RESUMO
RNA interference (RNAi) is the major antiviral mechanism in plants and invertebrates, but the absence of detectable viral (v)siRNAs in mammalian cells upon viral infection has questioned the functional relevance of this pathway in mammalian immunity. We designed a series of peptides specifically targeting enterovirus A71 (EV-A71)-encoded protein 3A, a viral suppressor of RNAi (VSR). These peptides abrogated the VSR function of EV-A71 in infected cells and resulted in the accumulation of vsiRNAs and reduced viral replication. These vsiRNAs were functional, as evidenced by RISC-loading and silencing of target RNAs. The effects of VSR-targeting peptides (VTPs) on infection with EV-A71 as well as another enterovirus, Coxsackievirus-A16, were ablated upon deletion of Dicer1 or AGO2, core components of the RNAi pathway. In vivo, VTP treatment protected mice against lethal EV-A71 challenge, with detectable vsiRNAs. Our findings provide evidence for the functional relevance of RNAi in mammalian immunity and present a therapeutic strategy for infectious disease.
Assuntos
Antivirais/farmacologia , Infecções por Enterovirus/virologia , RNA Viral/antagonistas & inibidores , Animais , Chlorocebus aethiops , Enterovirus Humano A , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/farmacologia , Interferência de RNA , RNA Interferente Pequeno/antagonistas & inibidores , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Human enteroviruses are the most common human pathogen with over 300 distinct genotypes. Previous work with poliovirus has suggested that it is possible to generate antibody responses in humans and animals that can recognize members of multiple enterovirus species. However, cross protective immunity across multiple enteroviruses is not observed epidemiologically in humans. Here we investigated whether immunization of mice or baboons with inactivated poliovirus or enterovirus virus-like-particles (VLPs) vaccines generates antibody responses that can recognize enterovirus D68 or A71. We found that mice only generated antibodies specific for the antigen they were immunized with, and repeated immunization failed to generate cross-reactive antibody responses as measured by both ELISA and neutralization assay. Immunization of baboons with IPV failed to generate neutralizing antibody responses against enterovirus D68 or A71. These results suggest that a multivalent approach to enterovirus vaccination is necessary to protect against enterovirus disease in vulnerable populations.
Assuntos
Anticorpos Antivirais , Reações Cruzadas , Infecções por Enterovirus , Vacina Antipólio de Vírus Inativado , Animais , Camundongos , Reações Cruzadas/imunologia , Anticorpos Antivirais/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/prevenção & controle , Infecções por Enterovirus/virologia , Vacina Antipólio de Vírus Inativado/imunologia , Vacina Antipólio de Vírus Inativado/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/imunologia , Anticorpos Neutralizantes/imunologia , Papio/imunologia , Humanos , Poliovirus/imunologia , Feminino , Formação de Anticorpos/imunologia , Enterovirus/imunologia , Camundongos Endogâmicos BALB C , Enterovirus Humano D/imunologiaRESUMO
RNA recombination in positive-strand RNA viruses is a molecular-genetic process, which permits the greatest evolution of the genome and may be essential to stabilizing the genome from the deleterious consequences of accumulated mutations. Enteroviruses represent a useful system to elucidate the details of this process. On the biochemical level, it is known that RNA recombination is catalyzed by the viral RNA-dependent RNA polymerase using a template-switching mechanism. For this mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. How a viral genome is trafficked to the site of genome replication and recombination, which is membrane associated and isolated from the cytoplasm, is not known. We hypothesized that genome translation was essential for colocalization of genomes for recombination. We show that complete inactivation of internal ribosome entry site (IRES)-mediated translation of a donor enteroviral genome enhanced recombination instead of impairing it. Recombination did not occur by a nonreplicative mechanism. Rather, sufficient translation of the nonstructural region of the genome occurred to support subsequent steps required for recombination. The noncanonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation. Our results support an eIF2A/eIF2D-dependent mechanism under conditions in which the eIF2-dependent mechanism is inactive. Detection of an IRES-independent mechanism for translation of the enterovirus genome provides an explanation for a variety of debated observations, including nonreplicative recombination and persistence of enteroviral RNA lacking an IRES. The existence of an eIF2A/eIF2D-dependent mechanism in enteroviruses predicts the existence of similar mechanisms in other viruses.
Assuntos
Infecções por Enterovirus , Enterovirus , Humanos , Enterovirus/fisiologia , Infecções por Enterovirus/virologia , Sítios Internos de Entrada Ribossomal , Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Interações Hospedeiro-PatógenoRESUMO
Enterovirus 71 (EV71) is a significant causative agent of hand, foot, and mouth disease, with potential serious neurologic complications or fatal outcomes. The lack of effective treatments for EV71 infection is attributed to its elusive pathogenicity. Our study reveals that human plasmacytoid dendritic cells (pDCs), the main type I IFN-producing cells, selectively express scavenger receptor class B, member 2 (SCARB2) and P-selectin glycoprotein ligand 1 (PSGL-1), crucial cellular receptors for EV71. Some strains of EV71 can replicate within pDCs and stimulate IFN-α production. The activation of pDCs by EV71 is hindered by Abs to PSGL-1 and soluble PSGL-1, whereas Abs to SCARB2 and soluble SCARB2 have a less pronounced effect. Our data suggest that only strains binding to PSGL-1, more commonly found in severe cases, can replicate in pDCs and induce IFN-α secretion, highlighting the importance of PSGL-1 in these processes. Furthermore, IFN-α secretion by pDCs can be triggered by EV71 or UV-inactivated EV71 virions, indicating that productive infection is not necessary for pDC activation. These findings provide new insights into the interaction between EV71 and pDCs, suggesting that pDC activation could potentially mitigate the severity of EV71-related diseases.
Assuntos
Células Dendríticas , Enterovirus Humano A , Interferon-alfa , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana , Células Dendríticas/imunologia , Células Dendríticas/virologia , Humanos , Enterovirus Humano A/imunologia , Enterovirus Humano A/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Lisossomal/imunologia , Interferon-alfa/metabolismo , Interferon-alfa/imunologia , Receptores Depuradores/metabolismo , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Replicação ViralRESUMO
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.
Assuntos
Infecções por Enterovirus , Enterovirus , Doença de Mão, Pé e Boca , Animais , Criança , Humanos , Camundongos , Anticorpos Antivirais , Modelos Animais de Doenças , Enterovirus/patogenicidade , Enterovirus/fisiologia , Enterovirus Humano A , Infecções por Enterovirus/patologia , Infecções por Enterovirus/virologia , Expressão Gênica , Doença de Mão, Pé e Boca/genética , Fosfatidilinositol 3-Quinases , VirulênciaRESUMO
Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease in children under 5 years old, which can result in severe neurological complications and even death. Due to limited treatments for EV71 infection, the identification of novel host factors and elucidation of mechanisms involved will help to counter this viral infection. N-terminal acetyltransferase 6 (NAT6) was identified as an essential host factor for EV71 infection with genome-wide CRISPR/Cas9 screening. NAT6 facilitates EV71 viral replication depending on its acetyltransferase activity but has little effect on viral release. In addition, NAT6 is also required for Echovirus 7 and coxsackievirus B5 infection, suggesting it might be a pan-enterovirus host factor. We further demonstrated that NAT6 is required for Golgi integrity and viral replication organelle (RO) biogenesis. NAT6 knockout significantly inhibited phosphatidylinositol 4-kinase IIIß (PI4KB) expression and PI4P production, both of which are key host factors for enterovirus infection and RO biogenesis. Further mechanism studies confirmed that NAT6 formed a complex with its substrate actin and one of the PI4KB recruiters-acyl-coenzyme A binding domain containing 3 (ACBD3). Through modulating actin dynamics, NAT6 maintained the integrity of the Golgi and the stability of ACBD3, thereby enhancing EV71 infection. Collectively, these results uncovered a novel mechanism of N-acetyltransferase supporting EV71 infection.IMPORTANCEEnterovirus 71 (EV71) is an important pathogen for children under the age of five, and currently, no effective treatment is available. Elucidating the mechanism of novel host factors supporting viral infection will reveal potential antiviral targets and aid antiviral development. Here, we demonstrated that a novel N-acetyltransferase, NAT6, is an essential host factor for EV71 replication. NAT6 could promote viral replication organelle (RO) formation to enhance viral replication. The formation of enterovirus ROs requires numerous host factors, including acyl-coenzyme A binding domain containing 3 (ACBD3) and phosphatidylinositol 4-kinase IIIß (PI4KB). NAT6 could stabilize the PI4KB recruiter, ACBD3, by inhibiting the autophagy degradation pathway. This study provides a fresh insight into the relationship between N-acetyltransferase and viral infection.
Assuntos
Enterovirus Humano A , Infecções por Enterovirus , Acetiltransferases N-Terminal , Fosfotransferases (Aceptor do Grupo Álcool) , Criança , Pré-Escolar , Humanos , 1-Fosfatidilinositol 4-Quinase/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antivirais , Coenzima A/metabolismo , Infecções por Coxsackievirus , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/virologia , Proteínas de Membrana/metabolismo , Acetiltransferases N-Terminal/metabolismo , Biogênese de Organelas , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Replicação Viral/fisiologiaRESUMO
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.
Assuntos
Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Fatores de Troca do Nucleotídeo Guanina , Homeostase , Resposta a Proteínas não Dobradas , Humanos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Infecções por Enterovirus/virologia , Infecções por Enterovirus/metabolismo , Apoptose , Enterovirus/fisiologia , Enterovirus/metabolismo , Células HeLa , Replicação Viral , Fator 1 de Ribosilação do ADP/metabolismo , Fator 1 de Ribosilação do ADP/genética , Células HEK293 , Interações Hospedeiro-Patógeno , Transdução de Sinais , eIF-2 Quinase/metabolismoRESUMO
The globally reemerging respiratory pathogen enterovirus D68 (EV-D68) is implicated in outbreaks of severe respiratory illness and associated with acute flaccid myelitis. However, there remains a lack of effective treatments for EV-D68 infection. In this work, we found that the host Toll-like receptor 7 (TLR7) proteins, which function as powerful innate immune sensors, were selectively elevated in expression in response to EV-D68 infection. Subsequently, we investigated the impact of Vesatolimod (GS-9620), a Toll-like receptor 7 agonist, on EV-D68 replication. Our findings revealed that EV-D68 infection resulted in increased mRNA levels of TLR7. Treatment with Vesatolimod significantly inhibited EV-D68 replication [half maximal effective concentration (EC50) = 0.1427 µM] without inducing significant cytotoxicity at virucidal concentrations. Although Vesatolimod exhibited limited impact on EV-D68 attachment, it suppressed RNA replication and viral protein synthesis after virus entry. Vesatolimod broadly inhibited the replication of circulating isolated strains of EV-D68. Furthermore, our findings demonstrated that treatment with Vesatolimod conferred resistance to both respiratory and neural cells against EV-D68 infection. Overall, these results present a promising strategy for drug development by pharmacologically activating TLR7 to initiate an antiviral state in EV-D68-infected cells selectively.IMPORTANCEConventional strategies for antiviral drug development primarily focus on directly targeting viral proteases or key components, as well as host proteins involved in viral replication. In this study, based on our intriguing discovery that enterovirus D68 (EV-D68) infection specifically upregulates the expression of immune sensor Toll-like receptor 7 (TLR7) protein, which is either absent or expressed at low levels in respiratory cells, we propose a potential antiviral approach utilizing TLR7 agonists to activate EV-D68-infected cells into an anti-viral defense state. Notably, our findings demonstrate that pharmacological activation of TLR7 effectively suppresses EV-D68 replication in respiratory tract cells through a TLR7/MyD88-dependent mechanism. This study not only presents a promising drug candidate and target against EV-D68 dissemination but also highlights the potential to exploit unique alterations in cellular innate immune responses induced by viral infections, selectively inducing a defensive state in infected cells while safeguarding uninfected normal cells from potential adverse effects associated with therapeutic interventions.
Assuntos
Antivirais , Enterovirus Humano D , Receptor 7 Toll-Like , Replicação Viral , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/metabolismo , Humanos , Replicação Viral/efeitos dos fármacos , Enterovirus Humano D/efeitos dos fármacos , Antivirais/farmacologia , Indóis/farmacologia , Infecções por Enterovirus/virologia , Imunidade Inata/efeitos dos fármacos , Linhagem Celular , Internalização do Vírus/efeitos dos fármacos , PteridinasRESUMO
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.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Quinase I-kappa B , NF-kappa B , Proteínas Virais , Replicação Viral , Animais , Humanos , Chlorocebus aethiops , Chaperona BiP do Retículo Endoplasmático/metabolismo , Enterovirus/crescimento & desenvolvimento , Enterovirus/imunologia , Enterovirus/metabolismo , Enterovirus/fisiologia , Infecções por Enterovirus/virologia , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/imunologia , Proteínas de Choque Térmico/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Quinase I-kappa B/metabolismo , Imunidade Inata , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , RNA Polimerase Dependente de RNA/metabolismo , Transdução de Sinais , Células Vero , Proteínas Virais/metabolismoRESUMO
Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory and neurologic disease [acute flaccid myelitis (AFM)]. Intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with US/IL/14-18952 (IL52), a clinical isolate from the 2014 EV-D68 epidemic, results in many of the pathogenic features of human AFM, including viral infection of the spinal cord, death of motor neurons, and resultant progressive paralysis. In distinction, CA/14-4231 (CA4231), another clinical isolate from the 2014 EV-D68 outbreak, does not cause paralysis in mice, does not grow in the spinal cord, and does not cause motor neuron loss following IM injection. A panel of chimeric viruses containing sequences from IL52 and CA4231 was used to demonstrate that VP1 is the main determinant of EV-D68 neurovirulence following IM injection of neonatal SW mice. VP1 contains four amino acid differences between IL52 and CA4231. Mutations resulting in substituting these four amino acids (CA4231 residues into the IL52 polyprotein) completely abolished neurovirulence. Conversely, mutations resulting in substituting VP1 IL52 amino acid residues into the CA4231 polyprotein created a virus that induced paralysis to the same degree as IL52. Neurovirulence following infection of neonatal SW mice with parental and chimeric viruses was associated with viral growth in the spinal cord. IMPORTANCE: Emerging viruses allow us to investigate mutations leading to increased disease severity. Enterovirus D68 (EV-D68), once the cause of rare cases of respiratory illness, recently acquired the ability to cause severe respiratory and neurologic disease. Chimeric viruses were used to demonstrate that viral structural protein VP1 determines growth in the spinal cord, motor neuron loss, and paralysis following intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with EV-D68. These results have relevance for predicting the clinical outcome of future EV-D68 epidemics as well as targeting retrograde transport as a potential strategy for treating virus-induced neurologic disease.
Assuntos
Proteínas do Capsídeo , Viroses do Sistema Nervoso Central , Modelos Animais de Doenças , Enterovirus Humano D , Infecções por Enterovirus , Mielite , Doenças Neuromusculares , Animais , Enterovirus Humano D/patogenicidade , Enterovirus Humano D/genética , Enterovirus Humano D/fisiologia , Mielite/virologia , Camundongos , Infecções por Enterovirus/virologia , Infecções por Enterovirus/patologia , Doenças Neuromusculares/virologia , Doenças Neuromusculares/patologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Viroses do Sistema Nervoso Central/virologia , Viroses do Sistema Nervoso Central/patologia , Humanos , Medula Espinal/virologia , Medula Espinal/patologia , Neurônios Motores/virologia , Neurônios Motores/patologia , Animais Recém-Nascidos , Virulência , Paralisia/virologiaRESUMO
Enterovirus D68 (EV-D68) is a picornavirus associated with severe respiratory illness and a paralytic disease called acute flaccid myelitis in infants. Currently, no protective vaccines or antivirals are available to combat this virus. Like other enteroviruses, EV-D68 uses components of the cellular autophagy pathway to rewire membranes for its replication. Here, we show that transcription factor EB (TFEB), the master transcriptional regulator of autophagy and lysosomal biogenesis, is crucial for EV-D68 infection. Knockdown of TFEB attenuated EV-D68 genomic RNA replication but did not impact viral binding or entry into host cells. The 3C protease of EV-D68 cleaves TFEB at the N-terminus at glutamine 60 (Q60) immediately post-peak viral RNA replication, disrupting TFEB-RagC interaction and restricting TFEB transport to the surface of the lysosome. Despite this, TFEB remained mostly cytosolic during EV-D68 infection. Overexpression of a TFEB mutant construct lacking the RagC-binding domain, but not the wild-type construct, blocks autophagy and increases EV-D68 nonlytic release in H1HeLa cells but not in autophagy-defective ATG7 KO H1HeLa cells. Our results identify TFEB as a vital host factor regulating multiple stages of the EV-D68 lifecycle and suggest that TFEB could be a promising target for antiviral development against EV-D68. IMPORTANCE: Enteroviruses are among the most significant causes of human disease. Some enteroviruses are responsible for severe paralytic diseases such as poliomyelitis or acute flaccid myelitis. The latter disease is associated with multiple non-polio enterovirus species, including enterovirus D68 (EV-D68), enterovirus 71, and coxsackievirus B3 (CVB3). Here, we demonstrate that EV-D68 interacts with a host transcription factor, transcription factor EB (TFEB), to promote viral RNA(vRNA) replication and regulate the egress of virions from cells. TFEB was previously implicated in the viral egress of CVB3, and the viral protease 3C cleaves TFEB during infection. Here, we show that EV-D68 3C protease also cleaves TFEB after the peak of vRNA replication. This cleavage disrupts TFEB interaction with the host protein RagC, which changes the localization and regulation of TFEB. TFEB lacking a RagC-binding domain inhibits autophagic flux and promotes virus egress. These mechanistic insights highlight how common host factors affect closely related, medically important viruses differently.
Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Enterovirus Humano D , Infecções por Enterovirus , Replicação Viral , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Humanos , Enterovirus Humano D/fisiologia , Enterovirus Humano D/metabolismo , Enterovirus Humano D/genética , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/virologia , Proteases Virais 3C/metabolismo , Lisossomos/metabolismo , RNA Viral/metabolismo , RNA Viral/genética , Proteínas Virais/metabolismo , Proteínas Virais/genética , Mielite/metabolismo , Mielite/virologia , Ligação Proteica , Células HEK293 , Doenças Neuromusculares , Viroses do Sistema Nervoso CentralRESUMO
Pyroptosis, a pro-inflammatory programmed cell death, has been implicated in the pathogenesis of coronavirus disease 2019 and other viral diseases. Gasdermin family proteins (GSDMs), including GSDMD and GSDME, are key regulators of pyroptotic cell death. However, the mechanisms by which virus infection modulates pyroptosis remain unclear. Here, we employed a mCherry-GSDMD fluorescent reporter assay to screen for viral proteins that impede the localization and function of GSDMD in living cells. Our data indicated that the main protease NSP5 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) blocked GSDMD-mediated pyroptosis via cleaving residues Q29 and Q193 of GSDMD. While another SARS-CoV-2 protease, NSP3, cleaved GSDME at residue G370 but activated GSDME-mediated pyroptosis. Interestingly, respiratory enterovirus EV-D68-encoded proteases 3C and 2A also exhibit similar differential regulation on the functions of GSDMs by inactivating GSDMD but initiating GSDME-mediated pyroptosis. EV-D68 infection exerted oncolytic effects on human cancer cells by inducing pyroptotic cell death. Our findings provide insights into how respiratory viruses manipulate host cell pyroptosis and suggest potential targets for antiviral therapy as well as cancer treatment.IMPORTANCEPyroptosis plays a crucial role in the pathogenesis of coronavirus disease 2019, and comprehending its function may facilitate the development of novel therapeutic strategies. This study aims to explore how viral-encoded proteases modulate pyroptosis. We investigated the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory enterovirus D68 (EV-D68) proteases on host cell pyroptosis. We found that SARS-CoV-2-encoded proteases NSP5 and NSP3 inactivate gasdermin D (GSDMD) but initiate gasdermin E (GSDME)-mediated pyroptosis, respectively. We also discovered that another respiratory virus EV-D68 encodes two distinct proteases 2A and 3C that selectively trigger GSDME-mediated pyroptosis while suppressing the function of GSDMD. Based on these findings, we further noted that EV-D68 infection triggers pyroptosis and produces oncolytic effects in human carcinoma cells. Our study provides new insights into the molecular mechanisms underlying virus-modulated pyroptosis and identifies potential targets for the development of antiviral and cancer therapeutics.
Assuntos
Endopeptidases , Enterovirus Humano D , Interações entre Hospedeiro e Microrganismos , Vírus Oncolíticos , Piroptose , SARS-CoV-2 , Humanos , Linhagem Celular Tumoral , COVID-19/metabolismo , COVID-19/terapia , COVID-19/virologia , Endopeptidases/genética , Endopeptidases/metabolismo , Enterovirus Humano D/enzimologia , Enterovirus Humano D/genética , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/virologia , Gasderminas/antagonistas & inibidores , Gasderminas/genética , Gasderminas/metabolismo , Terapia Viral Oncolítica , Vírus Oncolíticos/enzimologia , Vírus Oncolíticos/genética , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
In December 2023, we observed through hospital-based surveillance a severe outbreak of enterovirus D68 infection in pediatric inpatients in Dakar, Senegal. Molecular characterization revealed that subclade B3, the dominant lineage in outbreaks worldwide, was responsible for the outbreak. Enhanced surveillance in inpatient settings, including among patients with neurologic illnesses, is needed.
Assuntos
Surtos de Doenças , Enterovirus Humano D , Infecções por Enterovirus , Infecções Respiratórias , Humanos , Senegal/epidemiologia , Enterovirus Humano D/genética , Enterovirus Humano D/classificação , Enterovirus Humano D/isolamento & purificação , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Infecções por Enterovirus/diagnóstico , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Pré-Escolar , Lactente , Criança , Filogenia , Masculino , Feminino , Doença Aguda/epidemiologia , Adolescente , Hospitais , História do Século XXIRESUMO
IMPORTANCE: Coxsackievirus A6 (CV-A6) is a major emerging pathogen associated with atypical hand, foot, and mouth disease and can cause serious complications such as encephalitis, acute flaccid paralysis, and neurorespiratory syndrome. Therefore, revealing the associated pathogenic mechanisms could benefit the control of CV-A6 infections. In this study, we demonstrate that the nonstructural 2CCV-A6 suppresses IFN-ß production, which supports CV-A6 infection. This is achieved by depleting RNA sensors such as melanoma differentiation-associated gene 5 and retinoic acid-inducible gene I (RIG-I) through the lysosomal pathway. Such a function is shared by 2CEV-A71 and 2CCV-B3 but not 2CCV-A16, suggesting the latter might have an alternative way to promote viral replication. This study broadens our understanding of enterovirus 2C protein regulation of the RIG-I-like receptor signaling pathway and reveals a novel mechanism by which CV-A6 and other enteroviruses evade the host innate immune response. These findings on 2C may provide new therapeutic targets for the development of effective inhibitors against CV-A6 and other enterovirus infections.
Assuntos
Infecções por Coxsackievirus , Humanos , Enterovirus Humano A/genética , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/virologia , Doença de Mão, Pé e Boca/virologia , Imunidade Inata , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/virologia , Interferon beta/metabolismoRESUMO
IMPORTANCE: Enterovirus D68 (EV-D68) is an emerging respiratory pathogen associated with acute flaccid myelitis. Currently, no approved vaccines or antiviral drugs are available. Here, we report four functionally independent neutralizing antigenic sites (I to IV) by analyses of neutralizing monoclonal antibody (MAb)-resistant mutants. Site I is located in the VP1 BC loop near the fivefold axis. Site II resides in the VP2 EF loop, and site III is situated in VP1 C-terminus; both sites are located at the south rim of the canyon. Site IV is composed of residue in VP2 ßB strand and residues in the VP3 BC loop and resides around the threefold axis. The developed MAbs targeting the antigenic sites can inhibit viral binding to cells. These findings advance the understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape and also have important implications for the development of novel EV-D68 vaccines.
Assuntos
Anticorpos Neutralizantes , Proteínas do Capsídeo , Enterovirus Humano D , Infecções por Enterovirus , Humanos , Capsídeo , Proteínas do Capsídeo/química , Enterovirus Humano D/genética , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologiaRESUMO
IMPORTANCE: EV71 poses a significant health threat to children aged 5 and below. The process of EV71 infection and replication is predominantly influenced by ubiquitination modifications. Our previous findings indicate that EV71 prompts the activation of host deubiquitinating enzymes, thereby impeding the host interferon signaling pathway as a means of evading the immune response. Nevertheless, the precise mechanisms by which the host employs ubiquitination modifications to hinder EV71 infection remain unclear. The present study demonstrated that the nonstructural protein 2Apro, which is encoded by EV71, exhibits ubiquitination and degradation mediated by the host E3 ubiquitin ligase SPOP. In addition, it is the first report, to our knowledge, that SPOP is involved in the host antiviral response.
Assuntos
Cisteína Endopeptidases , Enterovirus Humano A , Infecções por Enterovirus , Interações entre Hospedeiro e Microrganismos , Ubiquitina-Proteína Ligases , Ubiquitina , Ubiquitinação , Proteínas Virais , Criança , Humanos , Enterovirus Humano A/enzimologia , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/virologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Cisteína Endopeptidases/metabolismoRESUMO
Enterovirus D68 (EV-D68), which causes severe respiratory diseases and irreversible central nervous system damage, has become a serious public health problem worldwide. However, the mechanisms by which EV-D68 exerts neurotoxicity remain unclear. Thus, we aimed to analyze the effects of EV-D68 infection on the cleavage, subcellular translocation, and pathogenic aggregation of TAR DNA-binding protein 43 kDa (TDP-43) in respiratory or neural cells. The results showed that EV-D68-encoded proteases 2A and 3C induced TDP-43 translocation and cleavage, respectively. Specifically, 3C cleaved residue 327Q of TDP-43. The 3C-mediated cleaved TDP-43 fragments had substantially decreased protein solubility compared with the wild-type TDP-43. Hence, 3C activity promoted TDP-43 aggregation, which exerted cytotoxicity to diverse human cells, including glioblastoma T98G cells. The effects of commercially available antiviral drugs on 3C-mediated TDP-43 cleavage were screened, and the results revealed lopinavir as a potent inhibitor of EV-D68 3C protease. Overall, these results suggested TDP-43 as a conserved host target of EV-D68 3C. This study is the first to provide evidence on the involvement of TDP-43 dysregulation in EV-D68 pathogenesis. IMPORTANCE Over the past decade, the incidence of enterovirus D68 (EV-D68) infection has increased worldwide. EV-D68 infection can cause different respiratory symptoms and severe neurological complications, including acute flaccid myelitis. Thus, elucidating the mechanisms underlying EV-D68 toxicity is important to develop novel methods to prevent EV-D68 infection-associated diseases. This study shows that EV-D68 infection triggers the translocalization, cleavage, and aggregation of TDP-43, an intracellular protein closely related to degenerative neurological disorders. The viral protease 3C decreased TDP-43 solubility, thereby exerting cytotoxicity to host cells, including human glioblastoma cells. Thus, counteracting 3C activity is an effective strategy to relieve EV-D68-triggered cell death. Cytoplasmic aggregation of TDP-43 is a hallmark of degenerative diseases, contributing to neural cell damage and central nervous system (CNS) disorders. The findings of this study on EV-D68-induced TDP-43 formation extend our understanding of virus-mediated cytotoxicity and the potential risks of TDP-43 dysfunction-related cognitive impairment and neurological symptoms in infected patients.
Assuntos
Proteínas de Ligação a DNA , Infecções por Enterovirus , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Enterovirus Humano D , Infecções por Enterovirus/fisiopatologia , Infecções por Enterovirus/virologia , Linhagem Celular Tumoral , Proteases Virais 3C/metabolismo , Agregação Patológica de Proteínas/genética , Lopinavir/farmacologia , Proteólise/efeitos dos fármacos , Inativação Gênica , Inibidores de Proteases/farmacologiaRESUMO
Enteroviruses are important human pathogens with diverse serotypes, posing a major challenge to develop vaccines for individual serotypes, the success of polio vaccines in controlling and eradicating polio, along with the recent emergence and high prevalence of enterovirus-caused infectious diseases, highlights the importance of enterovirus vaccine development. Given our previous report on enteroviruses weakened by the 2 A S/T125A mutation, we assessed the potential of the EV-A71 2A-125A mutant as a vaccine candidate to address this challenge. We found that the 2A-125A mutant caused transient mild symptoms, low viral loads, and no significant pathological changes mild pathological changes in hSCARB2-KI mice, producing long-lasting cross-neutralizing antibodies against two EV-A71 wild strains. Pre-exposure to the 2A-125A mutant substantially protected against the EV-A71 Isehara wild-type strain, causing minor pathologies, significantly reducing muscle and lung inflammation, and preventing neurological damage, with reduced viral loads in vivo. Pre-exposure also distinctly suppressed the expression of pro-inflammatory cytokines, correlating to the severity of clinical symptoms. Collectively, the EV-A71 2A-125A mutant was attenuated and could generate a robust and protective immune response, suggesting its potential as a vaccine candidate and global solution for specific enterovirus vaccine development.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Enterovirus Humano A , Infecções por Enterovirus , Vacinas Atenuadas , Carga Viral , Vacinas Virais , Animais , Enterovirus Humano A/imunologia , Enterovirus Humano A/genética , Infecções por Enterovirus/prevenção & controle , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Camundongos , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Vacinas Virais/imunologia , Vacinas Virais/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/genética , Humanos , Desenvolvimento de Vacinas , Feminino , Mutação , CitocinasRESUMO
Enterovirus (EV) infections have various symptoms and severe complications, including death. To determine EV prevalence and EV types in Slovenia, data on over 25 000 EV RNA tests for diagnostics and surveillance from 2014 to 2023 were analyzed. Altogether, 3733 cerebrospinal fluid (CSF) and 21 297 respiratory (sentinel and clinical) samples were tested for EV RNA. EV typing was performed on all residual EV-positive CSF samples and on subset of respiratory specimens. Altogether, 1238 samples tested positive for EV RNA: 238 (6.4%) CSF and 1000 (4.7%) respiratory samples. EV-positive patients were predominantly male (p < 0.001). Many EV-positive CSF samples were from infants under 3 months (33.1%), whereas most EV-positive respiratory samples were from children 1 to 2 years old (49.2%). Echovirus 30 (E-30) was most frequent in CSF (33.0%), followed by CV-B5 (13.8%) and E-6 (13.8%). CV-A6 was most frequent in respiratory samples (16.0%), followed by EV-D68 (7.6%) and CV-A5 (7.4%). EV types in CSF and respiratory samples show diverse dynamics, with some outbreaks indicated. A significant difference was found in the EV detection rate between CSF and respiratory samples by age. Various EV types were characterized, showing that some EV types are more neurotropic or cause more severe infections.
Assuntos
Infecções por Enterovirus , Enterovirus , Epidemiologia Molecular , Humanos , Eslovênia/epidemiologia , Lactente , Masculino , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Infecções por Enterovirus/líquido cefalorraquidiano , Feminino , Pré-Escolar , Enterovirus/genética , Enterovirus/isolamento & purificação , Enterovirus/classificação , Criança , Adolescente , RNA Viral/genética , RNA Viral/líquido cefalorraquidiano , Infecções Respiratórias/virologia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/líquido cefalorraquidiano , Recém-Nascido , Adulto , Adulto Jovem , Prevalência , Líquido Cefalorraquidiano/virologia , Genótipo , Pessoa de Meia-Idade , Idoso , FilogeniaRESUMO
Enterovirus D68 (EV-D68) is an emerging agent for which data on the susceptible adult population is scarce. We performed a 6-year analysis of respiratory samples from influenza-like illness (ILI) admitted during 2014-2020 in 4-10 hospitals in the Valencia Region, Spain. EV-D68 was identified in 68 (3.1%) among 2210 Enterovirus (EV)/Rhinovirus (HRV) positive samples. Phylogeny of 59 VP1 sequences showed isolates from 2014 clustering in B2 (6/12), B1 (5/12), and A2/D1 (1/12) subclades; those from 2015 (n = 1) and 2016 (n = 1) in B3 and A2/D1, respectively; and isolates from 2018 in A2/D3 (42/45), and B3 (3/45). B1 and B2 viruses were mainly detected in children (80% and 67%, respectively); B3 were equally distributed between children and adults; whereas A2/D1 and A2/D3 were observed only in adults. B3 viruses showed up to 16 amino acid changes at predicted antigenic sites. In conclusion, two EV-D68 epidemics linked to ILI hospitalized cases occurred in the Valencia Region in 2014 and 2018, with three fatal outcomes and one ICU admission. A2/D3 strains from 2018 were associated with severe respiratory infection in adults. Because of the significant impact of non-polio enteroviruses in ILI and the potential neurotropism, year-round surveillance in respiratory samples should be pursued.