Neurotropic Enterovirus Research Articles

Vascular endothelial growth factor receptor 2 as a potential host target for the inhibition of enterovirus replication.

As the first clinical case was identified in the United States, EV-A71, a significant neurotropic enterovirus, has been a common cause of hand, foot, and mouth disease (HFMD) in infants and young children. Developing an effective antiviral agent for EV-A71 and other human enteroviruses is crucial, as these viral pathogens consistently cause outbreaks in humans. In this study, we demonstrated that multiple inhibitors against VEGFRs effectively reduced EV-A71 replication, with Pazopanib emerging as the top candidate. Furthermore, Pazopanib also attenuated the replication of other enteroviruses, including CVA10, CVB1, EV-D70, and HRV-A, displaying broad-spectrum anti-enterovirus activity. Given that Pazopanib targets various VEGFRs, we narrowed the focus to VEGFR2 using knockdown and overexpression experiments. Transcriptomic analysis suggests that Pazopanib's potential downstream targets involve the TSAd-Src-PI3K-Akt pathway. Our work may contribute to identifying targets for antiviral inhibitors and advancing treatments for human enterovirus infections.

Doxycycline inhibits neurotropic enterovirus proliferation in vitro

Human enteroviruses (EVs) represent a global public health concern due to their association with a range of serious pediatric illnesses. Despite the high morbidity and mortality exerted by EVs, no broad-spectrum antivirals are currently available. Herein, we presented evidence that doxycycline can inhibit in vitro replication of various neurotropic EVs, including enterovirus A71 (EV-A71), enterovirus D68 (EV-D68), and coxsackievirus (CV)-A6, in a dose-dependent manner. Further investigations indicated that the drug primarily acted at the post-entry stage of virus infection in vitro, with inhibitory effects reaching up to 89 % for EV-A71 when administered two hours post-infection. These findings provide valuable insights for the development of antiviral drugs against EV infections.

Modulation of plasmacytoid dendritic cell and CD4+ T cell differentiation accompanied by upregulation of the cholinergic anti-inflammatory pathway induced by enterovirus 71.

Enterovirus 71 (EV71) is a neurotropic enterovirus associated with hand, foot, and mouth disease (HFMD) fatalities. In this study, we investigated the impact of EV71 on plasmacytoid dendritic cells (pDCs) and CD4+ T cells. The results showed that pDCs were promptly activated, secreting interferon (IFN)-α and inducing CD4+ T cell proliferation and differentiation during early EV71 infection. This initiated adaptive immune responses and promoted proinflammatory cytokine production by CD4+ T cells. Over time, viral nucleic acids and proteins were synthesized in pDCs and CD4+ T cells. Concurrently, the cholinergic anti-inflammatory pathway (CAP) was activated, exhibiting an anti-inflammatory role. With constant viral stimulation, pDCs and CD4+ T cells showed reduced differentiation and cytokine secretion. Defects in pDCs were identified as a key factor in CD4+ T cell tolerance. CAP had a more significant regulatory effect on CD4+ T cells than on pDCs and was capable of inhibiting inflammation in these cells.

Neurotropic enteroviruses (Picornaviridae: Enterovirus): predominant types, basis of neurovirulence.

Enteroviruses are one of the most common causative agents of infectious diseases of the central nervous system. They are characterized by genetic variability, the ability to infect a wide range of cells, including brain microglial cells and astrocytes, and persist in the central nervous system tissue, causing delayed and chronic diseases. The review presents data on the basis of neurovirulence of non-polio enteroviruses and the most common pathogens causing enteroviral neuroinfections.

Amino acid variation at VP1-145 of enterovirus A71 determines the viral infectivity and receptor usage in a primary human intestinal model.

With the near eradication of polio worldwide, polio-like illness (as is increasingly caused by EV-A71 infections) is of emerging concern. EV-A71 is indeed the most neurotropic enterovirus that poses a major threat globally to public health and specifically in infants and young children. Our findings will contribute to the understanding of the virulence and the pathogenicity of this virus. Further, our data also supports the identification of potential therapeutic targets against severe EV-A71 infection especially among infants and young children. Furthermore, our work highlights the key role of HSPG-binding mutations in the disease outcome of EV-A71. Additionally, EV-A71 is not able to infect the gut (the primary replication site in humans) in traditionally used animal models. Thus, our research highlights the need for human-based models to study human viral infections.Graphical Abstract.

Acute flaccid myelitis in Switzerland - association with enterovirus D68.

Poliomyelitis-like acute flaccid myelitis associated with enterovirus D68 (EV-D68) has emerged globally during the past decade. Here we describe the first documented case reported from Switzerland, and a second, suspected case occurring in temporal association. AFM occurs primarily in children, is usually heralded by a febrile, respiratory prodrome followed by acute-onset, usually asymmetrical, limb weakness with some predilection for the upper extremities, and respiratory muscle compromise in one third of reported cases. There is no specific therapy and the majority of cases result in permanent neurological sequelae. A comprehensive diagnostic workup and timely reporting to the health authorities are essential. Surveillance of respiratory and stool samples for EV-D68 and other neurotropic enteroviruses is in place in several European countries and warrants consideration in Switzerland. This could entail the extension of the poliomyelitis surveillance program of the Federal Office of Public Health by monitoring and enteroviral typing of respiratory samples from patients with acute flaccid paralysis.

RNA Structure in the 5' Untranslated Region of Enterovirus D68 Strains with Differing Neurovirulence Phenotypes.

Enterovirus-D68 (EV-D68) is a positive-sense single-stranded RNA virus within the family Picornaviridae. EV-D68 was initially considered a respiratory virus that primarily affected children. However, in 2014, EV-D68 outbreaks occurred causing the expected increase in respiratory illness cases, but also an increase in acute flaccid myelitis cases (AFM). Sequencing of 2014 outbreak isolates revealed variations in the 5' UTR of the genome compared to the historical Fermon strain. The structure of the 5' UTR RNA contributes to enterovirus virulence, including neurovirulence in poliovirus, and could contribute to neurovirulence in contemporary EV-D68 strains. In this study, the secondary and tertiary structures of 5' UTR RNA from the Fermon strain and 2014 isolate KT347251.1 are analyzed and compared. Secondary structures were determined using SHAPE-MaP and TurboFold II and tertiary structures were predicted using 3dRNAv2.0. Comparison of RNA structures between the EV-D68 strains shows significant remodeling at the secondary and tertiary levels. Notable secondary structure changes occurred in domains II, IV and V. Shifts in the secondary structure changed the tertiary structure of the individual domains and the orientation of the domains. Our comparative structural models for EV-D68 5' UTR RNA highlight regions of the molecule that could be targeted for treatment of neurotropic enteroviruses.

Effects and mechanism of Aβ1−42 on EV-A71 replication

Backgroundβ-Amyloid (Aβ) protein is a pivotal pathogenetic factor in Alzheimer’s disease (AD). However, increasing evidence suggests that the brain has to continuously produce excessive Aβ to efficaciously prevent pathogenic micro-organism infections, which induces and accelerates the disease process of AD. Meanwhile, Aβ exhibits activity against herpes simplex virus type 1 (HSV-1) and influenza A virus (IAV) replication, but not against other neurotropic viruses. Enterovirus A71 (EV-A71) is the most important neurotropic enterovirus in the post-polio era. Given the limitation of existing research on the relationship between Aβ and other virus infections, this study aimed to investigate the potent activity of Aβ on EV-A71 infection and extended the potential function of Aβ in other unenveloped viruses may be linked to Alzheimer's disease or infectious neurological diseases.MethodsAβ peptides 1–42 are a major pathological factor of senile plaques in Alzheimer’s disease (AD). Thus, we utilized Aβ1–42 as a test subject to perform our study. The production of monomer Aβ1–42 and their high-molecular oligomer accumulations in neural cells were detected by immunofluorescence assay, ELISA, or Western blot assay. The inhibitory activity of Aβ1–42 peptides against EV-A71 in vitro was detected by Western blot analysis or qRT-PCR. The mechanism of Aβ1–42 against EV-A71 replication was analyzed by time-of-addition assay, attachment inhibition assay, pre-attachment inhibition analysis, viral-penetration inhibition assay, TEM analysis of virus agglutination, and pull-down assay.ResultsWe found that EV-A71 infection induced Aβ production and accumulation in SH-SY5Y cells. We also revealed for the first time that Aβ1–42 efficiently inhibited the RNA level of EV-A71 VP1, and the protein levels of VP1, VP2, and nonstructural protein 3AB in SH-SY5Y, Vero, and human rhabdomyosarcoma (RD) cells. Mechanistically, we demonstrated that Aβ1–42 primarily targeted the early stage of EV-A71 entry to inhibit virus replication by binding virus capsid protein VP1 or scavenger receptor class B member 2. Moreover, Aβ1–42 formed non-enveloped EV-A71 particle aggregates within a certain period and bound to the capsid protein VP1, which partially caused Aβ1–42 to prevent viruses from infecting cells.ConclusionsOur findings unveiled that Aβ1–42 effectively inhibited nonenveloped EV-A71 by targeting the early phase of an EV-A71 life cycle, thereby extending the potential function of Aβ in other non-envelope viruses linked to infectious neurological diseases.

SLC35B2 Acts in a Dual Role in the Host Sulfation Required for EV71 Infection

As an important neurotropic enterovirus, enterovirus 71 (EV71) is occasionally associated with severe neurological diseases and high mortality rates in infants and young children. Understanding the interaction between host factors and EV71 will play a vital role in developing antivirals and optimizing vaccines. Here, we performed a genome-wide CRISPR-Cas9 knockout screen and revealed that scavenger receptor class B member 2 (SCARB2), solute carrier family 35 member B2 (SLC35B2), and beta-1,3-glucuronyltransferase 3 (B3GAT3) are essential in facilitating EV71 replication. Subsequently, the exploration of molecular mechanisms suggested that the knockout of SLC35B2 or B3GAT3, not SCARB2, led to a remarkable decrease in the binding of EV71 to cells and internalization into cells. Furthermore, we found that the infection efficiency for EV71 was positively correlated with the level of host cell sulfation, not simply with the amount of heparan sulfate, suggesting that an unidentified sulfated protein(s) must contribute to EV71 infection. In support of this idea, we screened possible sulfated proteins among the proteinous receptors for EV71 and confirmed that SCARB2 could uniquely interact with both tyrosyl protein sulfotransferases in humans. We then performed mass spectrometric analysis of SCARB2, identifying five sites with tyrosine sulfation. The function verification test indicated that there were more than five tyrosine-sulfated sites on SCARB2. Finally, we constructed a model for EV71 entry in which both heparan sulfate and SCARB2 are regulated by SLC35B2 and act cooperatively to support viral binding, internalization, and uncoating. Taken together, this is the first time that we performed the pooled CRISPR-Cas9 genetic screening to investigate the interplay of host cells and EV71. Furthermore, we found that a novel host factor, SLC35B2, played a dual role in regulating the overall sulfation comprising heparan sulfate sulfation and protein tyrosine sulfation, which are critical for EV71 entry. IMPORTANCE As the most important nonpolio neurotropic enterovirus lacking specific treatments, EV71 can transmit to the central nervous system, leading to severe and fatal neurological complications in infants and young children. The identification of new factors that facilitate or inhibit EV71 replication is crucial to uncover the mechanisms of viral infection and pathogenesis. To date, only a few host factors involved in EV71 infection have been characterized. Herein, we conducted a genome-wide CRISPR-Cas9 functional knockout (GeCKO) screen for the first time to study EV71 in HeLa cells. The screening results are presented as a ranked list of candidates, including 518 hits in the positive selection that facilitate EV71 replication and 1,044 hits in the negative selection that may be essential for cell growth and survival or for suppressing EV71 infection. We subsequently concentrated on the top three hits in the positive selection: SCARB2, SLC35B2, and B3GAT3. The knockout of any of these three genes confers strong resistance against EV71 infection. We confirmed that EV71 infection is codependent on two receptors, heparan sulfate and SCARB2. We also identified a host entry factor, SLC35B2, indirectly facilitating EV71 infection through regulation of the host cell sulfation, and determined a novel posttranslational modification, protein tyrosine sulfation existing in SCARB2. This study revealed that EV71 infectivity exhibits a significant positive correlation with the level of cellular sulfation regulated by SLC35B2. Due to the sulfation pathway being required for many distinct viruses, including but not limited to EV71 and respiratory syncytial virus (RSV), which were tested in this study, SLC35B2 represents a target of broad-spectrum antiviral therapy.

Return of the Neurotropic Enteroviruses: Co-Opting Cellular Pathways for Infection.

Enteroviruses are among the most common human infectious agents. While infections are often mild, the severe neuropathogenesis associated with recent outbreaks of emerging non-polio enteroviruses, such as EV-A71 and EV-D68, highlights their continuing threat to public health. In recent years, our understanding of how non-polio enteroviruses co-opt cellular pathways has greatly increased, revealing intricate host–virus relationships. In this review, we focus on newly identified mechanisms by which enteroviruses hijack the cellular machinery to promote their replication and spread, and address their potential for the development of host-directed therapeutics. Specifically, we discuss newly identified cellular receptors and their contribution to neurotropism and spread, host factors required for viral entry and replication, and recent insights into lipid acquisition and replication organelle biogenesis. The comprehensive knowledge of common cellular pathways required by enteroviruses could expose vulnerabilities amenable for host-directed therapeutics against a broad spectrum of enteroviruses. Since this will likely include newly arising strains, it will better prepare us for future epidemics. Moreover, identifying host proteins specific to neurovirulent strains may allow us to better understand factors contributing to the neurotropism of these viruses.

Molecular Pathogenicity of Enteroviruses Causing Neurological Disease.

Enteroviruses are single-stranded positive-sense RNA viruses that primarily cause self-limiting gastrointestinal or respiratory illness. In some cases, these viruses can invade the central nervous system, causing life-threatening neurological diseases including encephalitis, meningitis and acute flaccid paralysis (AFP). As we near the global eradication of poliovirus, formerly the major cause of AFP, the number of AFP cases have not diminished implying a non-poliovirus etiology. As the number of enteroviruses linked with neurological disease is expanding, of which many had previously little clinical significance, these viruses are becoming increasingly important to public health. Our current understanding of these non-polio enteroviruses is limited, especially with regards to their neurovirulence. Elucidating the molecular pathogenesis of these viruses is paramount for the development of effective therapeutic strategies. This review summarizes the clinical diseases associated with neurotropic enteroviruses and discusses recent advances in the understanding of viral invasion of the central nervous system, cell tropism and molecular pathogenesis as it correlates with host responses.

Viral engagement with host receptors blocked by a novel class of tryptophan dendrimers that targets the 5-fold-axis of the enterovirus-A71 capsid.

Enterovirus A71 (EV-A71) is a non-polio neurotropic enterovirus with pandemic potential. There are no antiviral agents approved to prevent or treat EV-A71 infections. We here report on the molecular mechanism by which a novel class of tryptophan dendrimers inhibits (at low nanomolar to high picomolar concentration) EV-A71 replication in vitro. A lead compound in the series (MADAL385) prevents binding and internalization of the virus but does not, unlike classical capsid binders, stabilize the particle. By means of resistance selection, reverse genetics and cryo-EM, we map the binding region of MADAL385 to the 5-fold vertex of the viral capsid and demonstrate that a single molecule binds to each vertex. By interacting with this region, MADAL385 prevents the interaction of the virus with its cellular receptors PSGL1 and heparan sulfate, thereby blocking the attachment of EV-A71 to the host cells.

Neurotropic enteroviruses co-opt "fair-weather-friend" commensal gut microbiota to drive host infection and central nervous system disturbances.

Some neurotropic enteroviruses hijack Trojan horse/raft commensal gut bacteria to render devastating biomimicking cryptic attacks on human/animal hosts. Such virus-microbe interactions manipulate hosts' gut-brain axes with accompanying infection-cycle-optimizing central nervous system (CNS) disturbances, including severe neurodevelopmental, neuromotor, and neuropsychiatric conditions. Co-opted bacteria thus indirectly influence host health, development, behavior, and mind as possible "fair-weather-friend" symbionts, switching from commensal to context-dependent pathogen-like strategies benefiting gut-bacteria fitness.

Seroepidemiological and phylogenetic characterization of neurotropic enteroviruses in Ireland, 2005-2014.

Enteroviruses (EVs) are associated with a broad spectrum of clinical presentation, including aseptic meningitis (AM), encephalitis, hand, foot and mouth disease, acute flaccid paralysis, and acute flaccid myelitis. Epidemics occur sporadically and are associated with increased cases of AM in children. The present study describes the seroepidemiological analysis of circulating EVs in Ireland from 2005 to 2014 and phylogenetic characterization of echovirus 30 (E-30), enterovirus A71 (EV-A71), and enterovirus D68 (EV-D68). EV VP1 genotyping was applied to viral isolates and clinical samples, including cerebrospinal fluid (CSF), and those isolates that remained untypeable by neutralising anti-sera. An increase in AM cases from 2010 to 2014 was associated with an E-30 genogroup variant VII and sequences clustered phylogenetically with those detected in AM outbreaks in France and Italy. EV-D68 viral RNA was not detected in CSF samples and no neurological involvement was reported. Three EV-A71 positive CSF samples were identified in patients presenting with AM. A phylogenetic analysis of respiratory-associated EV-D68 and EV-A71 cases in circulation was performed to determine baseline epidemiological data. EV-D68 segregated with clades B and B(1) and EV-A71 clustered as subgenogroup C2. The EV VP1 genotyping method was more sensitive than neutralising anti-sera methods by virus culture and importantly demonstrated concordance between EV genotypes in faecal and CSF samples which should facilitate EV screening by less invasive sampling approaches in AM presentations.

VP1 residues around the five-fold axis of enterovirus A71 mediate heparan sulfate interaction

Enterovirus A71 (EV-A71) is a neurotropic enterovirus that uses heparan sulfate as an attachment receptor. The molecular determinants of EV-A71-heparan sulfate interaction are unknown. With In silico heparin docking and mutagenesis of all possible lysine residues in VP1, we identified that K162, K242 and K244 are responsible for heparin interaction and inhibition. EV-A71 mutants with K242A and K244A rapidly acquired compensatory mutations, T100K or E98A, and Q145R-T237N respectively, which restored the heparin-binding phenotype. Both VP1-98 and VP1-145 modulates heparin binding. Heparin-binding phenotype was completely abolished with VP1-E98-E145, but was restored by an E98K or E145Q substitution. During cell culture adaptation, EV-A71 rapidly acquired K98 or Q/G145 to restore the heparin-binding phenotype. Together with next-generation sequencing analysis, our results implied that EV-A71 has high genetic plasticity by modulating positively-charged residues at the five-fold axis during in vitro heparin adaptation. Our finding has impact on EV-A71 vaccine production, evolutionary studies and pathogenesis.

Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication

Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets.

Enterovirus encephalitis including Enterovirus 71 and D68

Abstract: Enteroviruses are small RNA viruses that belong to several species groups (A to H and J) in the genus Enterovirus and family Piconarviridae. Central nervous system (CNS) involvement in enterovirus infections is well known although it is a relatively rare complication. Several neurological syndromes have been described including aseptic meningitis, acute flaccid paralysis (myelitis), and the more serious encephalitis. Perhaps the best known neurotropic enterovirus for more than a century is the poliovirus, although other enteroviruses have also been associated with neurological syndromes. Except perhaps for poliovirus and enterovirus A71 (EV-A71), many reports were anecdotal and causative links yet to be firmly established, partly because of the difficulty of demonstrating virus in the cerebrospinal fluid or CNS tissues. Based on poliovirus studies, enteroviral neuroinvasion had been long thought to be via haematogenous and peripheral nerve routes. Recent evidence from transgenic mouse models has confirmed poliovirus neuroinvasion by peripheral nerves. Human and animal studies have provided good evidence that EV-A71 may enter the CNS via spinal and cranial motor nerves by retrograde axonal transport but other neural pathways other than the motor pathway may help spread virus within the CNS. Brain MRI scans in echovirus 7, coxsackievirus A16, EV-A71 and enterovirus D68 (EV-D68) encephalitides often show similar lesions in the anterior horns of the spinal cord, dorsal brainstem and cerebellar dentate nucleus. If confirmed by autopsy and other studies, these findings could suggest a common pathway for enteroviral neuroinvasion via peripheral motor nerves. The neuron in the CNS is the main viral target so neuronal viral cytolysis/necrosis is probably responsible for CNS injury for most enteroviruses. However, other mechanisms of cell injury such as apoptosis and immunopathogenesis may have a role and should be investigated. With better molecular techniques and MRI facilities, the diagnosis of enterovirus encephalitis should improve in the future. A greater understanding of the pathology and neuropathogenesis of non-poliovirus encephalitides is important with the imminent worldwide eradication of poliovirus, and the emergence of enteroviruses such as EV-A71 and EV-D68, which capable of causing epidemic encephalitides.

Neurotropic Enterovirus Infections in the Central Nervous System.

Enteroviruses are a group of positive-sense single stranded viruses that belong to the Picornaviridae family. Most enteroviruses infect humans from the gastrointestinal tract and cause mild symptoms. However, several enteroviruses can invade the central nervous system (CNS) and result in various neurological symptoms that are correlated to mortality associated with enteroviral infections. In recent years, large outbreaks of enteroviruses occurred worldwide. Therefore, these neurotropic enteroviruses have been deemed as re-emerging pathogens. Although these viruses are becoming large threats to public health, our understanding of these viruses, especially for non-polio enteroviruses, is limited. In this article, we review recent advances in the trafficking of these pathogens from the peripheral to the central nervous system, compare their cell tropism, and discuss the effects of viral infections in their host neuronal cells.

Understanding Enterovirus 71 Neuropathogenesis and Its Impact on Other Neurotropic Enteroviruses.

Enterovirus A71 (EV-A71) belongs to the species group A in the Enterovirus genus within the Picornaviridae family. EV-A71 usually causes self-limiting hand, foot and mouth disease or herpangina but rarely causes severe neurological complications such as acute flaccid paralysis and encephalomyelitis. The pathology and neuropathogenesis of these neurological syndromes is beginning to be understood. EV-A71 neurotropism for motor neurons in the spinal cord and brainstem, and other neurons, is mainly responsible for central nervous system damage. This review on the general aspects, recent developments and advances of EV-A71 infection will focus on neuropathogenesis and its implications on other neurotropic enteroviruses, such as poliovirus and the newly emergent Enterovirus D68. With the imminent eradication of poliovirus, EV-A71 is likely to replace it as an important neurotropic enterovirus of worldwide importance.

Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone.

RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.