In vitro reconstitution reveals membrane clustering and RNA recruitment by the enteroviral AAA+ ATPase 2C.

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2C's association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divide the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.

The early communication stages between serine proteases and enterovirus capsids in the race for viral disintegration.

Serine proteases are important environmental contributors of enterovirus biocontrol. However, the structural features of molecular interaction accounting for the susceptibility of enteroviruses to proteases remains unexplained. Here, we describe the molecular mechanisms involved in the recruitment of serine proteases to viral capsids. Among the virus types used, coxsackievirus A9 (CVA9), but not CVB5 and echovirus 11 (E11), was inactivated by Subtilisin A in a host-independent manner, while Bovine Pancreatic Trypsin (BPT) only reduced CVA9 infectivity in a host-dependent manner. Predictive interaction models of each protease with capsid protomers indicate the main targets as internal disordered protein (IDP) segments exposed either on the 5-fold vertex (DE loop VP1) or at the 5/2-fold intersection (C-terminal end VP1) of viral capsids. We further show that a functional binding protease/capsid depends on both the strength and the evolution over time of protease-VP1 complexes, and lastly on the local adaptation of proteases on surrounding viral regions. Finally, we predicted three residues on CVA9 capsid that trigger cleavage by Subtilisin A, one of which may act as a sensor residue contributing to enzyme recognition on the DE loop. Overall, this study describes an important biological mechanism involved in enteroviruses biocontrol.

Exploration of Potential Broad-Spectrum Antiviral Targets in the Enterovirus Replication Element: Identification of Six Distinct 5' Cloverleaves.

Enterovirus genomic replication initiates at a predicted RNA cloverleaf (5'CL) at the 5' end of the RNA genome. The 5'CL contains one stem (SA) and three stem-loops (SLB, SLC, SLD). Here, we present an analysis of 5'CL conservation and divergence for 209 human health-related serotypes from the enterovirus genus, including enterovirus and rhinovirus species. Phylogenetic analysis indicates six distinct 5'CL serotypes that only partially correlate with the species definition. Additional findings include that 5'CL sequence conservation is higher between the EV species than between the RV species, the 5'CL of EVA and EVB are nearly identical, and RVC has the lowest 5'CL conservation. Regions of high conservation throughout all species include SA and the loop and nearby bases of SLB, which is consistent with known protein interactions at these sites. In addition to the known protein binding site for the Poly-C binding protein in the loop of SLB, other conserved consecutive cytosines in the stems of SLB and SLC provide additional potential interaction sites that have not yet been explored. Other sites of conservation, including the predicted bulge of SLD and other conserved stem, loop, and junction regions, are more difficult to explain and suggest additional interactions or structural requirements that are not yet fully understood. This more intricate understanding of sequence and structure conservation and variability in the 5'CL may assist in the development of broad-spectrum antivirals against a wide range of enteroviruses, while better defining the range of virus isotypes expected to be affected by a particular antiviral.

Enterovirus 3A protein disrupts endoplasmic reticulum homeostasis through interaction with GBF1.

Enteroviruses are single-stranded, positive-sense RNA viruses causing endoplasmic reticulum (ER) stress to induce or modulate downstream signaling pathways known as the unfolded protein responses (UPR). However, viral and host factors involved in the UPR related to viral pathogenesis remain unclear. In the present study, we aimed to identify the major regulator of enterovirus-induced UPR and elucidate the underlying molecular mechanisms. We showed that host Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1), which supports enteroviruses replication, was a major regulator of the UPR caused by infection with enteroviruses. In addition, we found that severe UPR was induced by the expression of 3A proteins encoded in human pathogenic enteroviruses, such as enterovirus A71, coxsackievirus B3, poliovirus, and enterovirus D68. The N-terminal-conserved residues of 3A protein interact with the GBF1 and induce UPR through inhibition of ADP-ribosylation factor 1 (ARF1) activation via GBF1 sequestration. Remodeling and expansion of ER and accumulation of ER-resident proteins were observed in cells infected with enteroviruses. Finally, 3A induced apoptosis in cells infected with enteroviruses via activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/C/EBP homologous protein (CHOP) pathway of UPR. Pharmaceutical inhibition of PERK suppressed the cell death caused by infection with enteroviruses, suggesting the UPR pathway is a therapeutic target for treating diseases caused by infection with enteroviruses.IMPORTANCEInfection caused by several plus-stranded RNA viruses leads to dysregulated ER homeostasis in the host cells. The mechanisms underlying the disruption and impairment of ER homeostasis and its significance in pathogenesis upon enteroviral infection remain unclear. Our findings suggested that the 3A protein encoded in human pathogenic enteroviruses disrupts ER homeostasis by interacting with GBF1, a major regulator of UPR. Enterovirus-mediated infections drive ER into pathogenic conditions, where ER-resident proteins are accumulated. Furthermore, in such scenarios, the PERK/CHOP signaling pathway induced by an unresolved imbalance of ER homeostasis essentially drives apoptosis. Therefore, elucidating the mechanisms underlying the virus-induced disruption of ER homeostasis might be a potential target to mitigate the pathogenesis of enteroviruses.

GRP78 recognizes EV-F 3D protein and activates NF-κB to repress virus replication by interacting with CHUK/IKBKB.

Enteroviruses are the causative agents associated with several human and animal diseases, posing a significant threat to human and animal health. As one of the host immune defense strategies, innate immunity plays a crucial role in defending against invading pathogens, where the host utilizes a variety of mechanisms to inhibit or eliminate the pathogen. Here, we report a new strategy for the host to repress enterovirus replication by the 78 kDa glucose-regulated protein (GRP78), also known as heat shock protein family A member 5 (HSPA5). The GRP78 recognizes the EV-encoded RNA-dependent RNA polymerases (RdRPs) 3D protein and interacts with the nuclear factor kappa B kinase complex (CHUK) and subunit beta gene (IKBKB) to facilitate the phosphorylation and nuclear translocation of NF-κB, which induces the production of inflammatory factors and leads to a broad inhibition of enterovirus replication. These findings demonstrate a new role of GRP78 in regulating host innate immunity in response to viral infection and provide new insights into the mechanism underlying enterovirus replication and NF-κB activation.IMPORTANCEGRP78 is known as a molecular chaperone for protein folding and plays a critical role in maintaining protein folding and participating in cell proliferation, cell survival, apoptosis, and metabolism. However, the functions of GRP78 to participate in enterovirus genome replication and innate immune responses are rarely documented. In this study, we explored the functions of the EV-3D-interacting protein GRP78 and found that GRP78 inhibits enterovirus replication by activating NF-κB through binding to EV-F 3D and interacting with the NF-κB signaling molecules CHUK/IKBKB. This is the first report that GRP78 interacts with CHUK/IKBKB to activate the NF-κB signaling pathway, which leads to the expression of the proinflammatory cytokines and inhibition of enterovirus replication. These results demonstrate a unique mechanism of virus replication regulation by GRP78 and provide insights into the prevention and treatment of viral 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.

NLRP3-dependent pyroptosis exacerbates coxsackievirus A16 and coxsackievirus A10-induced inflammatory response and viral replication in SH-SY5Y cells.

Coxsackievirus A16 (CV-A16) and coxsackievirus A10 (CV-A10), more commonly etiological agents of hand, foot and mouth disease (HFMD), are capable of causing severe neurological syndromes with high fatalities, but their neuropathogenesis has rarely been studied. Mounting evidence indicated that pyroptosis is an inflammatory form of cell death that might be widely involved in the pathogenic mechanisms of neurotropic viruses. Our study was designed to examine the effects of NLRP3-mediated pyroptosis in CV-A16- and CV-A10-induced inflammatory neuropathologic formation. In this work, it was showed that SH-SY5Y cells were susceptible to CV-A16 and CV-A10, and meanwhile their infections could result in a decreasing cell viability and an increasing LDH release as well as Caspase1 activation. Moreover, CV-A16 and CV-A10 infections triggered NLRP3-mediated pyroptosis and promoted the release of inflammatory cytokines. Additionally, activated NLRP3 accelerated the pyroptosis formation and aggravated the inflammatory response, but inhibited NLRP3 had a dampening effect on the above situation. Finally, it was further revealed that NLRP3 agonist enhanced the viral replication, but NLRP3 inhibitor suppressed the viral replication, suggesting that NLRP3-driven pyroptosis might support CV-A16 and CV-A10 production in SH-SY5Y cells. Together, our findings demonstrated a mechanism by which CV-A16 and CV-A10 induce inflammatory responses by evoking NLRP3 inflammasome-regulated pyroptosis, which in turn further stimulated the viral replication, providing novel insights into the pathogenesis of CV-A16 and CV-A10 infections.

Modulation of nucleotide metabolism by picornaviruses.

Viruses actively reprogram the metabolism of the host to ensure the availability of sufficient building blocks for virus replication and spreading. However, relatively little is known about how picornaviruses-a large family of small, non-enveloped positive-strand RNA viruses-modulate cellular metabolism for their own benefit. Here, we studied the modulation of host metabolism by coxsackievirus B3 (CVB3), a member of the enterovirus genus, and encephalomyocarditis virus (EMCV), a member of the cardiovirus genus, using steady-state as well as 13C-glucose tracing metabolomics. We demonstrate that both CVB3 and EMCV increase the levels of pyrimidine and purine metabolites and provide evidence that this increase is mediated through degradation of nucleic acids and nucleotide recycling, rather than upregulation of de novo synthesis. Finally, by integrating our metabolomics data with a previously acquired phosphoproteomics dataset of CVB3-infected cells, we identify alterations in phosphorylation status of key enzymes involved in nucleotide metabolism, providing insight into the regulation of nucleotide metabolism during infection.

The Effects of Mesenchymal Stem Cells Loaded with Oncolytic Coxsackievirus A21 on Mouse Models of Colorectal Cancer.

BACKGROUND: Cancer is a major cause of death worldwide. Colorectal cancer is the second most common type. Additional treatments like chemotherapy and radiation therapy may be recommended. Developing new techniques is vital due to drug resistance and a lack of targeted therapies. OBJECTIVE: In this study, the effects of mesenchymal stem cells (MSCs) loaded with oncolytic Coxsackievirus A21 (CVA21) on a mouse model of CRC were investigated. METHODS: The therapeutic potency of MSCs loaded with oncolytic CVA21 were evaluated in an experimental mouse model of colorectal cancer which received an injection CT26 cells per mouse subcutaneously. Splenocyte proliferation index, lactate dehydrogenase (LDH) assay, nitric oxide (NO) production assessment, and cytokine assay (IFN-γ, IL-4, IL-10, and TGF-ß) in the splenocyte supernatant were all used to evaluate the impact of MSCs loaded with CVA21. RESULTS: The results of this study showed that the treatment of a mouse model of colorectal cancer with MSCs loaded with oncolytic CVA21 could significantly suppress the tumor growth, which was accompanied by stimulation of splenocytes proliferation index, an increase of NO and LDH. Also, MSCs loaded with oncolytic CVA21 increased the secretion of IFN-γ and decreased the secretion of IL-4, IL-10, and TGF-ß. CONCLUSION: The results of the current study suggest that MSCs loaded with oncolytic CVA21 therapy for the CRC mouse model may have some potential advantages. On the other hand, the results of the study showed that, in addition to activating the acquired immune system, the use of MSCs loaded with oncolytic CVA21 also stimulates the innate immune system by increasing level of nitric oxide.

Emerging concerns of blood-brain barrier dysfunction caused by neurotropic enteroviral infections.

Enteroviruses (EVs), comprise a genus in the Picornaviridae family, which have been shown to be neurotropic and can cause various neurological disorders or long-term neurological condition, placing a huge burden on society and families. The blood-brain barrier (BBB) is a protective barrier that prevents dangerous substances from entering the central nervous system (CNS). Recently, numerous EVs have been demonstrated to have the ability to disrupt BBB, and further lead to severe neurological damage. However, the precise mechanisms of BBB disruption associated with these EVs remain largely unknown. In this Review, we focus on the molecular mechanisms of BBB dysfunction caused by EVs, emphasizing the invasiveness of enterovirus A71 (EVA71), which will provide a research direction for further treatment and prevention of CNS disorders.

Pathogenicity and landscape of differential gene expression in mice orally infected with clinical coxsackievirus A6 (CA6).

Hand, foot, and mouth disease (HFMD) is caused by more than 20 pathogenic enteroviruses belonging to the Picornaviridae family and Enterovirus genus. Since the introduction of the enterovirus-71 (EV71) vaccine in 2016, the number of HFMD cases caused by EV71 has decreased. However, cases of infections caused by other enteroviruses, such as coxsackievirus A6 (CA6) and coxsackievirus A10, have been increasing accordingly. In this study, we used a clinical isolate of CA6 to establish an intragastric infection mouse model using 7-day-old mice to mimic the natural transmission route, by which we investigated the differential gene expression profiles associated with virus infection and pathogenicity. After intragastric infection, mice exhibited hind limb paralysis symptoms and weight loss, similar to those reported for EV71 infection in mice. The skeletal muscle was identified as the main site of virus replication, with a peak viral load reaching 2.31 × 107 copies/mg at 5 dpi and increased infiltration of inflammatory cells. RNA sequencing analysis identified differentially expressed genes (DEGs) after CA6 infection. DEGs in the blood, muscle, brain, spleen, and thymus were predominantly enriched in immune system responses, including pathways such as Toll-like receptor signaling and PI3K-Akt signaling. Our study has unveiled the genes involved in the host immune response during CA6 infection, thereby enhancing our comprehension of the pathological mechanism of HFMD.IMPORTANCEThis study holds great significance for the field of hand, foot, and mouth disease (HFMD). It not only delves into the disease's etiology, transmission pathways, and severe complications but also establishes a novel mouse model that mimics the natural coxsackievirus A6 infection process, providing a pivotal platform to delve deeper into virus replication and pathogenic mechanisms. Additionally, utilizing RNA-seq technology, it unveils the dynamic gene expression changes during infection, offering valuable leads for identifying novel therapeutic drug targets. This research has the potential to enhance our understanding of HFMD, offering fresh perspectives for disease prevention and treatment and positively impacting children's health worldwide.

Anti- IL-22 neutralizing antibodies decrease inflammation lesions and reduce mortality in enterovirus 71-infected mice.

Hand, foot, and mouth disease (HFMD) can cause fatal encephalitis in 0-5-year-old infants and children. There is no effective antiviral drug available to treat HFMD caused by enterovirus 71 (EV71). Our study investigates the relationship between levels of IL-22 expression and the severity of disease after EV71 infection in a mouse model. Anti-IL-22 neutralizing antibodies were tested in EV71-infected mice of different ages. Our results show that anti-IL-22 neutralizing antibodies can effectively reduce mortality in EV71-infected mice. Anti-IL-22 neutralizing antibody effectively reduced various EV71-associated symptoms indicating promising potential of this therapeutic effector in patients with EV71-associated HFMD.

Toll-like receptor 3 (TLR3) and the development of type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) is a chronic, progressive autoimmune disease characterized by metabolic decompensation often leading to dehydration and ketoacidosis. Viral agents seem to play an important role in triggering the autoimmune destruction that leads to the development of T1DM. Among several viral strains investigated so far, the enterovirus family has been consistently associated with the onset of T1DM in humans. One of the mediators of viral damage is the double-stranded RNA (dsRNA) generated during replication and transcription of viral RNA and DNA. The Toll-like receptor 3 (TLR3) gene codes for an endoplasmic receptor of the pattern-recognition receptors (PRRs) family that recognizes dsRNA, plays an important role in the innate immune response triggered by viral infection. Binding of dsRNA to the TLR3 triggers the release of proinflammatory cytokines, such as interferons, which exhibit potent antiviral action; thus, protecting uninfected cells and inducing apoptosis of infected ones. Therefore, the TLR3 gene is a good candidate for the development of T1DM. Within this context, the objective of the present review was to address the role of the TLR3 gene in the development of T1DM. Arch Endocrinol Metab. 2015;59(1):4-12.

Enteroviruses as a possible cause of myocarditis, pericarditis and dilated cardiomyopathy in Belém, Brazil

We attempted to assess the role of enteroviruses in the etiology of myocarditis (MC), pericarditis (PC) and dilated cardiomyopathy (DCM) among 15 in-patients at a public hospital in Belém, Brazil, from November 1992 to December 1993. We obtained stool specimens and throat swabs from each patient (particularly acute cases) and, when possible, acute and convalescent serum samples for both isolation and serological procedures. MC, PC and DCM ocurred in 10, 2 and 3 patients, respectively, mostly in the 0- to 10- year age group. Neutralizing antibody seroconversions were detected as follows: one for Coxsackievirus (Cox) B2 in one patient suffering from MC, and two for Cox B4, in patients with DCM and MC. In addition, antibody titers of 1/320 against Cox B2 and Cox B4 were noted in two other patients, one suffering from PC and the other presenting MC. Isolation of echovirus (ECHO) serotype 1 was recorded ina a patient with MC, without either seroconversion or high antibody levels for Cox B 1 to 6. These results indicate that enteroviruses may be involved in the etiology of MC, PC and DCM in the Amazon region.