization with the viral RNA. Furthermore, sub-cellular distribution analysis showed that PCBP1 is located in ER-derived membrane, in where virus replication occurred in the cytoplasm of EV71-infected cells, suggesting PCBP1 is recruited in a membrane-associated replication complex. In addition, we found that the binding of PCBP1 to 5'UTR resulted in enhancing EV71 viral protein expression and virus production so as to facilitate viral replication. Thus, we revealed a novel mechanism in which PCBP1 as a positive regulator involved in regulation of EV71 replication in the host specialized membrane-associated replication complex, which provides an insight into cellular factors involved in EV71 replication.