Hyperglycemia facilitates EV71 replication: Insights into miR-206-mediated regulation of G3BP2 promoting EV71 IRES activity.

Background: Neurotropic virus infections actively manipulate host cell metabolism to enhance virus neurovirulence. Although hyperglycemia is common during severe infections, its specific role remains unclear. This study investigates the impact of hyperglycemia on the neurovirulence of enterovirus 71 (EV71), a neurovirulent virus relying on internal ribosome entry site (IRES)-mediated translation for replication. Methods: Utilizing hSCARB2-transgenic mice, we explore the effects of hyperglycemia in EV71 infection and elucidate the underlying mechanisms. Results: Remarkably, administering insulin alone to reduce hyperglycemia in hSCARB2-transgenic mice results in a decrease in brainstem encephalitis and viral load. Conversely, induced hyperglycemia exacerbates neuropathogenesis, highlighting the pivotal role of hyperglycemia in neurovirulence. Notably, miR-206 emerges as a crucial mediator induced by viral infection, with its expression further heightened by hyperglycemia and concurrently repressed by insulin. The use of antagomiR-206 effectively mitigates EV71-induced brainstem encephalitis and reduces viral load. Mechanistically, miR-206 facilitates IRES-driven virus replication by repressing the stress granule protein G3BP2. Conclusions: Novel therapeutic approaches against severe EV71 infections involve managing hyperglycemia and targeting the miR-206-stress granule pathway to modulate virus IRES activity.

Inhibition of ESCRT-independent extracellular vesicles biogenesis suppresses enterovirus 71 replication and pathogenesis in mice.

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease (HFMD), neurological complications, and even fatalities in infants. Clinically, the increase of extracellular vesicles (EVs) in EV71 patients' serum was highly associated with the severity of HFMD. EV71 boosts EVs biogenesis in an endosomal sorting complex required for transport (ESCRT)-dependent manner to facilitate viral replication. Yet, the impact of EVs-derived from ESCRT-independent pathway on EV71 replication and pathogenesis is highly concerned. Here, we assessed the effects of EV71-induced EVs from ESCRT-independent pathway on viral replication and pathogenesis by GW4869, a neutral sphingomyelinase inhibitor. Detailly, in EV71-infected mice, blockade of the biogenesis of tissue-derived EVs in the presence of GW4869 restored body weight loss, attenuated clinical scores, and improved survival rates. Furthermore, GW4869 dampens EVs biogenesis to reduce viral load and pathogenesis in multiple tissues of EV71-infected mice. Consistently, GW4869 treatment in a human intestinal epithelial HT29 cells decreased the biogenesis of EVs, in which the progeny EV71 particle was cloaked, leading to the reduction of viral infection and replication. Collectively, GW4869 inhibits EV71-induced EVs in an ESCRT-independent pathway and ultimately suppresses EV71 replication and pathogenesis. Our study provides a novel strategy for the development of therapeutic agents in the treatment for EV71-associated HFMD.

Enterovirus 71 Activates Plasmacytoid Dendritic Cell-Dependent PSGL-1 Binding Independent of Productive Infection.

Enterovirus 71 (EV71) is a significant causative agent of hand, foot, and mouth disease, with potential serious neurologic complications or fatal outcomes. The lack of effective treatments for EV71 infection is attributed to its elusive pathogenicity. Our study reveals that human plasmacytoid dendritic cells (pDCs), the main type I IFN-producing cells, selectively express scavenger receptor class B, member 2 (SCARB2) and P-selectin glycoprotein ligand 1 (PSGL-1), crucial cellular receptors for EV71. Some strains of EV71 can replicate within pDCs and stimulate IFN-α production. The activation of pDCs by EV71 is hindered by Abs to PSGL-1 and soluble PSGL-1, whereas Abs to SCARB2 and soluble SCARB2 have a less pronounced effect. Our data suggest that only strains binding to PSGL-1, more commonly found in severe cases, can replicate in pDCs and induce IFN-α secretion, highlighting the importance of PSGL-1 in these processes. Furthermore, IFN-α secretion by pDCs can be triggered by EV71 or UV-inactivated EV71 virions, indicating that productive infection is not necessary for pDC activation. These findings provide new insights into the interaction between EV71 and pDCs, suggesting that pDC activation could potentially mitigate the severity of EV71-related diseases.

High mobility group box 1 knockdown inhibits EV71 replication and attenuates cell pyroptosis through TLR4/NF-κB/NLRP3 axis.

Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD) in children. Nowadays, there are still no effective antiviral drugs for EV71 infection. High mobility group box 1 (HMGB1) is reported to be highly expressed in HFMD patients. However, the role and underlying mechanism of HMGB1 in EV71-associated HFMD are still unclear. HMGB1 expression was detected using RT-qPCR and western blot assays. Loss- and gain-function experiments were performed to evaluate the effects of HMGB1 on EV71-infected cells. The virus titer was examined by TCID50. CCK-8 and flow cytometry assays were applied to detect the cell viability and cell cycle. Oxidative stress was determined by relative commercial kits. HMGB1 level was elevated in the serum of EV71-infected patients with HFMD and EV71-induced RD cells. EV71 infection induced the transfer of HMGB1 from the nucleus into the cytoplasm. HMGB1 knockdown inhibited virus replication, viral protein (VP1) expression and promoted antiviral factor expression. In addition, the inhibition of HMGB1 improved cell viability, protected against S phase arrest, and inhibited EV71-induced cell injury and oxidative stress, whereas HMGB1 overexpression showed the opposite effects. In terms of mechanism, HMGB1 overexpression activated the TLR4/NF-κB/NLRP3 signaling pathway and promoted cell pyroptosis. The inhibition of TLR4 and NF-κB reversed the effects of HMGB1 overexpression on virus replication, oxidative stress, and pyroptosis. In conclusion, HMGB1 knockdown inhibits EV71 replication and attenuates pyroptosis through TLR4/NF-κB/NLRP3 axis.

N-terminal acetyltransferase 6 facilitates enterovirus 71 replication by regulating PI4KB expression and replication organelle biogenesis.

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease in children under 5 years old, which can result in severe neurological complications and even death. Due to limited treatments for EV71 infection, the identification of novel host factors and elucidation of mechanisms involved will help to counter this viral infection. N-terminal acetyltransferase 6 (NAT6) was identified as an essential host factor for EV71 infection with genome-wide CRISPR/Cas9 screening. NAT6 facilitates EV71 viral replication depending on its acetyltransferase activity but has little effect on viral release. In addition, NAT6 is also required for Echovirus 7 and coxsackievirus B5 infection, suggesting it might be a pan-enterovirus host factor. We further demonstrated that NAT6 is required for Golgi integrity and viral replication organelle (RO) biogenesis. NAT6 knockout significantly inhibited phosphatidylinositol 4-kinase IIIß (PI4KB) expression and PI4P production, both of which are key host factors for enterovirus infection and RO biogenesis. Further mechanism studies confirmed that NAT6 formed a complex with its substrate actin and one of the PI4KB recruiters-acyl-coenzyme A binding domain containing 3 (ACBD3). Through modulating actin dynamics, NAT6 maintained the integrity of the Golgi and the stability of ACBD3, thereby enhancing EV71 infection. Collectively, these results uncovered a novel mechanism of N-acetyltransferase supporting EV71 infection.IMPORTANCEEnterovirus 71 (EV71) is an important pathogen for children under the age of five, and currently, no effective treatment is available. Elucidating the mechanism of novel host factors supporting viral infection will reveal potential antiviral targets and aid antiviral development. Here, we demonstrated that a novel N-acetyltransferase, NAT6, is an essential host factor for EV71 replication. NAT6 could promote viral replication organelle (RO) formation to enhance viral replication. The formation of enterovirus ROs requires numerous host factors, including acyl-coenzyme A binding domain containing 3 (ACBD3) and phosphatidylinositol 4-kinase IIIß (PI4KB). NAT6 could stabilize the PI4KB recruiter, ACBD3, by inhibiting the autophagy degradation pathway. This study provides a fresh insight into the relationship between N-acetyltransferase and viral infection.

Recent Advances in Enterovirus A71 Infection and Antiviral Agents.

Enterovirus A71 (EV-A71) is one of the major causative agents of hand, foot, and mouth disease (HFMD) that majorly affects children. Most of the time, HFMD is a mild disease but can progress to severe complications, such as meningitis, brain stem encephalitis, acute flaccid paralysis, and even death. HFMD caused by EV-A71 has emerged as an acutely infectious disease of highly pathogenic potential in the Asia-Pacific region. In this review, we introduced the properties and life cycle of EV-A71, and the pathogenesis and the pathophysiology of EV-A71 infection, including tissue tropism and host range of virus infection, the diseases caused by the virus, as well as the genes and host cell immune mechanisms of major diseases caused by enterovirus 71 (EV-A71) infection, such as encephalitis and neurologic pulmonary edema. At the same time, clinicopathologic characteristics of EV-A71 infection were introduced. There is currently no specific medication for EV-A71 infection, highlighting the urgency and significance of developing suitable anti-EV-A71 agents. This overview also summarizes the targets of existing anti-EV-A71 agents, including virus entry, translation, polyprotein processing, replication, assembly and release; interferons; interleukins; the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase B signaling pathways; the oxidative stress pathway; the ubiquitin-proteasome system; and so on. Furthermore, it overviews the effects of natural products, monoclonal antibodies, and RNA interference against EV-A71. It also discusses issues limiting the research of antiviral drugs. This review is a systematic and comprehensive summary of the mechanism and pathological characteristics of EV-A71 infection, the latest progress of existing anti-EV-A71 agents. It would provide better understanding and guidance for the research and application of EV-A71 infection and antiviral inhibitors.

Enterovirus 71 leads to abnormal mitochondrial dynamics in human neuroblastoma SK-N-SH cells.

EV71, a significant pathogen causing hand-foot-mouth disease, is associated with severe neurological complications such as brain stem encephalitis, aseptic meningitis, and acute flaccid paralysis. While the role of mitochondrial dynamics in regulating the replication of numerous viruses is recognized, its specific involvement in EV71 remains unclear. This study aimed to elucidate the role of mitochondrial dynamics in human neuroblastoma SK-N-SH cells during EV71 infection. Utilizing laser confocal microscopy and transmission electron microscopy, we observed that EV71 infection induced mitochondrial elongation and damage to cristae structures, concurrently accelerating mitochondrial movement. Furthermore, we identified the reduction in the expression of dynamin-related protein 1 (Drp1) and optic atrophy protein 1 (Opa1) and the increased expression of Mitofusion 2 (Mfn2) upon EV71 infection. Notably, EV71 directly stimulated the generation of mitochondrial reactive oxygen species (ROS), leading to a decline in mitochondrial membrane potential and ATP levels. Remarkably, the application of melatonin, a potent mitochondrial protector, inhibited EV71 replication by restoring Drp1 expression. These findings collectively indicate that EV71 induces alterations in mitochondrial morphology and dynamics within SK-N-SH cells, potentially impairing mitochondrial function and contributing to nervous system dysfunction. The restoration of proper mitochondrial dynamics may hold promise as a prospective approach to counteract EV71 infection.

Proteomic and phosphoproteomic analysis of responses to enterovirus A71 infection reveals novel targets for antiviral and viral replication.

Hand, foot, and mouth disease (HFMD) is a common infectious disease in infants and children, especially those under five years of age. EV-A71 is a common pathogen that causes HFMD and the primary pathogen leading to severe or fatal HFMD, which is characterized by neurological complications. However, the underlying mechanisms of EV-A71 pathogenesis remain largely unknown. In this report, we used proteomic and phosphorylated proteomic methods to characterize the proteome and phosphoproteome profiles of EV-A71-infected human neuroblastoma SK-N-SH cells. More than 7744 host proteins and 10069 phosphorylation modification sites were successfully quantified. Among them, 974 proteins and 3648 phosphorylation modification sites were regulated significantly during EV-A71 infection. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that EV-A71 altered cell biological processes, including protein synthesis, RNA splicing and metabolism in SK-N-SH cells. Notably, based on the prediction of upregulated kinases during EV-A71 infection, we identified specific kinase inhibitors approved by the FDA, with ceralasertib, bosutinib, flavin mononucleotide, minocycline, pimasertib and acetylcysteine inhibiting EV-A71 infection. Finally, EV-A71 proteins were found to be phosphorylated during infection, with one site (S184 on 3D polymerase) observed to be crucial for viral replication because a S184A mutation knocked out viral replication. The results improve our understanding of the host response to EV-A71 infection of neuroblastoma cells and provide potential targets for developing anti-EV-A71 strategies.

Ubiquitin E3 ligase SPOP is a host negative regulator of enterovirus 71-encoded 2A protease.

IMPORTANCE: EV71 poses a significant health threat to children aged 5 and below. The process of EV71 infection and replication is predominantly influenced by ubiquitination modifications. Our previous findings indicate that EV71 prompts the activation of host deubiquitinating enzymes, thereby impeding the host interferon signaling pathway as a means of evading the immune response. Nevertheless, the precise mechanisms by which the host employs ubiquitination modifications to hinder EV71 infection remain unclear. The present study demonstrated that the nonstructural protein 2Apro, which is encoded by EV71, exhibits ubiquitination and degradation mediated by the host E3 ubiquitin ligase SPOP. In addition, it is the first report, to our knowledge, that SPOP is involved in the host antiviral response.

Evaluation of 3D Human Intestinal Organoids as a Platform for EV-A71 Antiviral Drug Discovery.

Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems and strategies to identify suitable pre-clinical candidates. Organoids represent a new and outstanding opportunity to test antiviral agents in a more physiologically relevant system. However, dedicated studies addressing the validation and direct comparison of organoids versus commonly used cell lines are lacking. Here, we described the use of human small intestinal organoids (HIOs) as a model to study antiviral treatment against human enterovirus 71 (EV-A71) infection and compared this model to EV-A71-infected RD cells. We used reference antiviral compounds such as enviroxime, rupintrivir, and 2'-C-methylcytidine (2'CMC) to assess their effects on cell viability, virus-induced cytopathic effect, and viral RNA yield in EV-A71-infected HIOs and cell line. The results indicated a difference in the activity of the tested compounds between the two models, with HIOs being more sensitive to infection and drug treatment. In conclusion, the outcome reveals the value added by using the organoid model in virus and antiviral studies.

Global quantitative proteomic analysis profiles of host protein expression in response to enterovirus A71 infection in bronchial epithelial cells based on tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS uncovers the key role of proteasome in virus replication.

Enterovirus A71 (EV-A71) is a neurotropic human pathogen which mainly caused hand, foot and mouth disease (HFMD) mostly in children under 5 years-old. Generally, EV-A71-associated HFMD is a relatively self-limiting febrile disease, but there will still be a small percentage of patients with rapid disease progression and severe neurological complications. To date, the underlying mechanism of EV-A71 inducing pathological injury of central nervous system (CNS) remains largely unclear. It has been investigated and discussed the changes of mRNA, miRNA and circRNA expression profile during infection by EV-A71 in our previous studies. However, these studies were only analyzed at the RNA level, not at the protein level. It's the protein levels that ultimately do the work in the body. Here, to address this, we performed a tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS approach to quantitatively identify cellular proteome changes at 24 h post-infection (hpi) in EV-A71-infected 16HBE cells. In total, 6615 proteins were identified by using TMT coupled with LC-MS/MS in this study. In the EV-A71- and mock-infected groups, 210 differentially expressed proteins were found, including 86 upregulated and 124 downregulated proteins, at 24 hpi. To ensure the validity and reliability of the proteomics data, 3 randomly selected proteins were verified by Western blot and Immunofluorescence analysis, and the results were consistent with the TMT results. Subsequently, functional enrichment analysis indicated that the up-regulated and down-regulated proteins were individually involved in various biological processes and signaling pathways, including metabolic process, AMPK signaling pathway, Neurotrophin signaling pathway, Viral myocarditis, GABAergic synapse, and so on. Moreover, among these enriched functional analysis, the "Proteasome" pathway was up-regulated, which has caught our attention. Inhibition of proteasome was found to obviously suppress the EV-A71 replication. Finally, further in-depth analysis revealed that these differentially expressed proteins contained distinct domains and localized in different subcellular components. Taken together, our data provided a comprehensive view of host cell response to EV-A71 and identified host proteins may lead to better understanding of the pathogenic mechanisms and host responses to EV-A71 infection, and also to the identification of new therapeutic targets for EV-A71 infection.

Identification of a novel acylthiourea-based potent broad-spectrum inhibitor for enterovirus 3D polymerase in vitro and in vivo.

Enterovirus infections have become a serious public health threat to young children, leading to hand-foot-and-mouth disease and more severe nervous system diseases. Due to the lack of licensed anti enterovirus drugs, we reported herein that a Tenovin-1 analog, acylthiourea-based 4-(tert-butyl)-N-((4-(4-(tert-butyl)benzamido)phenyl)carbamothioyl) benzamide (AcTU), displayed low nanomolar anti-EV-A71 activity with an EC50 of 1.0 nM in RD cells. Moreover, AcTU exhibited nanomolar to picomolar inhibitory activity against a series of enteroviruses including EV-D68, CV-A21, CV-A16 and CV-B1 (EC50 = 0.75-17.15 nM). Mechanistic studies indicated that AcTU inhibited enterovirus proliferation by targeting 3D polymerase. In addition, AcTU displayed moderate pharmacokinetic properties in rats (F = 7.4%, T1/2 = 3.26 h), and in vivo protection studies demonstrated that AcTU orally administered at 0.6 mg/kg/d was highly protective against lethal EV-A71 challenge in mice, potentially reducing mortality from 100% to 20% as well as alleviating symptoms. These results suggested that AcTU could be a potent clinical candidate for the treatment of enterovirus infections.

Mechanosensitive extrusion of Enterovirus A71-infected cells from colonic organoids.

Enterovirus A71 causes severe disease upon systemic infection, sometimes leading to life-threatening neurological dysfunction. However, in most cases infection is asymptomatic and limited to the gastrointestinal tract, where virus is amplified for transmission. Picornaviruses have previously been shown to exit infected cells via either cell lysis or secretion of vesicles. Here we report that entire Enterovirus A71-infected cells are specifically extruded from the apical surface of differentiated human colon organoids, as observed by confocal microscopy. Differential sensitivity to chemical and peptide inhibitors demonstrated that extrusion of virus-infected cells is dependent on force sensing via mechanosensitive ion channels rather than apoptotic cell death. When isolated and used as inoculum, intact virus-containing extruded cells can initiate new infections. In contrast, when mechanical force sensing is inhibited, large amounts of free virus are released. Thus, extrusion of live, virus-infected cells from intact epithelial tissue is likely to benefit both the integrity of host tissues and the protected spread of this faecal-oral pathogen within and between hosts.

Inhibitory effects and mechanisms of proanthocyanidins against enterovirus 71 infection.

Proanthocyanidins (PC), a natural flavonoid compound, was reported to possess a variety of pharmacological activities such as anti-tumor and anti-viral effects. In this study, the anti-Enterovirus 71 (EV71) activities and mechanisms of PC were investigated both in vitro and in vivo. The results showed that PC possessed anti-EV71 activities in different cell lines with low toxicity. PC can block both the adsorption and entry processes of EV71 via directly binding to virus VP1 protein. PC may competitively interfere with the binding of VP1 to its receptor SCARB2. PC can also regulate three different MAPK signaling pathways to reduce EV71 infection and attenuate virus induced inflammatory responses. Importantly, intramuscular therapy of EV71-infected mice with PC markedly improved their survival and attenuated the severe clinical symptoms. Therefore, the natural compound PC has potential to be developed into a novel anti-EV71 agent targeting viral VP1 protein and MAPK pathways.

Suppression of innate and acquired immunity in severe hand foot and mouth disease caused by EV71 infections in children.

Hand, foot, and mouth disease (HFMD) is a common children infectious disease caused by human enteroviruses. Most of the cases have minimal symptoms, however, some patients may develop serious neurological, cardiac complications, or even death. The pathological mechanism leading to severe HFMD is not clearly understood, and the immunological status of the individual patient may play an important role. Transcriptomes of peripheral blood mononuclear cells from EV71-infected patients (n = 45) and healthy controls (n = 36) were examined. Immune pathways were up-regulated in patients with mild disease symptoms (n = 11, M) compared to the healthy controls (n = 36, H), demonstrating an effective anti-viral response upon EV71 infection. However, in patients with severe symptoms (n = 23, S) as well as severe patients following treatment (n = 11, A), their innate and acquired immune pathways were down-regulated, indicating a global immunity suppression. Such immune suppression characteristics could thus provide an opportunity for early EV-71 infection prognosis prediction. Based on our cohort, an SVM model using RNA-seq expression levels of five genes (MCL1, ZBTB37, PLEKHM1P, IFNAR2 and YEATS2) was developed and achieved a high ROC-AUC (91·3%) in predicting severe HFMD. Meanwhile, qPCR fold-changes method was performed based three genes (MCL1, IFNAR2 and YEATS2) on additional cohort. This qPCR method achieved a ROC-AUC of 78.6% in predicting severe HFMD, which the patients could be distinguished in 2-3 h. Therefore, our models demonstrate the possibility of HFMD severity prediction based on the selected biomarkers that predict severe HFMD effectively.

TRAF3 activates STING-mediated suppression of EV-A71 and target of viral evasion.

Innate immunity represents one of the main host responses to viral infection.1-3 STING (Stimulator of interferon genes), a crucial immune adapter functioning in host cells, mediates cGAS (Cyclic GMP-AMP Synthase) sensing of exogenous and endogenous DNA fragments and generates innate immune responses.4 Whether STING activation was involved in infection and replication of enterovirus remains largely unknown. In the present study, we discovered that human enterovirus A71 (EV-A71) infection triggered STING activation in a cGAS dependent manner. EV-A71 infection caused mitochondrial damage and the discharge of mitochondrial DNA into the cytosol of infected cells. However, during EV-A71 infection, cGAS-STING activation was attenuated. EV-A71 proteins were screened and the viral protease 2Apro had the greatest capacity to inhibit cGAS-STING activation. We identified TRAF3 as an important factor during STING activation and as a target of 2Apro. Supplement of TRAF3 rescued cGAS-STING activation suppression by 2Apro. TRAF3 supported STING activation mediated TBK1 phosphorylation. Moreover, we found that 2Apro protease activity was essential for inhibiting STING activation. Furthermore, EV-D68 and CV-A16 infection also triggered STING activation. The viral protease 2Apro from EV-D68 and CV-A16 also had the ability to inhibit STING activation. As STING activation prior to EV-A71 infection generated cellular resistance to EV-A71 replication, blocking EV-A71-mediated STING suppression represents a new anti-viral target.

Murine models of neonatal susceptibility to a clinical strain of enterovirus A71.

Enterovirus A71 (EV-A71) is neurotropic and one of the primary enteric pathogens responsible for severe central nervous system infection in infants and young children. Neonatal mice are ideal models for studying the pathogenesis of infection caused by EV-A71. In this study, we assessed the susceptibility of neonatal BALB/c, C57BL/6, ICR, Kunming, and NIH mice to a clinically isolated EV-A71 strain. One-day-old mice were challenged with a clinical isolate of EV-A71 via intraperitoneal injection, then observed for 13 days for mortality, body-weight changes, and limb paralysis. RT-qPCR was performed to quantify viral RNA in the brain, spinal cord, skeletal muscle, and lungs of BALB/c and C57BL/6 mice. The expression of murine scavenger receptor class B member 2 (mSCARB2) was measured by western blotting. Finally, lesions were assessed by histological examination. We found that neonatal BALB/c and C57BL/6 mice were both susceptible to EV-A71, leading to decreased survival rate, greater body weight loss, and prominent hind-limb paralysis. Tissue viral loads of C57BL/6J mice were markedly higher than those of BALB/c mice, indicating that EV-A71 replicated more efficiently in C57BL/6 mice. Increased expression of mSCARB2 was observed 5 days after infection in C57BL/6 mice, which coincided with the peak in EV-A71 replication. Histological examination indicated that infection caused obvious pathogenic lesions. In conclusion, C57BL/6 are most susceptible to infection caused by EV-A71 and can be used as a model for studying its pathogenesis and test therapeutic options.

Temporal Proteomic and Phosphoproteomic Analysis of EV-A71-Infected Human Cells.

Human enterovirus A71 (EV-A71), a member of the Picornaviridae family, is one of the main etiological viruses that lead to hand, foot, and mouth disease (HFMD). We utilized a multiplex tandem mass tag-based quantitative proteomic technique to monitor the alternation of the whole cell proteome and phosphoproteome of human rhabdomyosarcoma cells over the course of EV-A71 infection. We successfully quantified more than 7000 host proteins and 17,000 phosphosites, of which 80 proteins and nearly 1700 phosphosites were significantly regulated upon viral infection. We found that Myc proto-oncogene protein level decreased significantly, benefiting EV-A71 replication. Multiple signaling pathways were regulated in phosphorylation events that converge for protein translation, cell cycle control, and cell survival. Numerous host factors targeted by virus proteins are phosphoproteins. These factors are involved in host translational initiation, unfolded protein response, endoplasmic reticulum stress, and stress granule formation, and their phosphorylation may play key roles in the virus life cycle. Notably, we identified three conserved phosphorylation sites on viral polyproteins that have not been previously reported. Our study provides valuable resources for a systematic understanding of the interaction between the host cells and the EV-A71 at the protein and the post-translational level.

Retrograde axonal transport of poliovirus and EV71 in motor neurons.

Poliovirus (PV) can spread through neural pathway to the central nervous system and replicates in motor neurons, which leads to poliomyelitis. Enterovirus 71 (EV71), which is closely related to PV, is one of the causative agents of hand-foot-and-mouth disease and can cause severe neurological diseases similar to poliomyelitis. Since PV is similar to EV71 in its motor neurotoxicity, we tried to understand if the results obtained with PV are of general applicability to EV71 and other viruses with similar characteristics. Using microfluidic devices, we demonstrated that both PV capsid and the PV genome undergo axonal retrograde transport with human PV receptor (hPVR), and the transported virus replicated in the soma of hPVR-expressing motor neurons. Similar to PV in hPVR-transgenic (Tg) mice, neural pathway ensuring spreading of EV71 has been shown in adult human scavenger receptor class B, member 2 (hSCARB2)-Tg mice. We have validated this finding in microfluidic devices by showing that EV71 is retrogradely transported together with hSCARB2 to the cell body where it replicates in an hSCARB2-dependent manner.

Host neuronal PRSS3 interacts with enterovirus A71 3A protein and its role in viral replication.

Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease associated with neurological complications in young children. Currently, there is no specific treatment for EV-A71 infection due to the inadequate information on viral biology and neuropathogenesis. Among enteroviruses, nonstructural 3A protein mediates the formation of replication organelles which plays a major role in viral RNA synthesis and assembly. Although enteroviral 3A proteins have been intensively studied, the data on EV-A71 3A, especially in neuronal cells, are still limited. In this study, PRSS3 (mesotrypsinogen, also known as brain trypsinogen) was identified as EV-A71 3A-interacting counterpart from the transfected human neuroblastoma SH-SY5Y cells by pull-down assay and liquid chromatography tandem mass spectrometry. It was confirmed that PRSS3 variant 3 derived from human SH-SY5Y cells had the physical interaction with EV-A71 3A. Importantly, the role of PRSS3 in EV-A71 replication was verified by overexpression and siRNA-mediated gene silencing approaches. The detailed mechanism of the PRSS3 involved in EV-A71 replication and neuropathogenesis warrants further experimental elucidation. In conclusion, this study has discovered a novel EV-A71 3A interacting protein that offers the opportunity to study the neuropathogenesis of the infection which paves the way for developing a specific and effective treatment for the disease.