Abstract
The genus Enterovirus (EV) of the family Picornaviridae includes poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. These diverse viruses cause a variety of diseases, including non-specific febrile illness, hand-foot-and-mouth disease, neonatal sepsis-like disease, encephalitis, paralysis and respiratory diseases. In recent years, several non-polio enteroviruses (NPEVs) have emerged as serious public health concerns. These include EV-A71, which has caused epidemics of hand-foot-and-mouth disease in Southeast Asia, and EV-D68, which recently caused a large outbreak of severe lower respiratory tract disease in North America. Infections with these viruses are associated with severe neurological complications. For decades, most research has focused on poliovirus, but in recent years, our knowledge of NPEVs has increased considerably. In this Review, we summarize recent insights from enterovirus research with a special emphasis on NPEVs. We discuss virion structures, host–receptor interactions, viral uncoating and the recent discovery of a universal enterovirus host factor that is involved in viral genome release. Moreover, we briefly explain the mechanisms of viral genome replication, virion assembly and virion release, and describe potential targets for antiviral therapy. We reflect on how these recent discoveries may help the development of antiviral therapies and vaccines.
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Introduction
Poliovirus is undoubtedly the best-known enterovirus and one of the most studied viruses. Since its identification in 1908, poliovirus has been intensively investigated to understand its life cycle and to control its spread and the debilitating disease it causes — poliomyelitis. The finding that poliovirus can be propagated in cultured, non-neural human cells not only led to the development of polio vaccines but also hugely contributed to the establishment of molecular virology as a new field. In fact, many important breakthroughs and concepts in molecular virology stem from studies on poliovirus. Poliovirus was the first animal RNA virus for which the complete genome sequence was determined1 and for which a reverse genetics system was established2. Furthermore, poliovirus3 and rhinovirus4 were the first animal RNA viruses for which the three-dimensional structure was solved by X-ray crystallography.
The genus Enterovirus of the Picornaviridae family consists of 13 species, of which seven contain human pathogens. Besides three poliovirus serotypes, these seven species contain more than 250 other types (Box 1). These non-polio enteroviruses (NPEVs) include many important pathogens, such as coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. Enteroviruses spread either through the faecal–oral route or via respiratory transmission. From their primary sites of replication in the gastrointestinal or respiratory tract, they can disseminate and infect other tissues and organs, including the central nervous system. Although most infections go unnoticed, NPEVs can cause a wide range of disorders with varying presentation and severity, most often in infants, young children and immunocompromised individuals. Coxsackieviruses, echoviruses and numbered enteroviruses are the main viral cause of aseptic meningitis. Furthermore, they can cause neonatal sepsis-like disease, encephalitis, acute flaccid paralysis (AFP), non-specific febrile illness, hand-foot-and-mouth disease (HFMD), herpangina, pleurodynia, pericarditis and myocarditis5. Coxsackieviruses B and echoviruses are also implicated as environmental factors in the aetiology of type 1 diabetes by persistently infecting and causing inflammation of pancreatic β-cells6. Rhinoviruses cause the common cold but can also trigger severe respiratory tract disease and exacerbations of asthma and chronic obstructive pulmonary disease (COPD)7. Despite their name and transmission route, enteroviruses are not associated with gastrointestinal illnesses. Apart from two inactivated enterovirus (EV)-A71 vaccines that were recently marketed in China, there are currently no effective measures to prevent or treat NPEV infections.
Some specific NPEVs have emerged as serious public health threats, particularly EV-A71 and EV-D68 (Box 2). EV-A71 is a major cause of HFMD, a disease that is usually mild and self-limiting, but serious and life-threatening neurological complications, such as brainstem encephalitis, meningitis and poliomyelitis-like paralysis, may occur. In the past 2 decades, large outbreaks of EV-A71 have occurred in Southeast Asia8,9. EV-D68 is an atypical enterovirus that replicates in the respiratory tract. EV-D68 has long been considered a rare pathogen, but it is now being increasingly detected during respiratory disease outbreaks worldwide. EV-D68 usually causes mild respiratory disease but can also cause severe bronchiolitis or pneumonia. In 2014, a nationwide outbreak of EV-D68 in the United States was associated with severe respiratory disease and a cluster of AFP and cranial nerve dysfunction in children10,11. Another well-known NPEV is coxsackievirus A24 variant, the major cause of large outbreaks and pandemics of acute haemorrhagic conjunctivitis, which affects millions of people5.
In this Review, we discuss our current understanding of enterovirus structure, host–receptor interactions, mechanisms of uncoating and the recent discovery of a universal enterovirus host factor that is involved in viral genome release. Moreover, we briefly explain the mechanism of viral genome replication and assembly and describe potential targets for antiviral therapy. We reflect on how these recent discoveries may help the development of antiviral therapies and vaccines.
The enterovirus life cycle
Enteroviruses are non-enveloped viruses of ~30 nm in size with a single-stranded RNA genome of positive polarity (+) RNA. The life cycle of all enteroviruses (Fig. 1) starts with binding to one or multiple cell surface receptors, resulting in receptor-mediated endocytosis. Enteroviruses can use different endocytic routes, depending on the serotype and cell type. Receptor binding and/or pH changes in the endosomal system induce virus uncoating, which is the release of the viral genome from the capsid into the cytoplasm via a pore in the endosomal membrane. Although different enteroviruses may use different receptors and entry pathways, many post-entry steps are highly conserved. After delivery to the cytosol, the viral RNA is translated into a single large polyprotein, which is proteolytically processed by viral proteinases 2Apro, 3Cpro and 3CDpro into ten proteins (capsid proteins VP0, VP1 and VP3 and replication proteins 2A–2 C and 3A–3D) and some stable and functional cleavage intermediates. Enterovirus genome replication takes place on virus-induced membrane structures termed replication organelles (ROs)12. Genome replication by the RNA-dependent RNA polymerase 3Dpol starts with the synthesis of a negative-strand copy of the incoming viral genome to generate a double-stranded RNA replication intermediate. The negative strand serves as a template for the synthesis of new positive strands. Newly synthesized viral RNAs either serve as a template for further translation and replication or are encapsidated into new virions. Enterovirus particles form by assembly of the structural capsid proteins VP0, VP1 and VP3 into protomers and pentamers. Together with a nascent viral RNA, pentamers form the provirion — a process that is intimately coupled with viral RNA replication. Finally, RNA-induced processing of VP0 into VP2 and VP4 yields mature virions.
Besides cleaving the viral polyprotein, viral proteinases 2Apro and 3Cpro cleave several host proteins to optimize virus translation, replication and virus spread and to suppress antiviral cellular responses. For example, they induce host shut-off (by cleaving cellular factors involved in transcription and cap-dependent translation of host mRNAs), disrupt nucleocytoplasmic transport (by cleaving nucleoporins), interrupt type I interferon and stress pathways (by cleaving RNA sensors and signalling proteins) and disrupt the cellular cytoskeleton (reviewed in refs13,14,15). Collectively, these changes induce dramatic changes in cellular morphology (that is, a cytopathic effect) and culminate in death and lysis of the cell (reviewed in ref.16). Although enteroviruses are described in textbooks as obligate lytic viruses that are released from ruptured cells, evidence is now accumulating that enteroviruses can also egress before cell lysis in membrane-bound structures that can accommodate multiple virions (reviewed in ref.17).
Targeting the viral life cycle
There is a great need for broad-range antiviral drugs to treat NPEV infections. Such drugs may also be instrumental in the global poliovirus eradication initiative to treat so-called chronic shedders18. Several enterovirus inhibitors have been identified and some have been tested in clinical trials, mainly to treat rhinovirus infections, but all have failed owing to lack of efficacy or toxicity issues19 (Table 1). Generally, antiviral compounds can target either viral proteins or essential host factors. Drugs targeting viral proteins usually have limited side effects, whereas the advantage of drugs that target host factors is that viruses are less likely to develop resistance. For many years, drug discovery has relied heavily on screens for antiviral activity in cell-based infectivity assays using large compound libraries, followed by stepwise chemical optimization. An advanced understanding of the structure and function of enterovirus proteins and essential host factors will aid broad-range antiviral drug development by allowing the implementation of dedicated high-throughput in vitro assays and the structure-guided development and optimization of drug candidates. Drug repurposing (that is, the use of compounds that have been developed to treat another condition) is an emerging strategy that can speed up antiviral drug development because safety and pharmacokinetic studies have already been performed. In the following sections, we discuss the various steps of the enterovirus life cycle and highlight examples of inhibitors at each stage.
Enterovirus structure
Rhinovirus 14 and poliovirus were the first animal RNA viruses for which three-dimensional structures were resolved3,4. Since then, numerous high-resolution structures have been determined, revealing that enterovirus particles share a similar architecture (Fig. 2a). Enterovirus particles are constructed of 60 repeating protomers, with each protomer consisting of the four structural proteins VP1, VP2, VP3 and VP4. Together they form the icosahedral shell with a pseudo T = 3 arrangement that encapsidates the viral genome. The small protein VP4 is myristoylated and located on the inside of the virion, whereas the surface of the particle is composed of subunits of VP1, VP2 and VP3, which each adopt the typical eight-stranded, antiparallel β-barrel fold. The surface of the capsid has several highly variable loops, which are readily accessible to the host immune system and account for the high antigenic diversity of enteroviruses. Most enteroviruses have a deep, circular surface depression or canyon encircling each fivefold axis of symmetry that frequently serves as the receptor binding site20.
Despite these similarities, some enteroviruses display unique structural features. The capsid of rhinovirus C (RV-C) contains protrusions on its surface, contrasting with most enterovirus particle surfaces, which are smoother and spherical21 (Fig. 2b). As a consequence, RV-C particles have shallow, narrow and non-continuous canyons, much resembling those of EV-D68 (ref.22). The biological consequences of such atypical canyons are yet to be determined. The floor of the canyon of all enteroviruses (with the exception of RV-C21) harbours a small, hydrophobic ‘pocket’ that is ordinarily filled with a lipid moiety or pocket factor, which is involved in regulating particle stability. Although not much structural variation exists within the Enterovirus genus, a high number of unique and distinctive structural features can be found among other picornavirus genera. For example, the Aichi virus contains poly-l-proline type II helices on its capsid23, whereas hepatitis A virus (HAV) is more angular, and its surface is devoid of any canyon or depression24. Similarly, Ljungan virus lacks a depression on its capsid surface but instead possesses marked protrusions that arise from the fivefold axes25. Owing to developments in structural biology (high-resolution cryo-electron microscopy (cryo-EM) and high-speed fixed-target X-ray crystallography at X-ray free-electron lasers)26, it is likely that the number of high-resolution structures will increase in the near future and enhance our understanding of the structural diversity within the picornavirus family.
One of the earliest explored strategies to inhibit enterovirus infections is to target the viral capsid proteins using so-called capsid binders. These molecules occupy the hydrophobic pocket and displace the pocket factor, thereby increasing particle stability and preventing receptor binding and/or genome release. The first series of capsid binders, known as the WIN compounds27, were chemically optimized to improve their antiviral and drug-like properties, which eventually resulted in the development of pleconaril. Several clinical trials have been performed with pleconaril and other classes of capsid binders; however, insufficient clinical benefits were observed. Additionally, the rapid emergence of drug-resistant viruses and the unusual structure of RV-C, with a canyon that lacks a pocket that can be targeted by capsid binders22, pose obstacles for the development of broad-range capsid binders.
Enterovirus entry receptors
One of the key determinants of viral tropism and pathogenesis is the availability of a specific cell surface receptor that can be used for viral attachment and entry. In 1989, poliovirus receptor (also known as CD155) (ref.28) and intercellular adhesion molecule 1 (ICAM1) (ref.29) were the first enterovirus receptors that were identified for poliovirus and major rhinoviruses, respectively. Since then, many NPEV receptors have been identified, of which the majority belong to the cell surface immunoglobulin-like or integrin receptor family (Table 2). The observation that enteroviruses can bind to a large repertoire of different receptors provides a likely explanation for the diverse spectrum of diseases that are caused by these viruses.
In this Review, we categorize receptors as either uncoating or attachment receptors. Uncoating receptors induce conformational changes in the viral capsid that mediate the uncoating process, whereas attachment receptors facilitate cell attachment and may promote virus uptake. Although uncoating receptors are usually proteins, it was recently shown that EV-D68 binds sialylated glycans via its canyon and that these cause the conformational changes that initiate uncoating30. This finding represents the first example of an enterovirus that can bind to carbohydrates to trigger uncoating. For influenza viruses, tropism is determined by the distribution of different sialic acids on the respiratory epithelium. Whether a similar correlation exists between the expression pattern of different sialic acids and EV-D68 tropism still needs to be explored. In addition, ICAM5, which is enriched in telencephalic grey matter, was identified as a second uncoating receptor, possibly explaining why EV-D68 is occasionally associated with AFP31. The reason why this virus engages two uncoating receptors is currently unknown. Besides uncoating receptors, attachment receptors can also be of importance for infection. For instance, binding to the attachment receptor complement decay-accelerating factor (DAF; also known as CD55) by group B coxsackieviruses activates signalling events in polarized epithelial cells that liberate the uncoating receptor (coxsackievirus–adenovirus receptor; CAR) from tight junctions, making CAR accessible to the virus32. Moreover, attachment factors can be determinants of viral tropism, which was highlighted by a recent study on coxsackievirus A24 variant, in which it was shown that adaptation to the attachment receptor sialic acid was associated with the emergence of acute haemorrhagic conjunctivitis pandemics33.
For EV-A71, two different receptors have been identified: scavenger receptor class B member 2 (SCARB2)34 and P-selectin glycoprotein ligand 1 (PSGL1)35. SCARB2 was shown to be the receptor of all EV-A71 strains studied thus far34,36, whereas PSGL1 was found to be the receptor for only some strains (EV-A71-PSGL1-binder (EV-A71-PB))35. In contrast to SCARB2, which was shown to induce uncoating36, it is still unclear how PSGL1 supports EV-A71-PB infection. Autopsies of fatal EV-A71 infection cases revealed viral antigens in the brainstem and spinal cord, but no specific cell type that is targeted by the virus has been identified thus far37. SCARB2 is expressed on neurons and glial cells38, and transgenic mice expressing human SCARB2 develop similar pathological features as humans39, suggesting that SCARB2 is the main determinant for the development of neurological disease. PSGL1 is primarily expressed on leukocytes, although expression in human neuronal tissue was also reported38. Despite this information, the role of PSGL1 in EV-A71-induced disease remains unclear.
Receptor identification is not only useful to better understand viral pathogenicity but also to develop suitable cell and/or animal model systems to study the biology of a virus and to identify antiviral drugs. This is exemplified by RV-C, a major cause of severe respiratory tract infections (Box 2). Despite its discovery more than 10 years ago40, our knowledge of this virus was greatly hampered owing to its inability to grow in standard cell culture systems41. The recent discovery of the respiratory epithelium-expressed cadherin-related family member 3 (CDHR3) as the protein receptor and the generation of a CDHR3-overexpressing cell line that supports RV-C infection can thus be considered important milestones in RV-C research42. In differentiated human bronchial epithelial cell cultures, RV-C was found to preferentially infect CDHR3-expressing ciliated cells, providing the first insight into RV-C tropism43. Intriguingly, a coding SNP in CDHR3 that was previously associated with an increased risk of severe childhood asthma exacerbation was shown to enhance RV-C infection in cell culture by increasing CDHR3 expression at the cell surface44.
Although many NPEV receptors have been identified, the interplay between different receptors and the exact mechanism by which these receptors mediate infection are still poorly understood. Further studies are necessary to improve our understanding of virus tropism and pathogenesis and to define new targets for intervention. Several strategies targeting virus–receptor interactions have been reported, including the use of soluble receptor fragments45 and antibodies that bind to receptors or to receptor-binding epitopes on the viral capsid46. However, these strategies will be limited to a narrow range of viruses that share a common receptor.
Enterovirus uncoating
Uncoating stages
The entry mechanism by which enveloped viruses fuse with the cellular membrane is relatively well understood; however, it is less clear how enteroviruses deliver their genome into the host cell. Although enteroviruses utilize a highly diverse set of receptors and endocytic routes, the uncoating mechanism of poliovirus and NPEVs follows a number of conserved steps, which gradually destabilize the virus particle and lead to genome release at the correct time and location. Early studies on poliovirus identified three distinct particle types that occur during the entry process (Fig. 3a) on the basis of differences in their sedimentation coefficients and antigenic properties: the 160S, 135S and 80S particles47. The 160S particle is the native, infectious form of the virion. The 135S particle (altered particle, or A-particle) is a destabilized, infectious particle type that is formed in vitro, but it remains unclear whether this intermediate also exists during the uncoating process within infected cells. The 80S particle is the empty virion, which has released its genome and is a non-infectious end product of the uncoating process. The structure of the native particle is dynamic and undergoes ‘breathing’, which is the recurrent expansion and shrinkage of the virion, accompanied by transient exposure of VP4 and the N-terminal extension of VP1 (ref.48). Cellular uncoating cues (that is, receptor binding or low endosomal pH) lock the virion in its expanded state, forming the A-particle. Unlike the native particle, this uncoating intermediate is hydrophobic owing to the irreversible externalization of the VP1 N terminus, which contains an amphipathic helix that allows the virion to interact with membranes49 (Fig. 3b). The A-particle is no longer bound to its receptor, lacks the internal protein VP4 (ref.50) and has a ~4% increased radius compared with the native virion47, probably to allow rearrangement of the genomic RNA in preparation of its release.
Uncoating cues
Although all enteroviruses undergo similar structural changes during uncoating, the cues driving these destabilizing events are diverse. For most enteroviruses, the first uncoating cue encountered during cell entry is receptor binding. These uncoating receptors bind in the canyon, where they engage the GH loop of VP1, causing collapse of the hydrophobic pocket and expulsion of the pocket factor51. While proteinaceous uncoating receptors directly engage the VP1 GH loop, a recent study describing the crystal structure of EV-D68 in complex with its sialic acid receptor30 revealed that sialic acid indirectly displaces the GH loop via a series of long range conformational changes. Although this unconventional mode of receptor binding triggered pocket factor expulsion, sialic acid did not induce A-particle formation, suggesting that additional uncoating cues are required to complete the uncoating process. In addition to receptor binding, some enteroviruses rely on endosomal acidification as a cue to initiate uncoating. As a low pH is restricted to specific cellular compartments, this chemical cue allows a virus to control the timing and site of genome release. Whereas endosomal acidification is the only known uncoating cue for minor-group rhinoviruses52, some other enteroviruses, like EV-A71 (ref.53), require simultaneous engagement with their receptor and a low pH to fully uncoat.
Pore formation
Numerous structural studies that revealed enterovirus uncoating intermediates have provided extensive information about the molecular events that occur between receptor binding and A-particle formation, but the subsequent process of RNA release is still poorly understood. Because enteroviruses lack an external membrane, the viral genome must be translocated across a membrane to reach the cytoplasm. Early studies on poliovirus genome delivery showed that virions interact with lipid membranes and induce the formation of ion-permeable channels54, a capacity that was found to be abrogated by mutations in VP4 (ref.55). Indeed, recombinant myristoylated VP4 of RV-16 was shown to independently induce membrane permeability56 by forming multimeric pores57. These pores have a channel size consistent with the size that is required for translocation of the single-stranded viral RNA genome57. Whether the virion is directly associated with this transmembrane channel during genome release has been unclear for a long time. Yet, an electron tomography study of poliovirus bound to receptor-decorated liposomes revealed approximately 50 Å-long ‘umbilical’ connectors linking the virion to the membrane58. It was proposed that these connectors represent genomic RNA and viral proteins, which might shield the RNA during translocation. Consistent with this observation, a recent study showed that the poliovirus genomic RNA is protected from degradation by co-endocytosed RNase during the entry process59, supporting a scenario in which a discrete channel connects the virion to the host cell membrane.
Genome release
Other major questions in studies of viral uncoating concern the exit site and orientation of the viral RNA during egress from the virus particle. So far, our understanding of the directionality of RNA release is based solely on a study of RV-A2, which revealed that the 3' end of the genome exits the virion before the 5' end60. The site of RNA release from the virion was originally proposed to be located at the fivefold axis61, but cryo-EM structures of uncoating intermediates (A-particles or empty particles) revealed holes in the capsid at the twofold axis and near the quasi-threefold axis51,62. The opening at the quasi-threefold axis was found to serve as a channel for externalization of the VP1 N terminus63. Indications of the site of RNA release are based on asymmetric cryo-EM reconstructions of heated poliovirus particles that are in the process of genome release64. Although the resolution was low (~50 Å), these structures implicated a region spanning the twofold and quasi-threefold axes. A limitation of most cell-free uncoating studies performed to date is that viral uncoating is triggered by numerous cues (for example, heat, acid or soluble receptor molecules) that act on the entire virion (that is, global stimulation), whereas uncoating during a natural infection is probably asymmetrical, because only one side of the virion interacts directly with the membrane. In a recent study, asymmetric coxsackievirus B3 uncoating was induced with receptors embedded in lipid bilayer nanodiscs, generating the first high-resolution structures of an enterovirus uncoating intermediate attached to a membrane65. Unlike particles formed upon global stimulation, this structure revealed an opening at the threefold axis. Together, these findings show that more studies employing lipid membranes, as well as high-resolution cryo-EM, are required to determine the exact site of RNA exit from the virion.
The pan-enterovirus host factor PLA2G16
Although receptor binding is the only known uncoating cue for many enteroviruses, genome release does not take place immediately after receptor binding on the cell surface but after a period of internalization into the cell66. For that reason, several studies have suggested the existence of an additional unknown uncoating cue that controls the moment at which the virion releases its genome65,67. A plausible candidate for this role is the host factor phospholipase A2 group XVI (PLA2G16), a lipid-modifying enzyme of which the physiological function remains poorly understood. PLA2G16 was identified as a universal enterovirus host factor that facilitates the displacement of viral genomes from virus-containing endocytic vesicles68. It is likely that PLA2G16 is involved in the formation, expansion or maintenance of the membrane pore, either by directly interacting with viral proteins or RNA, or indirectly, by creating a hospitable lipid environment that favours pore formation. For instance, lipids modified by PLA2G16 may affect membrane fluidity or interact with pore components, thereby facilitating their insertion into the endosomal membrane. Interestingly, some (non-Enterovirus) picornaviruses possess a non-structural 2A protein with homology to PLA2G16. Although these 2A proteins have never been found in association with virions, the possibility exists that these proteins perform a similar function in virus entry as PLA2G16. Analogously, most parvoviruses have a capsid protein that contains a phospholipase A2 domain, which is thought to modify the membrane of endocytic vesicles to allow viral genome release into the cytoplasm69. These findings suggest that various non-enveloped viruses have independently evolved to employ lipid-modifying enzymes for virus entry via mechanisms that are not yet fully understood.
Targeting the uncoating stage of enterovirus infection could be a promising strategy in the development of antiviral drugs against NPEVs. In addition to pocket factor analogues, which stabilize the virion, molecules that prevent genome translocation by targeting viral proteins or host factors that are involved in pore formation might be promising candidates. The pan-enterovirus host factor PLA2G16 could be an excellent drug target, because all enteroviruses tested to date require this factor and because the activity of PLA2G16 is dispensable for the survival of mice68. Because no selective PLA2G16 inhibitors have been reported thus far, developing specific in vitro assays to screen for inhibitors of its phospholipase activity could be a promising strategy to develop broadly acting anti-enterovirus drugs.
Translation and genome replication
Following its delivery to the cytosol, the viral genome is directly translated by ribosomes, yielding capsid proteins and viral proteins that mediate genome replication. Translation is mediated by an internal ribosome entry site (IRES) and requires a subset of the canonical translation machinery as well as several IRES trans-acting factors (ITAFs) (reviewed in refs70,71). The mechanism of RNA replication is well conserved among all enteroviruses (reviewed in ref.72). The core enzyme of the replication machinery is the viral RNA-dependent RNA polymerase 3Dpol. Replication is initiated at higher-order RNA elements in the 5′ and 3′ UTRs and requires a primer, the viral peptide 3B (also known as VPg) coupled to two uridines. VPg uridylylation requires a structural RNA element located in the coding region of the genome. 3Dpol operates in conjunction with several viral proteins, including 3CDpro and 3AB, which together bind to structural RNA elements involved in priming, and 2C, which has ATPase-dependent helicase and ATPase-independent RNA chaperone activities. Besides viral proteins, several host RNA-binding proteins are involved in genome replication, including poly(rC)-binding protein 2 (PCBP2), polyadenylate-binding protein 1 (PABP1) and heterogeneous nuclear ribonucleoprotein C (HNRNPC).
Replication occurs on virus-induced, tubulovesicular ROs, which are thought to originate from endoplasmic reticulum (ER) and/or Golgi apparatus membranes. RO formation requires the viral membrane-associated proteins 2BC and 3A73 and a specific set of host factors, but the exact composition and mechanism of formation of ROs remains unknown. 3A interacts with acyl-CoA-binding domain-containing protein 3 (ACBD3) to recruit the lipid kinase phosphatidylinositol 4-kinase-β (PI4KB) to membranes, leading to an accumulation of phosphatidylinositol-4-phosphate (PtdIns4P) lipids. PtdIns4P attracts oxysterol-binding protein (OSBP) to form membrane contact sites between the ER and ROs and mediate a PtdIns4P-driven accumulation of cholesterol at ROs (reviewed in refs12,74). Increased levels of PtdIns4P and/or cholesterol are important for RO formation and efficient genome replication, possibly by facilitating proper processing of the viral polyprotein75,76,77,78. 3A also binds Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), but its exact role in replication is unclear. The roles of 2BC, 2B and 2C in membrane reorganization are less well understood. It has been suggested that 2BC contributes to PI4KB recruitment79. 2B has viroporin activity80,81, but its exact function in RO formation is unknown. 2C binds reticulon 3, an ER protein that promotes membrane curvature, but the importance of this interaction for RO formation has not been investigated82. Some host factors appear to be important for a subgroup of enteroviruses. For example, valosin-containing protein (VCP) was identified as a host factor required for poliovirus replication but not coxsackievirus B3 replication83. Of note, several other host proteins have been implicated in enterovirus replication, but their roles in replication are often poorly understood. Additionally, enteroviruses trigger activation of autophagy. Although autophagosomes have been suggested to have a role in genome replication84, it is currently thought that virus-induced autophagosomes are primarily involved in non-lytic virion release (see below).
The mechanisms of genome translation and replication are highly conserved between enteroviruses and are therefore attractive targets for the development of broadly acting antiviral drugs, which can target viral enzymes, specific host factors or essential cellular processes (Table 1). Several inhibitors targeting viral enzymes 3Cpro and 3Dpol have been described. 3Cpro inhibitors include peptidomimetics that target its active site, such as rupintrivir. 3Dpol inhibitors include nucleoside analogues (for example, the anticancer drug gemcitabine (Gemzar)85 and non-nucleoside inhibitors (for example, GPC-N114 (ref.86)), which target a conserved RNA template channel in 3Dpol). Additionally, a number of 2C inhibitors have been reported, but their mode of action is poorly understood. Among them is the antidepressant drug fluoxetine (Prozac)87,88, which inhibits enterovirus B and enterovirus D members. Fluoxetine was successfully used to treat an immunocompromised child with chronic enteroviral encephalitis89, underscoring the potential of drug repurposing in antiviral therapy. The recently elucidated structure of EV-A71 2C90 will likely encourage soaking or co-crystallization studies and provide more insight into the mode of action of 2C inhibitors, allowing optimization through structure-guided drug design. In addition, inhibitors of host factors, including compounds targeting PI4KB and OSBP, inhibit a broad range of enteroviruses. Unfortunately, PI4KB inhibition was found to be associated with adverse side effects91,92. OSBP inhibitors include the antifungal drug itraconazole (Sporanox)93, which was shown to have antiviral activity in mice94.
Virion assembly and release
Assembly
The enterovirus life cycle ends with the formation of new infectious progeny, which is a highly complex, stepwise process that is poorly understood95. In a first step, heat shock protein 90 (HSP90) associates with the myristoylated capsid precursor P1, catalysing the proteolytic processing by 3CDpro to yield VP0, VP1 and VP3 (ref.96). This yields a protomeric particle that subsequently self-oligomerizes to form pentameric particles. These pentamers are stabilized by their myristate moieties97,98 and, for some enteroviruses, by association with glutathione99,100. The current model is that in a next step, pentameric particles condense around replicating viral RNA to form the provirion. Concurrently with encapsidation and as a last quality control step, the viral RNA mediates VP0 cleavage into VP2 and VP4, generating the mature virus particle101.
Although many RNA viruses encode at least one RNA packaging signal to provide specificity and precision during viral assembly, packaging signals have not been identified thus far in the genomes of enteroviruses. For enteroviruses, it is currently believed that encapsidation specificity is governed by direct protein–protein interaction between 2C, as part of the replication complex, and the viral capsid protein VP3 without an apparent involvement of an RNA packaging signal102. Intriguingly, parechovirus 1 and foot-and-mouth disease virus (FMDV) were recently shown to contain multiple short regions of ordered RNA structures that bind capsid proteins, dispersed throughout their genomes103,104. Whether enterovirus or other picornavirus genomes also contain such structures needs to be investigated.
Enterovirus assembly can be inhibited by geldanamycin105, an HSP90 inhibitor, but this inhibitor has cytotoxic effects and has mainly been investigated for its antitumour activity. Glutathione levels can be manipulated in several ways, but this is unlikely to be an effective antiviral strategy because only a subset of enteroviruses depends on glutathione for efficient assembly99,100,106.
Virion release
Enteroviruses are typically considered cytolytic viruses that kill and lyse their host cell for release. However, several studies have revealed that enteroviruses can also be released in a non-lytic manner in vesicles that mediate en bloc transmission of virions17,107,108,109,110. The current model is that enteroviruses trigger autophagy and upregulate the formation of double-membrane autophagosomes that engulf clusters of virions84. Fusion of these autophagosomes with lysosomes is inhibited by the virus to prevent degradation of their contents. Instead, fusion of the outer membrane of virion-containing, double-membrane autophagosomes with the plasma membrane releases single-membrane vesicular structures filled with virions to the extracellular milieu. The membranes of these extracellular vesicles are enriched in the lipid phosphatidylserine, which assists uptake in cells, likely via phosphatidylserine receptors109. Nevertheless, the bona fide virus receptor is still needed for infection, implying that at some point, virions are released from the extracellular vesicles109. Non-lytic release may be a much more widely adopted transmission strategy of picornaviruses than previously thought and has also been implicated for HAV, which evades neutralizing antibodies by cloaking itself in exosome-like structures111,112. Thus, non-lytic release may enhance infection efficiency, but its exact role in enterovirus dissemination and pathogenesis remains to be explored. The seemingly widespread role of autophagy in virion release by enteroviruses and other picornaviruses makes this a potential broad-range antiviral target. However, given the central role of autophagy in cellular and organismal homeostasis, it seems unlikely that autophagy inhibitors are sufficiently safe for clinical application.
Advances in enterovirus vaccine development
Besides antiviral drugs, vaccines are of great importance in combating enterovirus infections. Both inactivated poliovirus vaccines (IPV) and live, attenuated oral poliovirus vaccines (OPV) have been instrumental in preventing poliomyelitis. The major advantages of OPV are that less virus is needed to induce protective immunity and that OPV limits virus spreading by inducing mucosal immunity. However, a major drawback is that OPV can revert and/or recombine with closely related Enterovirus C members, leading to vaccine-associated paralytic poliomyelitis and circulating vaccine-derived poliovirus113. Currently, safe and effective OPVs are being developed, involving various attenuating traits, including destabilizing mutations in the IRES114, mutations in 3Dpol that improve proofreading activity115,116 and lower RNA recombination capacity117, and synonymous mutations in the capsid-coding region that alter codon pair bias118.
Theoretically, inactivated or live, attenuated vaccines can be developed against any NPEV, as exemplified by the EV-A71 vaccine that is currently marketed in China9. Development of a polyvalent, broad-spectrum vaccine against many different enterovirus serotypes was considered unfeasible for a long time. However, recently it was shown that combining 50 inactivated rhinovirus types into a single vaccine elicited neutralizing antibodies against 49 types, suggesting that universal, polyvalent, inactivated enterovirus vaccines will be developed119. Another promising approach is the use of virus-like particles (VLPs)120, which assemble upon co-expression of capsid proteins and 3CDpro, allowing the manufacture of vaccines of viruses that are difficult to culture, for example, Rhinovirus C members or viruses that are restricted to high-containment facilities (poliovirus in the post-eradication era). Enterovirus VLPs can differ slightly from native virions in their structure, antigenic properties and stability, owing to the absence of a stabilizing genome. However, this problem can be circumvented by introducing specific stabilizing capsid mutations121. Recently, stabilized poliovirus VLPs were efficiently produced in plants — which represent a cheap, safe and high-level expression system — and immunization of mice with these VLPs provided protection against a poliovirus challenge122. In conclusion, several strategies can be used to develop potent vaccines against NPEVs.
Conclusions and future perspectives
The large group of NPEVs contains many human pathogens that cause a wide spectrum of illnesses, including severe neurological and respiratory diseases. In this Review, we described the current knowledge of the different stages of the life cycle of enteroviruses, most of which is derived from studies on poliovirus. Although these studies have provided valuable insights into the enterovirus life cycle, many important questions need to be addressed. For instance, future studies should elucidate the mechanism of viral genome translocation across the endosomal membrane and the contribution of PLA2G16 to this process; clarify the early post-entry events and identify where the viral RNA is first translated and replicated; and establish the exact origin and function of ROs. In addition, how the viral RNA is encapsidated and which viral and cellular factors are involved in this process, how the viral RNA is organized inside the virion, and how virus-containing extracellular vesicles are formed and what their physiological function is are all important questions that need to be addressed. Recently, we have seen major advances in technologies that may be instrumental in solving these questions. Several new microscopy techniques are becoming increasingly available, for example, three-dimensional scanning electron microscopy approaches that allow the reconstruction of entire cells and the visualization of virus-induced intracellular changes at nanoscale resolutions. Moreover, in situ cryo-electron tomography, an imaging modality that allows structural analysis of macromolecular complexes in their physiological microenvironment, may be applied to gain more insight into viral replication complexes and their association with ROs. CRISPR–Cas9 gene knockout screens can be used to identify essential host factors, and nuclease-inactive Cas9 can be used to bind specific viral RNA sequences, allowing RNA tracking in living cells. Novel advances in mass spectrometry, including phospho-proteomics and crosslink-proteomics, lipidomics and metabolomics, may shed new light on our understanding of virus-induced changes in host cell biology and signalling that are essential for virus replication and/or propagation. Future NPEV research should focus on receptor usage and tropism, pathogenesis, transmission dynamics, epidemiology and evolution. Evolution studies are also needed to better understand how poliovirus evolved from EV-C coxsackievirus123, which is an essential question in view of the polio eradication campaign.
Intensified efforts should be undertaken to develop preventive and therapeutic options to control NPEV infections. New insights into the viral life cycle, combined with rapid developments in (computational) drug discovery, are expected to spur the development of novel, broad-range enterovirus inhibitors. The successful development of highly effective antiviral drugs against another (+) RNA virus — hepatitis C virus — implies that it should be possible to develop antiviral drugs against enteroviruses, provided that sufficient investments are made. Besides treating NPEV infection, broad-range drugs may also contribute to the eradication of poliovirus. Research on this devastating human pathogen has laid important foundations for the field of molecular virology. Although poliovirus may soon be eradicated, fundamental research on NPEVs is likely to have far-reaching implications that go beyond the field of picornaviruses.
Change history
03 May 2018
In the version of this Review originally published, co-author Hendrik Jan Thibaut’s name was incorrectly indexed as “Jan Thibaut, H”. It should have appeared as “Thibaut, HJ”. This has now been corrected in all versions of the Review. The publisher apologizes to the authors and to readers for this error.
References
Racaniello, V. R. & Baltimore, D. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc. Natl Acad. Sci. USA 78, 4887–4891 (1981).
Racaniello, V. & Baltimore, D. Cloned poliovirus complementary DNA is infectious in mammalian cells. Science 214, 916–919 (1981).
Hogle, J., Chow, M. & Filman, D. Three-dimensional structure of poliovirus at 2.9 A resolution. Science 229, 1358–1365 (1985).
Rossmann, M. G. et al. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature 317, 145–153 (1985).
Pons-Salort, M., Parker, E. P. K. & Grassly, N. C. The epidemiology of non-polio enteroviruses. Curr. Opin. Infect. Dis. 28, 479–487 (2015).
Hober, D. & Sauter, P. Pathogenesis of type 1 diabetes mellitus: interplay between enterovirus and host. Nat. Rev. Endocrinol. 6, 279–289 (2010).
Thibaut, H. J. et al. Toward antiviral therapy/prophylaxis for rhinovirus-induced exacerbations of chronic obstructive pulmonary disease: challenges, opportunities, and strategies. Rev. Med. Virol. 26, 21–33 (2016).
Ooi, M. H., Wong, S. C., Lewthwaite, P., Cardosa, M. J. & Solomon, T. Clinical features, diagnosis, and management of enterovirus 71. Lancet Neurol. 9, 1097–1105 (2010).
Yi, E.-J., Shin, Y.-J., Kim, J.-H., Kim, T.-G. & Chang, S.-Y. Enterovirus 71 infection and vaccines. Clin. Exp. Vaccine Res. 6, 4–14 (2017).
Messacar, K. et al. A cluster of acute flaccid paralysis and cranial nerve dysfunction temporally associated with an outbreak of enterovirus D68 in children in CO, USA. Lancet 385, 1662–1671 (2015).
Holm-Hansen, C. C., Midgley, S. E. & Fischer, T. K. Global emergence of enterovirus D68: a systematic review. Lancet Infect. Dis. 16, e64–e75 (2016).
van der Schaar, H. M., Dorobantu, C. M., Albulescu, L., Strating, J. R. P. M. & van Kuppeveld, F. J. M. Fat(al) attraction: picornaviruses usurp lipid transfer at membrane contact sites to create replication organelles. Trends Microbiol. 24, 535–546 (2016).
Lloyd, R. E. Enterovirus control of translation and RNA granule stress responses. Viruses 8, 1–16 (2016).
Lloyd, R. E. Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses. Virology 479–480, 457–474 (2015).
Feng, Q., Langereis, M. A. & van Kuppeveld, F. J. M. Induction and suppression of innate antiviral responses by picornaviruses. Cytokine Growth Factor Rev. 25, 577–585 (2014).
Harris, K. G. & Coyne, C. B. Death waits for no man — does it wait for a virus? How enteroviruses induce and control cell death. Cytokine Growth Factor Rev. 25, 587–596 (2014).
Lai, J. K. F., Sam, I.-C. & Chan, Y. F. The autophagic machinery in Enterovirus infection. Viruses 449, 35–44 (2016).
Kew, O. M., Sutter, R. W., de Gourville, E. M., Dowdle, W. R. & Pallansch, M. A. Vaccine-derived polioviruses and the endgame strategy for global polio eradication. Annu. Rev. Microbiol. 59, 587–635 (2005).
Benschop, K. S. M., van der Avoort, H. G. A. M., Duizer, E. & Koopmans, M. P. G. Antivirals against enteroviruses: a critical review from a public-health perspective. Antivir. Ther. 20, 121–130 (2015).
Rossmann, M. G., He, Y. & Kuhn, R. J. Picornavirus-receptor interactions. Trends Microbiol. 10, 324–331 (2002).
Liu, Y. et al. Atomic structure of a rhinovirus C, a virus species linked to severe childhood asthma. Proc. Natl Acad. Sci. USA 113, 8997–9002 (2016).This study describes the first cryo-EM structure of a member of the rhinovirus C species and provides a molecular explanation for the insensitivity of this virus for capsid-binding antivirals.
Liu, Y. et al. Structure and inhibition of EV-D68, a virus that causes respiratory illness in children. Science 347, 71–74 (2015).This study describes the crystal structure and pleconaril sensitivity of EV-D68, an emerging respiratory pathogen associated with AFP.
Zhu, L. et al. Structure of human Aichi virus and implications for receptor binding. Nat. Microbiol. 1, 16150 (2016).
Wang, X. et al. Hepatitis A virus and the origins of picornaviruses. Nature 517, 85–88 (2014).
Zhu, L. et al. Structure of Ljungan virus provides insight into genome packaging of this picornavirus. Nat. Commun. 6, 8316 (2015).
Roedig, P. et al. High-speed fixed-target serial virus crystallography. Nat. Methods 14, 805–810 (2017).
Thibaut, H. J., De Palma, A. M. & Neyts, J. Combating enterovirus replication: state-of-the-art on antiviral research. Biochem. Pharmacol. 83, 185–192 (2012).
Mendelsohn, C. L., Wimmer, E. & Racaniello, V. R. Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily. Cell 56, 855–865 (1989).
Greve, J. M. et al. The major human rhinovirus receptor is ICAM-1. Cell 56, 839–847 (1989).
Liu, Y. et al. Sialic acid-dependent cell entry of human enterovirus D68. Nat. Commun. 6, 8865 (2015).This work identifies the binding site of sialic acid on EV-D68 and reveals that sialic binding initiates uncoating via pocket factor displacement.
Wei, W. et al. ICAM-5/telencephalin is a functional entry receptor for enterovirus D68. Cell Host Microbe 20, 631–641 (2016).This work identifies ICAM5 as a protein receptor for EV-D68 and shows that ICAM5 can trigger virus uncoating.
Coyne, C. B. & Bergelson, J. M. Virus-induced Abl and Fyn kinase signals permit coxsackievirus entry through epithelial tight junctions. Cell 124, 119–131 (2006).
Baggen, J. et al. A role of enhanced receptor engagement in the evolution of a pandemic acute haemorrhagic conjunctivitis virus. Proc. Natl Acad. Sci. USA 115, 397–402 (2018).
Yamayoshi, S. et al. Scavenger receptor B2 is a cellular receptor for enterovirus 71. Nat. Med. 15, 798–801 (2009).
Nishimura, Y. et al. Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71. Nat. Med. 15, 794–797 (2009).
Yamayoshi, S., Ohka, S., Fujii, K. & Koike, S. Functional comparison of SCARB2 and PSGL1 as receptors for enterovirus 71. J. Virol. 87, 3335–3347 (2013).
Lei, X., Cui, S., Zhao, Z. & Wang, J. Etiology, pathogenesis, antivirals and vaccines of hand, foot, and mouth disease. Natl Sci. Rev. 2, 268–284 (2015).
Jiao, X. Y., Guo, L., Huang, D. Y., Chang, X. L. & Qiu, Q. C. Distribution of EV71 receptors SCARB2 and PSGL-1 in human tissues. Virus Res. 190, 40–52 (2014).
Fujii, K. et al. Transgenic mouse model for the study of enterovirus 71 neuropathogenesis. Proc. Natl Acad. Sci. USA 110, 14753–14758 (2013).
Lamson, D. et al. MassTag polymerase chain reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza like illness in New York state during 2004–2005. J. Infect. Dis. 194, 1398–1402 (2006).
Bochkov, Y. A. et al. Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C. Nat. Med. 17, 627–632 (2011).
Bochkov, Y. A. et al. Cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus C binding and replication. Proc. Natl Acad. Sci. USA 112, 5485–5490 (2015).This work identifies CDHR3 as the first receptor for rhinovirus C and reveals a link between a SNP that intensifies respiratory illness to rhinovirus C susceptibility.
Griggs, T. F. et al. Rhinovirus C targets ciliated airway epithelial cells. Respir. Res. 18, 84 (2017).
Bønnelykke, K. et al. A genome-wide association study identifies CDHR3 as a susceptibility locus for early childhood asthma with severe exacerbations. Nat. Genet. 46, 51–55 (2014).
Turner, R. B. et al. Efficacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection. JAMA 281, 1797–1804 (1999).
Kuo, R. L. & Shih, S. R. Strategies to develop antivirals against enterovirus 71. Virol. J. 10, 1 (2013).
Belnap, D. M. et al. Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus. J. Virol. 74, 1342–1354 (2000).
Li, Q., Yafal, A. G., Lee, Y. M., Hogle, J. & Chow, M. Poliovirus neutralization by antibodies to internal epitopes of VP4 and VP1 results from reversible exposure of these sequences at physiological temperature. J. Virol. 68, 3965–3970 (1994).
Fricks, C. E. & Hogle, J. M. Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding. J. Virol. 64, 1934–1945 (1990).
Crowell, R. L. & Philipson, L. Specific alterations of coxsackievirus B3 eluted from HeLa cells. J. Virol. 8, 509–515 (1971).
Wang, X. et al. A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat. Struct. Mol. Biol. 19, 424–429 (2012).This study describes the crystal structure of EV-A71 empty particles, revealing a mechanism in which receptor binding causes pocket collapse via engagement of the VP1 GH loop.
Prchla, E., Kuechler, E., Blaas, D. & Fuchs, R. Uncoating of human rhinovirus serotype 2 from late endosomes. J. Virol. 68, 3713–3723 (1994).
Hussain, K. M., Leong, K. L. J., Ng, M. M.-L. & Chu, J. J. H. The essential role of clathrin-mediated endocytosis in the infectious entry of human enterovirus 71. J. Biol. Chem. 286, 309–321 (2011).
Tosteson, M. T. & Chow, M. Characterization of the ion channels formed by poliovirus in planar lipid membranes. J. Virol. 71, 507–511 (1997).
Danthi, P., Tosteson, M., Li, Q.-H. & Chow, M. Genome delivery and ion channel properties are altered in VP4 mutants of poliovirus. J. Virol. 77, 5266–5274 (2003).
Davis, M. P. et al. Recombinant VP4 of human rhinovirus induces permeability in model membranes. J. Virol. 82, 4169–4174 (2008).
Panjwani, A. et al. Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore. PLoS Pathog. 10, e1004294 (2014).
Strauss, M., Levy, H. C., Bostina, M., Filman, D. J. & Hogle, J. M. RNA transfer from poliovirus 135 S particles across membranes is mediated by long umbilical connectors. J. Virol. 87, 3903–3914 (2013).
Groppelli, E. et al. Picornavirus RNA is protected from clevage by ribonucleases during virion uncoating and transfer across cellular and model membranes. PLoS Pathog. 13, e1006197 (2017).
Harutyunyan, S. et al. Viral uncoating is directional: exit of the genomic RNA in a common cold virus starts with the poly-(A) tail at the 3′-end. PLoS Pathog. 9, e1003270 (2013).This study is the first to describe that the genome of an enterovirus (RV-2) is released in a fixed orientation, with the 3′ end egressing first.
Hadfield, A. T. et al. The refined structure of human rhinovirus 16 at 2.15 A resolution: implications for the viral life cycle. Structure 5, 427–441 (1997).
Levy, H. C., Bostina, M., Filman, D. J. & Hogle, J. M. Catching a virus in the act of RNA release: a novel poliovirus uncoating intermediate characterized by cryo-electron microscopy. J. Virol. 84, 4426–4441 (2010).This study is the first to reveal an opening near the twofold axis of an enterovirus uncoating intermediate, pointing towards a new possible site of genome egress.
Ren, J. et al. Picornavirus uncoating intermediate captured in atomic detail. Nat. Commun. 4, 1929 (2013).
Bostina, M., Levy, H., Filman, D. J. & Hogle, J. M. Poliovirus RNA is released from the capsid near a twofold symmetry axis. J. Virol. 85, 776–783 (2011).
Lee, H. et al. The novel asymmetric entry intermediate of a picornavirus captured with nanodiscs. Sci. Adv. 2, e1501929 (2016).This work describes the first structure of an enterovirus uncoating intermediate that is induced asymmetrically, by presenting receptor molecules embedded in lipid nanodiscs.
Brandenburg, B. et al. Imaging poliovirus entry in live cells. PLoS Biol. 5, 1543–1555 (2007).
Strauss, M., Schotte, L., Karunatilaka, K. S., Filman, D. J. & Hogle, J. M. Cryo-electron microscopy structures of expanded poliovirus with VHHs sample the conformational repertoire of the expanded state. J. Virol. 91, 1–22 (2017).
Staring, J. et al. PLA2G16 represents a switch between entry and clearance of Picornaviridae. Nature 541, 412–416 (2017).Using genome-wide haploid screens, this study identified PLA2G16 as an enterovirus-wide host factor required for genome release, as well as a novel antiviral sensing mechanism involving galectin 8.
Zádori, Z. et al. A viral phospholipase A2 is required for parvovirus infectivity. Dev. Cell 1, 291–302 (2001).
Lozano, G. & Martínez-Salas, E. Structural insights into viral IRES-dependent translation mechanisms. Curr. Opin. Virol. 12, 113–120 (2015).
Lee, K. M., Chen, C. J. & Shih, S. R. Regulation mechanisms of viral IRES-driven translation. Trends Microbiol. 25, 546–561 (2017).
Paul, A. V. & Wimmer, E. Initiation of protein-primed picornavirus RNA synthesis. Virus Res. 206, 12–26 (2015).
Suhy, D. A., Giddings, T. H. & Kirkegaard, K. Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles. J. Virol. 74, 8953–8965 (2000).
Strating, J. R. P. M. & van Kuppeveld, F. J. M. Viral rewiring of cellular lipid metabolism to create membranous replication compartments. Curr. Opin. Cell Biol. 47, 24–33 (2017).
Ilnytska, O. et al. Enteroviruses harness the cellular endocytic machinery to remodel the host cell cholesterol landscape for effective viral replication. Cell Host Microbe 14, 281–293 (2013).
Ford Siltz, L. A. et al. New small-molecule inhibitors effectively blocking picornavirus replication. J. Virol. 88, 11091–11107 (2014).
Arita, M. Mechanism of poliovirus resistance to host phosphatidylinositol-4 kinase III β inhibitor. ACS Infect. Dis. 2, 140–148 (2016).
Melia, C. et al. Escaping host factor PI4KB inhibition: enterovirus genomic RNA replication in the absence of replication organelles. Cell Rep. 21, 587–599 (2017).
Arita, M. Phosphatidylinositol-4 kinase III beta and oxysterol-binding protein accumulate unesterified cholesterol on poliovirus-induced membrane structure. Microbiol. Immunol. 58, 239–256 (2014).
Agirre, A., Barco, A., Carrasco, L. & Nieva, J. L. Viroporin-mediated membrane permeabilization: pore formation by nonstructural poliovirus 2B protein. J. Biol. Chem. 277, 40434–40441 (2002).
van Kuppeveld, F. J. M., de Jong, A. S., Melchers, W. J. G. & Willems, P. H. G. M. Enterovirus protein 2B po(u)res out the calcium: a viral strategy to survive? Trends Microbiol. 13, 41–44 (2005).
Tang, W.-F. et al. Reticulon 3 binds the 2C protein of enterovirus 71 and is required for viral replication. J. Biol. Chem. 282, 5888–5898 (2007).
Arita, M., Wakita, T. & Shimizu, H. Valosin-containing protein (VCP/p97) is required for poliovirus replication and is involved in cellular protein secretion pathway in poliovirus infection. J. Virol. 86, 5541–5553 (2012).
Jackson, W. T. et al. Subversion of cellular autophagosomal machinery by RNA viruses. PloS Biol. 3, 861–871 (2005).
Song, J. H. et al. Antiviral activity of gemcitabine against human rhinovirus in vitro and in vivo. Antiviral Res. 145, 6–13 (2017).
van der Linden, L. et al. The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family. PloS Pathog. 11, e1004733 (2015).
Zuo, J. et al. Fluoxetine is a potent inhibitor of coxsackievirus replication. Antimicrob. Agents Chemother. 56, 4838–4844 (2012).
Ulferts, R. et al. Selective serotonin reuptake inhibitor fluoxetine inhibits replication of human enteroviruses B and D by targeting viral protein 2C. Antimicrob. Agents Chemother. 57, 1952–1956 (2013).
Gofshteyn, J., Cárdenas, A. M. & Bearden, D. Treatment of chronic enterovirus encephalitis with fluoxetine in a patient with X-linked agammaglobulinemia. Pediatr. Neurol. 64, 94–98 (2016).
Guan, H. et al. Crystal structure of 2C helicase from enterovirus 71. Sci. Adv. 3, e1602573 (2017).
Spickler, C. et al. Phosphatidylinositol 4-kinase III beta is essential for replication of human rhinovirus and its inhibition causes a lethal phenotype in vivo. Antimicrob. Agents Chemother. 57, 3358–3368 (2013).
Lamarche, M. J. et al. Anti-hepatitis C virus activity and toxicity of type III phosphatidylinositol-4-kinase beta inhibitors. Antimicrob. Agents Chemother. 56, 5149–5156 (2012).
Strating, J. R. P. M. et al. Itraconazole inhibits enterovirus replication by targeting the oxysterol-binding protein. Cell Rep. 10, 600–615 (2015).
Shim, A. et al. Therapeutic and prophylactic activity of itraconazole against human rhinovirus infection in a murine model. Sci. Rep. 6, 23110 (2016).
Jiang, P., Liu, Y., Ma, H.-C., Paul, A. V. & Wimmer, E. Picornavirus morphogenesis. Microbiol. Mol. Biol. Rev. 78, 418–437 (2014).
Geller, R., Vignuzzi, M., Andino, R. & Frydman, J. Evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance. Genes Dev. 21, 195–205 (2007).
Paul, A. V., Schultz, A., Pincus, S. E., Oroszlan, S. & Wimmer, E. Capsid protein VP4 of poliovirus is N-myristoylated. Proc. Natl Acad. Sci. USA 84, 7827–7831 (1987).
Chow, M. et al. Myristylation of picornavirus capsid protein VP4 and its structural significance. Nature 327, 482–486 (1987).
Thibaut, H. J. et al. Binding of glutathione to enterovirus capsids is essential for virion morphogenesis. PloS Pathog. 10, e1004039 (2014).
Ma, H. C. et al. An interaction between glutathione and the capsid is required for the morphogenesis of C-cluster enteroviruses. PloS Pathog. 10, e1004052 (2014).
Basavappa, R. et al. Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of the empty capsid assembly intermediate at 2.9 A resolution. Protein Sci. 3, 1651–1669 (1994).
Liu, Y. et al. Direct interaction between two viral proteins, the nonstructural protein 2CATPase and the capsid protein VP3, is required for enterovirus morphogenesis. PloS Pathog. 6, e1001066 (2010).
Shakeel, S. et al. Genomic RNA folding mediates assembly of human parechovirus. Nat. Commun. 8, 5 (2017).
Logan, G. et al. Deep sequencing of foot-and-mouth disease virus reveals RNA sequences involved in genome packaging. J. Virol. 92, e01159–17 (2017).
Tsou, Y.-L. et al. Heat shock protein 90: role in enterovirus 71 entry and assembly and potential target for therapy. PloS ONE 8, e77133 (2013).
Smith, A. D. & Dawson, H. Glutathione is required for efficient production of infectious picornavirus virions. Virology 353, 258–267 (2006).
Robinson, S. M. et al. Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers. PloS Pathog. 10, e1004045 (2014).
Bird, S. W., Maynard, N. D., Covert, M. W. & Kirkegaard, K. Nonlytic viral spread enhanced by autophagy components. Proc. Natl Acad. Sci. USA 111, 13081–13086 (2014).
Chen, Y. H. et al. Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses. Cell 160, 619–630 (2015).This work shows that poliovirus particles can be packaged into phosphatidylserine-enriched vesicles that allow en bloc transmission between cells.
Too, I. H. K. et al. Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway. Sci. Rep. 6, 36983 (2016).
McKnight, K. L. et al. Protein composition of the hepatitis A virus quasi-envelope. Proc. Natl Acad. Sci. USA 114, 6587–6592 (2017).
Feng, Z. et al. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 496, 367–371 (2013).
Bandyopadhyay, A. S., Garon, J., Seib, K. & Orenstein, W. A. Polio vaccination: past, present and future. Future Microbiol. 10, 791–808 (2015).
Knowlson, S. et al. New strains intended for the production of inactivated polio vaccine at low-containment after eradication. PloS Pathog. 11, e1005316 (2015).
Pfeiffer, J. K. & Kirkegaard, K. A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity. Proc. Natl Acad. Sci. USA 100, 7289–7294 (2003).
Vignuzzi, M., Stone, J. K., Arnold, J. J., Cameron, C. E. & Andino, R. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature 439, 344–348 (2006).
Xiao, Y. et al. RNA Recombination Enhances Adaptability and Is Required for Virus Spread and Virulence. Cell Host Microbe 19, 493–503 (2016).
Coleman, J. R. et al. Virus attenuation by genome-scale changes in codon pair bias. Science 320, 1784–1787 (2008).
Lee, S. et al. A polyvalent inactivated rhinovirus vaccine is broadly immunogenic in rhesus macaques. Nat. Commun. 7, 12838 (2016).
Fox, H., Knowlson, S., Minor, P. D. & Macadam, A. J. Genetically thermo-stabilised, immunogenic poliovirus empty capsids; a strategy for non-replicating vaccines. PloS Pathog. 13, e1006117 (2017).
Adeyemi, O. O., Nicol, C., Stonehouse, N. J. & Rowlands, D. J. Increasing type-1 poliovirus capsid stability by thermal selection. J. Virol. 91, e01586–16 (2017).
Marsian, J. et al. Plant-made polio type 3 stabilized VLPs — a candidate synthetic polio vaccine. Nat. Commun. 8, 245 (2017).
Jiang, P. et al. Evidence for emergence of diverse polioviruses from C-cluster coxsackie A viruses and implications for global poliovirus eradication. Proc. Natl Acad. Sci. USA 104, 9457–9462 (2007).
Ho, M. et al. An epidemic of enterovirus 71 infection in Taiwan. N. Engl. J. Med. 341, 929–935 (1999).
Liu, S.-L. et al. Comparative epidemiology and virology of fatal and nonfatal cases of hand, foot and mouth disease in mainland China from 2008 to 2014. Rev. Med. Virol. 25, 115–128 (2015).
Xing, W. et al. Hand, foot, and mouth disease in China, 2008-12: an epidemiological study. Lancet Infect. Dis. 14, 308–318 (2014).
Greninger, A. L. et al. A novel outbreak enterovirus D68 strain associated with acute flaccid myelitis cases in the USA (2012–2014): a retrospective cohort study. Lancet Infect. Dis. 3099, 10–12 (2015).
Messacar, K. et al. Acute flaccid myelitis: a clinical review of US cases 2012–2015. Ann. Neurol. 80, 326–338 (2016).
Pevear, D. C., Tull, T. M., Seipel, M. E. & Groarke, J. M. Activity of pleconaril against enteroviruses. Antimicrob. Agents Chemother. 43, 2109–2115 (1999).
De Palma, A. M. et al. Potential use of antiviral agents in polio eradication. Emerg. Infect. Dis. 14, 545–551 (2008).
Tijsma, A. et al. The capsid binder vapendavir and the novel protease inhibitor SG85 inhibit enterovirus 71 replication. Antimicrob. Agents Chemother. 58, 6990–6992 (2014).
Sun, L. et al. Antiviral activity of broad-spectrum and enterovirus-specific inhibitors against clinical isolates of enterovirus D68. Antimicrob. Agents Chemother. 59, 7782–7785 (2015).
Rhoden, E., Zhang, M., Nix, W. A. & Oberste, M. S. In vitro efficacy of antiviral compounds against enterovirus D68. Antimicrob. Agents Chemother. 59, 1–13 (2015).
Mello, C. et al. Multiple classes of antiviral agents exhibit in vitro activity against human rhinovirus type C. Antimicrob. Agents Chemother. 58, 1546–1555 (2014).
Patick, A. K. et al. In vitro antiviral activity of AG7088, a potent inhibitor of human rhinovirus 3 C protease. Antimicrob. Agents Chemother. 43, 2444–2450 (1999).
Feil, S. C. et al. An orally available 3-ethoxybenzisoxazole capsid binder with clinical activity against human rhinovirus. ACS Med. Chem. Lett. 3, 303–307 (2012).
Rhoden, E., Liu, H.-M., Wang-Chern, S. & Oberste, M. S. Anti-poliovirus activity of protease inhibitor AG-7404, and assessment of in vitro activity in combination with antiviral capsid inhibitor compounds. Antiviral Res. 98, 186–191 (2013).
Buontempo, P. J. et al. SCH 48973: a potent, broad-spectrum, antienterovirus compound. Antimicrob. Agents Chemother. 41, 1220–1225 (1997).
Collett, M. S. et al. Antiviral activity of pocapavir in a randomized, blinded, placebo-controlled human oral poliovirus vaccine challenge model. J. Infect. Dis. 215, 335–343 (2017).
De Colibus, L. et al. More-powerful virus inhibitors from structure-based analysis of HEV71 capsid-binding molecules. Nat. Struct. Mol. Biol. 21, 282–288 (2014).
Patick, A. K. et al. In vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3 C protease. Antimicrob. Agents Chemother. 49, 2267–2275 (2005).
Ma, G.-H. et al. Identification and biochemical characterization of DC07090 as a novel potent small molecule inhibitor against human enterovirus 71 3 C protease by structure-based virtual screening. Eur. J. Med. Chem. 124, 981–991 (2016).
Kang, H. et al. Synergistic antiviral activity of gemcitabine and ribavirin against enteroviruses. Antiviral Res. 124, 1–10 (2015).
Deng, C.-L. et al. Inhibition of enterovirus 71 by adenosine analog NITD008. J. Virol. 88, 11915–11923 (2014).
Crotty, S. et al. The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. Nat. Med. 6, 1375–1379 (2000).
Andersen, D., Murray, B., Robins, R. & North, J. In vitro antiviral activity of ribavirin against picornaviruses. Antivir. Chem. Chemother. 3, 361–370 (1992).
Harrison, D. N., Gazina, E. V., Purcell, D. F., Anderson, D. A. & Petrou, S. Amiloride derivatives inhibit coxsackievirus B3 RNA replication. J. Virol. 82, 1465–1473 (2008).
Ulferts, R. et al. Screening of a library of FDA-approved drugs identifies several enterovirus replication inhibitors that target viral protein 2C. Antimicrob. Agents Chemother. 60, 2627–2638 (2016).
Hadaschik, D., Klein, M., Zimmermann, H., Eggers, H. J. & Nelsen-Salz, B. Dependence of echovirus 9 on the enterovirus RNA replication inhibitor 2-(alpha-Hydroxybenzyl)-benzimidazole maps to nonstructural protein 2C. J. Virol. 73, 10536–10539 (1999).
van der Schaar, H. M. et al. A novel, broad-spectrum inhibitor of enterovirus replication that targets host cell factor phosphatidylinositol 4-kinase IIIbeta. Antimicrob. Agents Chemother. 57, 4971–4981 (2013).
Arita, M., Dobrikov, G., Purstinger, G. & Galabov, A. S. Allosteric regulation of phosphatidylinositol 4-kinase III beta by an antipicornavirus compound MDL-860. ACS Infect. Dis. 3, 585–594 (2017).
Torney, H. L., Dulworth, J. K. & Steward, D. L. Antiviral activity and mechanism of action of 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860). Antimicrob. Agents Chemother. 22, 635–638 (1982).
Albulescu, L. et al. Broad-range inhibition of enterovirus replication by OSW-1, a natural compound targeting OSBP. Antiviral Res. 117, 110–114 (2015).
Tan, Y. W., Hong, W. J. & Chu, J. J. H. Inhibition of enterovirus VP4 myristoylation is a potential antiviral strategy for hand, foot and mouth disease. Antiviral Res. 133, 191–195 (2016).
Yamayoshi, S. et al. Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus 71. J. Virol. 86, 5686–5696 (2012).
Nishimura, Y. & Shimizu, H. Cellular receptors for human enterovirus species A. Front. Microbiol. 3, 105 (2012).
Yamayoshi, S., Fujii, K. & Koike, S. Receptors for enterovirus 71. Emerg. Microbes Infect. 3, e53 (2014).
Royston, L. & Tapparel, C. Rhinoviruses and respiratory enteroviruses: not as simple as ABC. Viruses 8, E16 (2016).
Baggen, J. et al. Enterovirus D68 receptor requirements unveiled by haploid genetics. Proc. Natl Acad. Sci. USA 113, 1399–1404 (2016).This study identifies both α2,3-linked and α2,6-linked sialic acids as EV-D68 receptors and reveals the existence of strains that do not require sialic acid.
Hofer, F. et al. Members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus. Proc. Natl Acad. Sci. USA 91, 1839–1842 (1994).
Bergelson, J. M. et al. Decay-accelerating factor (CD55), a glycosylphosphatidylinositol-anchored complement regulatory protein, is a receptor for several echoviruses. Proc. Natl Acad. Sci. USA 91, 6245–6248 (1994).
Martino, T. A. et al. The coxsackie-adenovirus receptor (CAR) is used by reference strains and clinical isolates representing all six serotypes of coxsackievirus group B and by swine vesicular disease virus. Virology 271, 99–108 (2000).
Williams, Ç. H. et al. Integrin αvβ6 Is an RGD-Dependent Receptor for Coxsackievirus A9. J. Virol. 78, 6967–6973 (2004).
Bergelson, J. M., Shepley, M. P., Chan, B. M., Hemler, M. E. & Finberg, R. W. Identification of the integrin VLA-2 as a receptor for echovirus 1. Science 255, 1718–1720 (1992).
Acknowledgements
Research in the authors’ laboratory is supported by grants from the Netherlands Organisation for Scientific Research (NWO-VICI-91812628 and NWO-ECHO-711.017.002 to F.J.M.v.K.) and from the European Union (Horizon2020 Marie Sklodowska Curie ETN ‘ANTIVIRALS’ grant agreement number 642434 to F.J.M.v.K.).
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Nature Reviews Microbiology thanks S. Hafenstein, Q. Li and S.-R. Shih for their contributions to the peer review of this work.
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J.B., H.J.T., J.R.P.M.S. and F.J.M.v.K. researched data for the article and substantially contributed to discussion of content, wrote the article and reviewed and edited the manuscript before submission.
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Glossary
- Acute flaccid paralysis
-
A type of paralysis that is characterized by a sudden weakness or loss of muscle tone.
- Hand-foot-and-mouth disease
-
A viral disease in which characteristic vesicular lesions appear on the hands, feet and inside the mouth.
- Herpangina
-
A viral infection of the mouth that is characterized by blisters in the throat.
- Pleurodynia
-
A viral disease that is characterized by a sudden pain in the muscles between the ribs.
- Pericarditis
-
An inflammation of the pericardium, the membrane that surrounds the heart and the beginning of the great vessels.
- Myocarditis
-
An inflammation of the heart muscle.
- Bronchiolitis
-
An inflammation of the bronchioles that is caused by a viral infection.
- Replication organelles
-
Virus-induced membranous structures that are generated from remodelled host membranes that accumulate in the cytoplasmic area of the cell and that are thought to support replication by scaffolding the replication machinery and occluding the viral RNA from antiviral defence systems.
- Protomers
-
Assembly intermediates of an enterovirus capsid that is composed of one copy of each of the structural proteins VP0, VP1 and VP3.
- Chronic shedders
-
Immunodeficient people who excrete vaccine-derived poliovirus for prolonged times.
- Myristoylation
-
A saturated fatty acid that is linked to the N terminus of structural protein VP4 and has a role in both the entry and assembly process.
- Poly-l-proline type II helices
-
Helical protein structures that consist of repeating proline residues and are frequently involved in protein–protein and protein–nucleic acid interactions.
- High-speed fixed-target X-ray crystallography at X-ray free-electron lasers
-
A novel method in which X-ray free-electron lasers that generate high-intensity X-ray pulses and sample fixation on a micro-patterned pore-containing chip are combined to allow very fast structure determination with limited beam time and sample material.
- Telencephalic grey matter
-
An area of the cerebral cortex that contains the nerve bodies.
- Sedimentation coefficients
-
Quantities that describe the sedimentation rate of a particle when centrifuged in a fluid medium, which depends on particle size, density and shape.
- GH loop
-
A structural loop in structural protein VP1 that forms the border between the hydrophobic pocket and the bottom of the canyon.
- Phospholipase A2 group XVI
-
(PLA2G16). A lipid-modifying enzyme that catalyses the release of fatty acids from phospholipids in adipose tissue and has been proposed to have acyltransferase activity.
- Phospholipase A2 domain
-
A domain with phospholipase A2 catalytic activity; that is, it cleaves off fatty acids from the second hydroxyl group of the glycerol backbone of a phospholipid.
- Internal ribosome entry site
-
(IRES). A highly structured RNA element in the 5′ UTR that allows cap-independent initiation of translation.
- IRES trans-acting factors
-
(ITAFs). Host RNA-binding proteins that bind to the enteroviral IRES and stimulate translation (for example, polypyrimidine tract-binding protein 2, poly(rC)-binding protein 2, polyadenylate-binding protein 1, La and serine/arginine-rich splicing factor 3).
- Poly(rC)-binding protein 2
-
(PCBP2). A host RNA-binding protein that functions as an ITAF to stimulate IRES-mediated translation and that binds to a replication element in the 5′ UTR to stimulate viral RNA replication.
- Polyadenylate-binding protein 1
-
(PABP1). A host protein that binds the viral poly(A) tail and interacts with poly(rC)-binding protein 2 to mediate circularization of the viral genome and initiate negative-strand RNA synthesis.
- Heterogeneous nuclear ribonucleoprotein C
-
(HNRNPC). A host protein that binds the 5′ and 3′ ends of enterovirus genomes and stabilizes interactions between them.
- Acyl-CoA-binding domain-containing 3
-
(ACBD3). A protein that regulates function and structure of the Golgi complex through its interaction with the integral membrane protein giantin.
- Phosphatidylinositol 4-kinase-β
-
(PI4KB). A Golgi-localized lipid kinase that phosphorylates phosphatidylinositol to yield phosphatidylinositol-4-phosphate.
- Oxysterol-binding protein
-
(OSBP). A protein that exchanges phosphatidylinositol-4-phosphate lipids and cholesterol at membrane contact sites.
- Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1
-
(GBF1). A guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) proteins that plays important roles in intracellular membrane transport and homeostasis.
- Autophagy
-
A cellular catabolic process in which intracellular parts of the cell are engulfed by membranes and targeted for breakdown, which yields energy or building blocks or clears aged or damaged parts of a cell.
- Soaking
-
An approach in crystallography in which a protein is crystallized, followed by addition of a ligand to form a co-crystal, allowing determination of the structure of the complex.
- Co-crystallization
-
An approach in crystallography in which a protein and its ligand are crystallized together, forming a co-crystal that is used to determine the structure of the complex.
- Glutathione
-
A tripeptide composed of glutamate, cysteine and glycine that has an important role in cellular redox reactions and as an antioxidant.
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Baggen, J., Thibaut, H.J., Strating, J.R.P.M. et al. The life cycle of non-polio enteroviruses and how to target it. Nat Rev Microbiol 16, 368–381 (2018). https://doi.org/10.1038/s41579-018-0005-4
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DOI: https://doi.org/10.1038/s41579-018-0005-4
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