Enterovirus D68 3C protease antagonizes type I interferon signaling by cleaving signal transducer and activator of transcription 1.

Following the successful control of poliovirus, the re-emergence of respiratory enterovirus D68 (EV-D68), a prominent non-polio enterovirus, has become a serious public health concern worldwide. Host innate immune responses are the primary defense against EV-D68 invasion; however, the mechanism underlying viral evasion of the antiviral activity of interferons (IFN) remains unclear. In this study, we found that EV-D68 inhibited type I IFN signaling by cleaving signal transducer and activator of transcription 1 (STAT1), a crucial factor in cellular responses to interferons and other cytokines. We observed that the prototype and circulating EV-D68 strains conserved their ability to induce STAT1 cleavage and attenuate IFN signal transduction. Further investigation revealed that EV-D68 3C protease cleaves STAT1 at the 131Q residue. Interestingly, not all enterovirus-encoded 3C proteases exhibited this ability. EV-D68 and poliovirus 3C proteases efficiently induced STAT1 cleavage; whereas, 3C proteases from EV-A71, coxsackievirus A16, and echoviruses did not. STAT1 cleavage also abolished the nuclear translocation capacity of STAT1 in response to IFN stimulation to activate downstream signaling elements. Overall, these results suggest that STAT1, targeted by viral protease 3C, is utilized by EV-D68 to subvert the host's innate immune response.IMPORTANCEEnterovirus D68 (EV-D68) has significantly transformed over the past decade, evolving from a rare pathogen to a potential pandemic pathogen. The interferon (IFN) signaling pathway is an important defense mechanism and therapeutic target for the host to resist viral invasion. Previous studies have reported that the EV-D68 virus blocks or weakens immune recognition and IFN production in host cells through diverse strategies; however, the mechanisms of EV-D68 resistance to IFN signaling have not been fully elucidated. Our study revealed that EV-D68 relies on its own encoded protease, 3C, to directly cleave signal transducer and activator of transcription 1 (STAT1), a pivotal transduction component in the IFN signaling pathway, disrupting the IFN-mediated antiviral response. Previous studies on human enteroviruses have not documented direct cleavage of the STAT1 protein to evade cellular immune defenses. However, not all enteroviral 3C proteins can cleave STAT1. These findings highlight the diverse evolutionary strategies different human enteroviruses employ to evade host immunity.

Licochalcone A regulates viral IRES activity to inhibit enterovirus replication.

Enterovirus D68 (EV-D68), belonging to the genus Enterovirus of the Picornavirus family, is an emerging pathogen that can cause neurological and respiratory diseases in children. However, there is little understanding of the pathogenesis of EV-D68, and no effective vaccine or drug for the prevention or treatment of the diseases caused by this virus is available. Autophagy is a cellular process that targets cytoplasmic proteins or organelles to the lysosomes for degradation. Enteroviruses strategically harness the host autophagy pathway to facilitate the completion of their life cycle. Therefore, we selected an autophagy compound library to screen for autophagy-related compounds that may affect viral growth. By using the neutralization screening assay, we identified a compound, 'licochalcone A' that significantly inhibited EV-D68 replication. To investigate the mechanism by which licochalcone A inhibits EV-D68 replication and to identify the viral life cycle stage it inhibits, the time-of-addition, viral attachment, viral entry, and dual-luciferase reporter assays were performed. The results of the time-of-addition assay showed that licochalcone A, a characteristic chalcone found in liquorice roots and widely used in traditional Chinese medicine, inhibits EV-D68 replication during the early stages of the viral life cycle, while those of the dual-luciferase reporter assay showed that licochalcone A does not regulate viral attachment and entry, but inhibits EV-D68 IRES-dependent translation. Licochalcone A also inhibited enterovirus A71 and coxsackievirus B3 but did not significantly inhibit dengue virus 2 or human coronavirus 229E replication. Licochalcone A regulates IRES translation to inhibit EV-D68 viral replication.

In vitro and in vivo models for the study of EV-D68 infection.

Enterovirus D68 (EV-D68) is one of hundreds of non-polio enteroviruses that typically cause cold-like respiratory illness. The first EV-D68 outbreak in the United States in 2014 aroused widespread concern among the public and health authorities. The infection was found to be associated with increased surveillance of acute flaccid myelitis, a neurological condition that causes limb paralysis in conjunction with spinal cord inflammation. In vitro studies utilising two-dimensional (2D) and three-dimensional (3D) culture systems have been employed to elucidate the pathogenic mechanism of EV-D68. Various animal models have also been developed to investigate viral tropism and distribution, pathogenesis, and immune responses during EV-D68 infection. EV-D68 infections have primarily been investigated in respiratory, intestinal and neural cell lines/tissues, as well as in small-size immunocompetent rodent models that were limited to a young age. Some studies have implemented strategies to overcome the barriers by using immunodeficient mice or virus adaptation. Although the existing models may not fully recapitulate both respiratory and neurological disease observed in human EV-D68 infection, they have been valuable for studying pathogenesis and evaluating potential vaccine or therapeutic candidates. In this review, we summarise the methodologies and findings from each experimental model and discuss their applications and limitations.

SIRT-1 is required for release of enveloped enteroviruses.

Enterovirus D68 (EV-D68) is a re-emerging enterovirus that causes acute respiratory illness in infants and has recently been linked to Acute Flaccid Myelitis. Here, we show that the histone deacetylase, SIRT-1, is essential for autophagy and EV-D68 infection. Knockdown of SIRT-1 inhibits autophagy and reduces EV-D68 extracellular titers. The proviral activity of SIRT-1 does not require its deacetylase activity or functional autophagy. SIRT-1's proviral activity is, we demonstrate, mediated through the repression of endoplasmic reticulum stress (ER stress). Inducing ER stress through thapsigargin treatment or SERCA2A knockdown in SIRT-1 knockdown cells had no additional effect on EV-D68 extracellular titers. Knockdown of SIRT-1 also decreases poliovirus and SARS-CoV-2 titers but not coxsackievirus B3. In non-lytic conditions, EV-D68 is primarily released in an enveloped form, and SIRT-1 is required for this process. Our data show that SIRT-1, through its translocation to the cytosol, is critical to promote the release of enveloped EV-D68 viral particles.

Enterovirus D68 VP3 Targets the Interferon Regulatory Factor 7 To Inhibit Type I Interferon Response.

Enterovirus D68 (EV-D68) is a globally emerging pathogen causing severe respiratory illnesses mainly in children. The protease from EV-D68 could impair type I interferon (IFN-I) production. However, the role of the EV-D68 structural protein in antagonizing host antiviral responses remains largely unknown. We showed that the EV-D68 structural protein VP3 interacted with IFN regulatory factor 7 (IRF7), and this interaction suppressed the phosphorylation and nuclear translocation of IRF7 and then repressed the transcription of IFN. Furthermore, VP3 inhibited the TNF receptor associated factor 6 (TRAF6)-induced ubiquitination of IRF7 by competitive interaction with IRF7. IRF7Δ305-503 showed much weaker interaction ability to VP3, and VP3Δ41-50 performed weaker interaction ability with IRF7. The VP3 from enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) was also found to interact with the IRF7 protein. These results indicate that the enterovirus structural protein VP3 plays a pivotal role in subverting host innate immune responses and may be a potential target for antiviral drug research. IMPORTANCE EV-D68 is a globally emerging pathogen that causes severe respiratory illnesses. Here, we report that EV-D68 inhibits innate immune responses by targeting IRF7. Further investigations revealed that the structural protein VP3 inhibited the TRAF6-induced ubiquitination of IRF7 by competitive interaction with IRF7. These results indicate that the control of IRF7 by VP3 may be a mechanism by which EV-D68 represses IFN-I production.

Telaprevir Treatment Reduces Paralysis in a Mouse Model of Enterovirus D68 Acute Flaccid Myelitis.

In 2014, 2016, and 2018, the United States experienced unprecedented spikes in pediatric cases of acute flaccid myelitis (AFM), which is a poliomyelitis-like paralytic illness. Accumulating clinical, immunological, and epidemiological evidence has identified enterovirus D68 (EV-D68) as a major causative agent of these biennial AFM outbreaks. There are currently no available FDA-approved antivirals that are effective against EV-D68, and the treatment for EV-D68-associated AFM is primarily supportive. Telaprevir is an food and drug administration (FDA)-approved protease inhibitor that irreversibly binds the EV-D68 2A protease and inhibits EV-D68 replication in vitro. Here, we utilize a murine model of EV-D68 associated AFM to show that early telaprevir treatment improves paralysis outcomes in Swiss Webster (SW) mice. Telaprevir reduces both viral titer and apoptotic activity in both muscles and spinal cords at early disease time points, which results in improved AFM outcomes in infected mice. Following intramuscular inoculation in mice, EV-D68 infection results in a stereotypic pattern of weakness that is reflected by the loss of the innervating motor neuron population, in sequential order, of the ipsilateral (injected) hindlimb, the contralateral hindlimb, and then the forelimbs. Telaprevir treatment preserved motor neuron populations and reduced weakness in limbs beyond the injected hindlimb. The effects of telaprevir were not seen when the treatment was delayed, and toxicity limited doses beyond 35 mg/kg. These studies are a proof of principle, provide the first evidence of benefit of an FDA-approved antiviral drug with which to treat AFM, and emphasize both the need to develop better tolerated therapies that remain efficacious when administered after viral infections and the development of clinical symptoms. IMPORTANCE Recent outbreaks of EV-D68 in 2014, 2016, and 2018 have resulted in over 600 cases of a paralytic illness that is known as AFM. AFM is a predominantly pediatric disease with no FDA-approved treatment, and many patients show minimal recovery from limb weakness. Telaprevir is an FDA-approved antiviral that has been shown to inhibit EV-D68 in vitro. Here, we demonstrate that a telaprevir treatment that is given concurrently with an EV-D68 infection improves AFM outcomes in mice by reducing apoptosis and viral titers at early time points. Telaprevir also protected motor neurons and improved paralysis outcomes in limbs beyond the site of viral inoculation. This study improves understanding of EV-D68 pathogenesis in the mouse model of AFM. This study serves as a proof of principle for the first FDA-approved drug that has been shown to improve AFM outcomes and have in vivo efficacy against EV-D68 as well as underlines the importance of the continued development of EV-D68 antivirals.

Animal Models of Enterovirus D68 Infection and Disease.

Human enterovirus D68 (EV-D68) is a globally reemerging respiratory pathogen that is associated with the development of acute flaccid myelitis (AFM) in children. Currently, there are no approved vaccines or treatments for EV-D68 infection, and there is a paucity of data related to the virus and host-specific factors that predict disease severity and progression to the neurologic syndrome. EV-D68 infection of various animal models has served as an important platform for characterization and comparison of disease pathogenesis between historic and contemporary isolates. Still, there are significant gaps in our knowledge of EV-D68 pathogenesis that constrain the development and evaluation of targeted vaccines and antiviral therapies. Continued refinement and characterization of animal models that faithfully reproduce key elements of EV-D68 infection and disease is essential for ensuring public health preparedness for future EV-D68 outbreaks.

Comparison of tissue tropism and host response to enteric and respiratory enteroviruses.

Enteroviruses (EVs) are among the most prevalent viruses worldwide. They are characterized by a high genetic and phenotypic diversity, being able to cause a plethora of symptoms. EV-D68, a respiratory EV, and EV-D94, an enteric EV, represent an interesting paradigm of EV tropism heterogeneity. They belong to the same species, but display distinct phenotypic characteristics and in vivo tropism. Here, we used these two viruses as well as relevant 3D respiratory, intestinal and neural tissue culture models, to highlight key distinctive features of enteric and respiratory EVs. We emphasize the critical role of temperature in restricting EV-D68 tissue tropism. Using transcriptomic analysis, we underscore fundamental differences between intestinal and respiratory tissues, both in the steady-state and in response to infection. Intestinal tissues present higher cell proliferation rate and are more immunotolerant than respiratory tissues. Importantly, we highlight the different strategies applied by EV-D94 and EV-D68 towards the host antiviral response of intestinal and respiratory tissues. EV-D68 strongly activates antiviral pathways while EV-D94, on the contrary, barely induces any host defense mechanisms. In summary, our study provides an insightful characterization of the differential pathogenesis of EV-D68 and EV-D94 and the interplay with their main target tissues.

Neutralizing Antibody Given after Paralysis Onset Reduces the Severity of Paralysis Compared to Nonspecific Antibody-Treated Controls in a Mouse Model of EV-D68-Associated Acute Flaccid Myelitis.

Enterovirus D68 (EV-D68) can cause mild to severe respiratory illness and is associated with a poliomyelitis-like paralytic syndrome called acute flaccid myelitis (AFM). Most cases of EV-D68-associated AFM occur in young children who are brought to the clinic after the onset of neurologic symptoms. There are currently no known antiviral therapies for AFM, and it is unknown whether antiviral treatments will be effective if initiated after the onset of neurologic symptoms (when patients are likely to present for medical care). We developed a "clinical treatment model" for AFM, in which individual EV-D68-infected mice are tracked and treated with an EV-D68-specific human-mouse chimeric monoclonal antibody after the onset of moderate paralysis. Mice treated with antibody had significantly better paralysis outcomes compared to nonspecific antibody-treated controls. Treatment did not reverse paralysis that was present at the time of treatment initiation but did slow the further loss of function, including progression of weakness to other limbs, as well as reducing viral titer in the muscle and spinal cords of treated animals. We observed the greatest therapeutic effect in EV-D68 isolates which were neutralized by low concentrations of antibody, and diminishing therapeutic effect in EV-D68 isolates which required higher doses of antibody for neutralization. This work supports the use of virus-specific immunotherapy for the treatment of AFM. It also suggests that patients who present with AFM should be treated as soon as possible if recent infection with EV-D68 is suspected.

Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes.

Enterovirus D68 (EV-D68) is an emerging pathogen which causes respiratory disease and is associated with an acute flaccid myelitis that predominately affects children. EV-D68 can infect motor neurons, causing cell death and a loss of motor control leading to flaccid paralysis. However, it remains unknown how viral particles gain entry into the central nervous system (CNS). Here, we show that three distinct densities of EV-D68 particle can be isolated from infected muscle and neural cell lines (RD and SH-SY5Y) using high-speed density centrifugation to separate cell supernatant. The lowest-density peak is composed of viral particles, which have adhered to the exterior surface of a small extracellular vesicle called an exosome. Analysis of prototypic (historic) and contemporary EV-D68 strains suggests that binding to exosomes is a ubiquitous characteristic of EV-D68. We further show that interaction with exosomes increases viral infectivity in a neural cell line. Analysis of the two higher-density peaks, which are not associated with exosomes, revealed that a significant amount of viral titer in the modern (2014) EV-D68 strains is found at 1.20 g/cm3, whereas this density has a very low viral titer in the prototypic Fermon strain. IMPORTANCE Despite the strong causal link between enterovirus D68 (EV-D68) and acute flaccid myelitis (AFM), it remains unclear how EV-D68 gains entry into the central nervous system and what receptors enable it to infect motor neurons. We show that EV-D68 particles can adhere to exosomes, placing EV-D68 among a handful of other picornaviruses which are known to interact with extracellular vesicles. Uptake and infection of permissive cells by virally contaminated exosomes would have major implications in the search for the EV-D68 receptor, as well as providing a possible route for viral entry into motor neurons. This work identifies a novel cellular entry route for EV-D68 and may facilitate the identification of genetic risk factors for development of AFM.

Host Restriction Factor A3G Inhibits the Replication of Enterovirus D68 by Competitively Binding the 5' Untranslated Region with PCBP1.

The host restriction factor APOBEC3G (A3G) inhibits an extensive variety of viruses, including retroviruses, DNA viruses, and RNA viruses. Our study shows that A3G inhibits enterovirus 71 (EV71) and coxsackievirus A16 (CA16) via competitively binding the 5' untranslated region (UTR) with the host protein poly(C)-binding protein 1 (PCBP1), which is required for the replication of multiple EVs. However, whether A3G inhibits other EVs in addition to EV71 and CA16 has not been investigated. Here, we demonstrate that A3G could inhibit the replication of EVD68, which requires PCBP1 for its replication, but not CA6, which does not require PCBP1 for replication. Further investigation revealed that the nucleic-acid-binding activity of A3G is required for EVD68 restriction, similar to the mechanism presented for EV71 restriction. Mechanistically, A3G competitively binds to the cloverleaf (1 to 123 nucleotides [nt]) and the stem-loop IV (234 to 446 nt) domains of the EVD68 5' UTR with PCBP1, thereby inhibiting the 5' UTR activity of EVD68; by contrast, A3G does not interact with CA6 5' UTR, resulting in no effect on CA6 replication. Moreover, the nonstructural protein 2C, encoded by EVD68, overcomes A3G suppression by inducing A3G degradation via the autophagy-lysosome pathway. Our findings revealed that A3G might have broad-spectrum antiviral activity against multiple EVs through this general mechanism, and they might provide important information for the development of an anti-EV strategy. IMPORTANCE As the two major pathogens causing hand, foot, and mouth disease (HFMD), enterovirus 71 (EV71) and coxsackievirus A16 (CA16) attract a lot of attention for the study of their pathogenesis, their involvement with cellular proteins, and so on. However, other EVs such as CA6 and EVD68 constantly occur sporadically or have spread worldwide in recent years. Therefore, more information related to these EVs is needed in order to develop a broad-spectrum anti-EV inhibitor. In this study, we first reveal that the protein poly(C)-binding protein 1 (PCBP1), involved in PV- and EV71 virus replication, is also required for the replication of EVD68, but not for the replication of CA6. Next, we found that the host-restriction factor A3G specifically inhibits the replication of EVD68, but not the replication of CA6, by competitively binding to the 5' UTR of EVD68 along with PCBP1. Our findings broaden knowledge related to EV replication and the interplay between EVs and host factors.

Functional and structural characterization of a two-MAb cocktail for delayed treatment of enterovirus D68 infections.

Enterovirus D68 (EV-D68) is an emerging pathogen associated with respiratory diseases and/or acute flaccid myelitis. Here, two MAbs, 2H12 and 8F12, raised against EV-D68 virus-like particle (VLP), show distinct preference in binding VLP and virion and in neutralizing different EV-D68 strains. A combination of 2H12 and 8F12 exhibits balanced and potent neutralization effects and confers broader protection in mice than single MAbs when given at onset of symptoms. Cryo-EM structures of EV-D68 virion complexed with 2H12 or 8F12 show that both antibodies bind to the canyon region of the virion, creating steric hindrance for sialic acid receptor binding. Additionally, 2H12 binding can impair virion integrity and trigger premature viral uncoating. We also capture an uncoating intermediate induced by 2H12 binding, not previously described for picornaviruses. Our study elucidates the structural basis and neutralizing mechanisms of the 2H12 and 8F12 MAbs and supports further development of the 2H12/8F12 cocktail as a broad-spectrum therapeutic agent against EV-D68 infections in humans.

Epidemiological dynamics of enterovirus D68 in the United States and implications for acute flaccid myelitis.

Acute flaccid myelitis (AFM) recently emerged in the United States as a rare but serious neurological condition since 2012. Enterovirus D68 (EV-D68) is thought to be a main causative agent, but limited surveillance of EV-D68 in the United States has hampered the ability to assess their causal relationship. Using surveillance data from the BioFire Syndromic Trends epidemiology network in the United States from January 2014 to September 2019, we characterized the epidemiological dynamics of EV-D68 and found latitudinal gradient in the mean timing of EV-D68 cases, which are likely climate driven. We also demonstrated a strong spatiotemporal association of EV-D68 with AFM. Mathematical modeling suggested that the recent dominant biennial cycles of EV-D68 dynamics may not be stable. Nonetheless, we predicted that a major EV-D68 outbreak, and hence an AFM outbreak, would have still been possible in 2020 under normal epidemiological conditions. Nonpharmaceutical intervention efforts due to the ongoing COVID-19 pandemic are likely to have reduced the sizes of EV-D68 and AFM outbreaks in 2020, illustrating the broader epidemiological impact of the pandemic.

Regulation of the proteostasis network during enterovirus infection: A feedforward mechanism for EV-A71 and EV-D68.

The proteostasis network guarantees successful protein synthesis, folding, transportation, and degradation. Mounting evidence has revealed that this network maintains proteome integrity and is linked to cellular physiology, pathology, and virus infection. Human enterovirus A71 (EV-A71) and EV-D68 are suspected causative agents of acute flaccid myelitis, a severe poliomyelitis-like neurologic syndrome with no known cure. In this context, further clarification of the molecular mechanisms underlying EV-A71 and EV-D68 infection is paramount. Here, we summarize the components of the proteostasis network that are intercepted by EV-A71 and EV-D68, as well as antivirals that target this network and may help develop improved antiviral drugs.

Enhanced Enterovirus D68 Replication in Neuroblastoma Cells Is Associated with a Cell Culture-Adaptive Amino Acid Substitution in VP1.

Since its emergence in the United States in 2014, enterovirus D68 (EV-D68) has been and is associated with severe respiratory diseases and acute flaccid myelitis. Even though EV-D68 has been shown to replicate in different neuronal cells in vitro, it is currently poorly understood which viral factors contribute to the ability to replicate efficiently in cells of the central nervous system and whether this feature is a clade-specific feature. Here, we determined the replication kinetics of clinical EV-D68 isolates from (sub)clades A, B1, B2, B3, and D1 in human neuroblastoma cells (SK-N-SH). Subsequently, we compared sequences to identify viral factors associated with increased viral replication. All clinical isolates replicated in SK-N-SH cells, although there was a large difference in efficiency. Efficient replication of clinical isolates was associated with an amino acid substitution at position 271 of VP1 (E271K), which was acquired during virus propagation in vitro Recognition of heparan sulfate in addition to sialic acids was associated with increased attachment, infection, and replication. Removal of heparan sulfate resulted in a decrease in attachment, internalization, and replication of viruses with E271K. Taken together, our study suggests that the replication kinetics of EV-D68 isolates in SK-N-SH cells is not a clade-specific feature. However, recognition of heparan sulfate as an additional receptor had a large effect on phenotypic characteristics in vitro. These observations emphasize the need to compare sequences from virus stocks with clinical isolates in order to retrieve phenotypic characteristics from original virus isolates.IMPORTANCE Enterovirus D68 (EV-D68) causes mild to severe respiratory disease and is associated with acute flaccid myelitis since 2014. Currently, the understanding of the ability of EV-D68 to replicate in the central nervous system (CNS), and whether it is associated with a specific clade of EV-D68 viruses or specific viral factors, is lacking. Comparing different EV-D68 clades did not reveal clade-specific phenotypic characteristics. However, we did show that viruses which acquired a cell culture-adapted amino acid substitution in VP1 (E271K) recognized heparan sulfate as an additional receptor. Recognition of heparan sulfate resulted in an increase in attachment, infection, and replication in neuroblastoma cells compared with viruses without this specific amino acid substitution. The ability of EV-D68 viruses to acquire cell culture-adaptive substitutions which have a large effect in experimental settings emphasizes the need to sequence virus stocks.

Mapping Attenuation Determinants in Enterovirus-D68.

Enterovirus (EV)-D68 has been associated with epidemics in the United Sates in 2014, 2016 and 2018. This study aims to identify potential viral virulence determinants. We found that neonatal type I interferon receptor knockout mice are susceptible to EV-D68 infection via intraperitoneal inoculation and were able to recapitulate the paralysis process observed in human disease. Among the EV-D68 strains tested, strain US/MO-14-18949 caused no observable disease in this mouse model, whereas the other strains caused paralysis and death. Sequence analysis revealed several conserved genetic changes among these virus strains: nucleotide positions 107 and 648 in the 5'-untranslated region (UTR); amino acid position 88 in VP3; 1, 148, 282 and 283 in VP1; 22 in 2A; 47 in 3A. A series of chimeric and point-mutated infectious clones were constructed to identify viral elements responsible for the distinct virulence. A single amino acid change from isoleucine to valine at position 88 in VP3 attenuated neurovirulence by reducing virus replication in the brain and spinal cord of infected mice.

Receptor-Binding Assays of Enterovirus D68.

Human enterovirus D68 (EV-D68) is a causative agent for acute respiratory infections and potentially central nervous system illnesses with increasing epidemiological significance. Recent studies have highlighted the role of sialic acids as a functional receptor for EV-D68 in vitro. However, further investigations are required to reveal its significance in actual infections in human.

Synthesis and antiviral effect of novel fluoxetine analogues as enterovirus 2C inhibitors.

Enteroviruses (EV) are a group of positive-strand RNA (+RNA) viruses that include many important human pathogens (e.g. poliovirus, coxsackievirus, echovirus, numbered enteroviruses and rhinoviruses). Fluoxetine was identified in drug repurposing screens as potent inhibitor of enterovirus B and enterovirus D replication. In this paper we are reporting the synthesis and the antiviral effect of a series of fluoxetine analogues. The results obtained offer a preliminary insight into the structure-activity relationship of its chemical scaffold and confirm the importance of the chiral configuration. We identified a racemic fluoxetine analogue, 2b, which showed a similar antiviral activity compared to (S)-fluoxetine. Investigating the stereochemistry of 2b revealed that the S-enantiomer exerts potent antiviral activity and increased the antiviral spectrum compared to the racemic mixture of 2b. In line with the observed antiviral effect, the S-enantiomer displayed a dose-dependent shift in the melting temperature in thermal shift assays, indicative for direct binding to the recombinant 2C protein.

Methyl-ß-cyclodextrin inhibits EV-D68 virus entry by perturbing the accumulation of virus particles and ICAM-5 in lipid rafts.

Enterovirus D68 (EV-D68) is a member of the Picornavirus family and a causative agent of respiratory diseases in children. The incidence of EV-D68 infection has increased worldwide in recent years. Thus far, there are no approved antiviral agents or vaccines for EV-D68. Here, we show that methyl-ß-cyclodextrin (MßCD), a common drug that disrupts lipid rafts, specifically inhibits EV-D68 infection without producing significant cytotoxicity at virucidal concentrations. The addition of exogenous cholesterol attenuated the anti-EV-D68 activity of MßCD. MßCD treatment had a weak influence on the attachment of viral particles to the cell membrane but significantly inhibited EV-D68 entry into host cells. We demonstrated that EV-D68 facilitated the translocation of the viral receptor ICAM-5 to membrane rafts in infected cells. The colocalization of viral particles with ICAM-5 in lipid rafts was thoroughly abolished in cells after treatment with MßCD. Finally, we showed that MßCD inhibited the replication of isolated circulating EV-D68 strains. In summary, our results demonstrate that MßCD suppresses EV-D68 replication by perturbing the accumulation of virus particles and ICAM-5 in lipid rafts. This mechanism represents a promising strategy for drug development.

Typical Stress Granule Proteins Interact with the 3' Untranslated Region of Enterovirus D68 To Inhibit Viral Replication.

Stress granules (SGs) are formed in the cytoplasm under environmental stress, including viral infection. Human enterovirus D68 (EV-D68) is a highly pathogenic virus which can cause serious respiratory and neurological diseases. At present, there is no effective drug or vaccine against EV-D68 infection, and the relationship between EV-D68 infection and SGs is poorly understood. This study revealed the biological function of SGs in EV-D68 infection. Our results suggest that EV-D68 infection induced the accumulation of SG marker proteins Ras GTPase-activated protein-binding protein 1 (G3BP1), T cell intracellular antigen 1 (TIA1), and human antigen R (HUR) in the cytoplasm of infected host cells during early infection but inhibited their accumulation during the late stage. Simultaneously, we revealed that EV-D68 infection induces HUR, TIA1, and G3BP1 colocalization, which marks the formation of typical SGs dependent on protein kinase R (PKR) and eIF2α phosphorylation. In addition, we found that TIA1, HUR, and G3BP1 were capable of targeting the 3' untranslated regions (UTRs) of EV-D68 RNA to inhibit viral replication. However, the formation of SGs in response to arsenite (Ars) gradually decreased as the infection progressed, and G3BP1 was cleaved in the late stage as a strategy to antagonize SGs. Our findings have important implications in understanding the mechanism of interaction between EV-D68 and the host while providing a potential target for the development of antiviral drugs.IMPORTANCE EV-D68 is a serious threat to human health, and there are currently no effective treatments or vaccines. SGs play an important role in cellular innate immunity as a target with antiviral effects. This manuscript describes the formation of SGs induced by EV-D68 early infection but inhibited during the late stage of infection. Moreover, TIA1, HUR, and G3BP1 can chelate a specific site of the 3' UTR of EV-D68 to inhibit viral replication, and this interaction is sequence and complex dependent. However, this inhibition can be antagonized by overexpression of the minireplicon. These findings increase our understanding of EV-D68 infection and may help identify new antiviral targets that can inhibit viral replication and limit the pathogenesis of EV-D68.