RESUMO
Coxsackievirus A16 (CVA16) causes hand, foot and mouth disease in infants and young children. However, no vaccine or anti-viral agent is currently available for CVA16. Here, the functions and working mechanisms of two CVA16-specific neutralizing monoclonal antibodies (MAbs), 9B5 and 8C4, are comprehensively investigated. Both 9B5 and 8C4 display potent neutralization in vitro and prophylactic and therapeutic efficacy in a mouse model of CVA16 infection. Mechanistically, 9B5 exerts neutralization primarily through inhibiting CVA16 attachment to cell surface via blockade of CVA16 binding to its attachment receptor, heparan sulfate, whereas 8C4 functions mainly at the post-attachment stage of CVA16 entry by interfering with the interaction between CVA16 and its uncoating receptor SCARB2. Cryo-EM studies show that 9B5 and 8C4 target distinct epitopes located at the 5-fold and 3-fold protrusions of CVA16 capsids, respectively, and exhibit differential binding preference to three forms of naturally occurring CVA16 particles. Moreover, 9B5 and 8C4 are compatible in formulating an antibody cocktail which displays the ability to prevent virus escape seen with individual MAbs. Together, our work elucidates the functional and structural basis of CVA16 antibody-mediated neutralization and protection, providing important information for design and development of effective CVA16 vaccines and antibody therapies.
Assuntos
Infecções por Coxsackievirus , Enterovirus Humano A , Enterovirus , Camundongos , Animais , Enterovirus Humano A/química , Anticorpos Neutralizantes , Capsídeo/química , Proteínas do Capsídeo/química , Enterovirus/químicaRESUMO
Even at low concentrations in environmental waters, some viruses are highly infective, making them a threat to human health. They are the leading cause of waterborne enteric diseases. In agriculture, plant viruses in irrigation and runoff water threat the crops. The low concentrations pose a challenge to early contamination detection. Thus, concentrating the virus particles into a small volume may be mandatory to achieve reliable detection in molecular techniques. This paper reviews the organic monoliths developments and their applications to concentrate virus particles from waters (waste, surface, tap, sea, and irrigation waters). Free-radical polymerization and polyaddition reactions are the most common strategies to prepare the monoliths currently used for virus concentration. Here, the routes for preparing and functionalizing both methacrylate and epoxy-based monoliths will be shortly described, following a revision of their retention mechanisms and applications in the concentration of enteric and plant viruses in several kinds of waters.
Assuntos
Cromatografia/métodos , Enterovirus/isolamento & purificação , Água Doce/virologia , Vírus de Plantas/isolamento & purificação , Polímeros/química , Ultrafiltração/métodos , Águas Residuárias/virologia , Irrigação Agrícola , Cromatografia/instrumentação , Enterovirus/química , Vírus de Plantas/química , Ultrafiltração/instrumentaçãoRESUMO
Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2Apro), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2Apro result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2Apro of genetic groups A and B hinder 2Apro as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2Apro cleave the substrate GAB1 at different sites, the 2Apro from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2Apro structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2Apro-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2Apro as a potential target for broad-spectrum anti-enteroviral inhibitors.
Assuntos
Antivirais/química , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Rhinovirus/enzimologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Cisteína Endopeptidases/genética , Enterovirus/química , Enterovirus/enzimologia , Fator de Iniciação Eucariótico 4G/metabolismo , Variação Genética , Células HeLa , Humanos , Conformação Proteica , Rhinovirus/química , Rhinovirus/genética , Especificidade por Substrato , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genéticaRESUMO
Cadherin Related Family Member 3 (CDHR3) is the identified and required cellular receptor for all virus isolates in the rhinovirus-C species (RV-C). Cryo-EM determinations recently resolved the atomic structure of RV-C15a, and subsequently, a complex of this virus bound to CDHR3 extracellular domain 1 (EC1), the N-terminal portion of this receptor responsible for virus interactions. The EC1 binds to a hypervariable sequence footprint on the virus surface, near the 3-fold axis of icosahedral symmetry. The key contacts involve discontinuous residues from 3 viral proteins, VP1, VP2 and VP3. That single cryo-EM EC1 structure, however, could not resolve whether the virus-receptor interface was structurally adaptable to accommodate multiple virus sequences. We now report the solution NMR determination of CDHR3 EC1, showing that this protein, in fact, is mostly inflexible, particularly in the virus-binding face. The new, higher resolution dataset identifies 3 cis-Pro residues in important loop regions, where they can influence both rigidity and overall protein conformation. The data also provide clarification about the residues involved in essential calcium ion binding, and a potential CDHR3 surface groove feature that may be involved in native protein interactions with cellular partners.
Assuntos
Caderinas/química , Enterovirus/química , Proteínas de Membrana/química , Proteínas Relacionadas a Caderinas , Enterovirus/classificação , Infecções por Enterovirus/virologia , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Virais/química , Ligação ViralRESUMO
JSPR is a single particle cryo-EM image processing and 3D reconstruction software developed in the Jiang laboratory at Purdue University. It began as a few refinement scripts for symmetric and asymmetric reconstructions of icosahedral viruses, but has grown into a comprehensive suite of tools for building ab initio reconstructions, high resolution refinements of viruses, protein complexes of arbitrary symmetries including helical tubes/filaments, and image file handling utilities. In this review, we will present examples achieved using JSPR and demonstrate recently implemented features of JSPR such as multi-aberration "alignments" and automatic optimization of masking for the assessment of map resolution using "true" FSC.
Assuntos
Microscopia Crioeletrônica/métodos , Imagem Individual de Molécula/métodos , Vírus/química , Bacteriófagos/química , Enterovirus/química , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Conformação Proteica , Software , Zika virus/químicaRESUMO
Enteroviruses are common agents of infectious disease that are spread by the fecal-oral route. They are readily inactivated by mild heat, which causes the viral capsid to disintegrate or undergo conformational change. While beneficial for the thermal treatment of food or water, this heat sensitivity poses challenges for the stability of enterovirus vaccines. The thermostability of an enterovirus can be modulated by the composition of the suspending matrix, though the effects of the matrix on virus stability are not understood. Here, we determined the thermostability of four enterovirus strains in solutions with various concentrations of NaCl and different pH values. The experimental findings were combined with molecular modeling of the protein interaction forces at the pentamer and the protomer interfaces of the viral capsids. While pH only had a modest effect on thermostability, increasing NaCl concentrations raised the breakpoint temperatures of all viruses tested by up to 20°C. This breakpoint shift could be explained by an enhancement of the van der Waals attraction forces at the two protein interfaces. In comparison, the (net repulsive) electrostatic interactions were less affected by NaCl. Depending on the interface considered, the breakpoint temperature shifted by 7.5 or 5.6°C per 100-kcal/(mol·Å) increase in protein interaction force.IMPORTANCE The genus Enterovirus encompasses important contaminants of water and food (e.g., coxsackieviruses), as well as viruses of acute public health concern (e.g., poliovirus). Depending on the properties of the surrounding matrix, enteroviruses exhibit different sensitivities to heat, which in turn influences their persistence in the environment, during food treatment, and during vaccine storage. Here, we determined the effect of NaCl and pH on the heat stability of different enteroviruses and related the observed effects to changes in protein interaction forces in the viral capsid. We demonstrate that NaCl renders enteroviruses thermotolerant and that this effect stems from an increase in van der Waals forces at different protein subunits in the viral capsid. This work sheds light on the mechanism by which salt enhances virus stability.
Assuntos
Proteínas do Capsídeo/química , Enterovirus/química , Modelos Moleculares , Animais , Linhagem Celular , Chlorocebus aethiops , Concentração de Íons de Hidrogênio , Estabilidade Proteica , Cloreto de Sódio , TemperaturaRESUMO
Infection by Rhinovirus-C (RV-C), a species of Picornaviridae Enterovirus, is strongly associated with childhood asthma exacerbations. Cellular binding and entry by all RV-C, which trigger these episodes, is mediated by the first extracellular domain (EC1) of cadherin-related protein 3 (CDHR3), a surface cadherin-like protein expressed primarily on the apical surfaces of ciliated airway epithelial cells. Although recombinant EC1 is a potent inhibitor of viral infection, there is no molecular description of this protein or its binding site on RV-C. Here we present cryo-electron microscopy (EM) data resolving the EC1 and EC1+2 domains of human CDHR3 complexed with viral isolate C15a. Structure-suggested residues contributing to required interfaces on both EC1 and C15a were probed and identified by mutagenesis studies with four different RV-C genotypes. In contrast to most other rhinoviruses, which bind intercellular adhesion molecule 1 receptors via a capsid protein VP1-specific fivefold canyon feature, the CDHR3 EC1 contacts C15a, and presumably all RV-Cs, in a unique cohesive footprint near the threefold vertex, encompassing residues primarily from viral protein VP3, but also from VP1 and VP2. The EC1+2 footprint on C15a is similar to that of EC1 alone but shows that steric hindrance imposed by EC2 would likely prevent multiprotein binding by the native receptor at any singular threefold vertex. Definition of the molecular interface between the RV-Cs and their receptors provides new avenues that can be explored for potential antiviral therapies.
Assuntos
Caderinas/química , Caderinas/metabolismo , Microscopia Crioeletrônica/métodos , Enterovirus/química , Enterovirus/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Virais/metabolismo , Proteínas Relacionadas a Caderinas , Enterovirus/classificação , Infecções por Enterovirus/virologia , Células HeLa , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Enterovirus A71 (EV-A71) causes hand, foot and mouth disease epidemics with neurological complications and fatalities. However, the neuropathogenesis of EV-A71 remains poorly understood. In mice, adaptation and virulence determinants have been mapped to mutations at VP2-149, VP1-145 and VP1-244. We investigate how these amino acids alter heparin-binding phenotype and shapes EV-A71 virulence in one-day old mice. We constructed six viruses with varying residues at VP1-98, VP1-145 (which are both heparin-binding determinants) and VP2-149 (based on the wild type 149K/98E/145Q, termed KEQ) to generate KKQ, KKE, KEE, IEE and IEQ variants. We demonstrated that the weak heparin-binder IEE was highly lethal in mice. The initially strong heparin-binding IEQ variant acquired an additional mutation VP1-K244E, which confers weak heparin-binding phenotype resulting in elevated viremia and increased virus antigens in mice brain, with subsequent high virulence. IEE and IEQ-244E variants inoculated into mice disseminated efficiently and displayed high viremia. Increasing polymerase fidelity and impairing recombination of IEQ attenuated the virulence, suggesting the importance of population diversity in EV-A71 pathogenesis in vivo. Combining in silico docking and deep sequencing approaches, we inferred that virus population diversity is shaped by electrostatic interactions at the five-fold axis of the virus surface. Electrostatic surface charges facilitate virus adaptation by generating poor heparin-binding variants for better in vivo dissemination in mice, likely due to reduced adsorption to heparin-rich peripheral tissues, which ultimately results in increased neurovirulence. The dynamic switching between heparin-binding and weak heparin-binding phenotype in vivo explained the neurovirulence of EV-A71.
Assuntos
Encéfalo/virologia , Proteínas do Capsídeo/metabolismo , Enterovirus Humano A/genética , Infecções por Enterovirus/virologia , Enterovirus/genética , Heparina/metabolismo , Fatores de Virulência/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Enterovirus/química , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/metabolismo , Heparina/química , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Endogâmicos ICR , Mutação , Fenótipo , Eletricidade Estática , Células Tumorais Cultivadas , Virulência , Fatores de Virulência/química , Fatores de Virulência/genética , Replicação ViralRESUMO
The hydrophobic pocket within viral capsid protein 1 is a target to combat the rhino- and enteroviruses (RV and EV) using small molecules. The highly conserved amino acids lining this pocket enable the development of antivirals with broad-spectrum of activity against numerous RVs and EVs. Inhibitor binding blocks: the attachment of the virion to the host cell membrane, viral uncoating, and/or production of infectious virus particles. Syntheses and biological studies of the most well-known antipicornaviral capsid binders have been reviewed and we propose next steps in this research.
Assuntos
Antivirais/farmacologia , Capsídeo/metabolismo , Enterovirus/efeitos dos fármacos , Antivirais/química , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Enterovirus/química , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
This study was conducted to evaluate the microbiological quality of a mangrove estuary in the Vitória Bay region, Espírito Santo, Brazil. We analyzed the presence and concentration of enteric viruses and thermotolerant coliforms in water, mussels (Mytella charruana and Mytella guyanensis), and oysters (Crassostrea rhizophorae), collected over a 13-month period. Human adenovirus, rotavirus A (RVA), and norovirus genogroup II were analyzed by quantitative PCR. The highest viral load was found in RVA-positive samples with a concentration of 3.0 × 104 genome copies (GC) L-1 in water samples and 1.3 × 105 GC g-1 in bivalves. RVA was the most prevalent virus in all matrices. Thermotolerant coliforms were quantified as colony-forming units (CFU) by the membrane filtration method. The concentration of these bacteria in water was in accordance with the Brazilian standard for recreational waters (< 250 CFU 100 mL-1) during most of the monitoring period (12 out of 13 months). However, thermotolerant coliform concentrations of 3.0, 3.1, and 2.6 log CFU 100 g-1 were detected in M. charruana, M. guyanensis, and C. rhizophorae, respectively. The presence of human-specific viruses in water and bivalves reflects the strong anthropogenic impact on the mangrove and serves as an early warning of waterborne and foodborne disease outbreaks resulting from the consumption of shellfish and the practice of water recreational activities in the region.
Assuntos
Bivalves/virologia , Crassostrea/virologia , Enterovirus/isolamento & purificação , Água do Mar/virologia , Frutos do Mar/virologia , Animais , Brasil , Enterovirus/química , Enterovirus/classificação , Enterovirus/genética , Infecções por Enterovirus/virologia , Estuários , Contaminação de Alimentos/análise , Temperatura Alta , HumanosRESUMO
Coxsackievirus A10 (CVA10), a human type-A Enterovirus (HEV-A), can cause diseases ranging from hand-foot-and-mouth disease to polio-myelitis-like disease. CVA10, together with some other HEV-As, utilizing the molecule KREMEN1 as an entry receptor, constitutes a KREMEN1-dependent subgroup within HEV-As. Currently, there is no vaccine or antiviral therapy available for treating diseases caused by CVA10. The atomic-resolution structure of the CVA10 virion, which is within the KREMEN1-dependent subgroup, shows significant conformational differences in the putative receptor binding sites and serotype-specific epitopes, when compared to the SCARB2-dependent subgroup of HEV-A, such as EV71, highlighting specific differences between the sub-groups. We also report two expanded structures of CVA10, an empty particle and uncoating intermediate at atomic resolution, as well as a medium-resolution genome structure reconstructed using a symmetry-mismatch method. Structural comparisons coupled with previous results, reveal an ordered signal transmission process for enterovirus uncoating, converting exo-genetic receptor-attachment inputs into a generic RNA release mechanism.
Assuntos
Enterovirus/química , Receptores Virais/metabolismo , Desenvelopamento do Vírus , Animais , Capsídeo/metabolismo , Microscopia Crioeletrônica , Enterovirus/ultraestrutura , Infecções por Enterovirus/patologia , Infecções por Enterovirus/virologia , Genoma Viral , Camundongos , Modelos MolecularesRESUMO
Hospital wastewater (HWW) represents a major source of the diffusion of many antibiotics and some toxic pathogenic microorganisms in the aquatic environment. Sanitation services play a critical role in controlling transmission of numerous waterborne pathogens, especially enteric human adenoviruses (HAdVs) that can cause acute gastroenteritis. This study intended to evaluate the human adenoviruses (HAdVs) detection rates, to determine the genotype of these viruses and to assess the efficiency of HAdVs removal in hospital pilot wastewater treatment plant (PWWTP) in Tunis City, Tunisia. Therefore, hospital wastewater samples (n = 102) were collected during the study year from the two biological wastewater treatment techniques: natural oxidizing ponds and the rotating biological disks or biodisks. Nested polymerase chain reaction (Nested PCR) was used to evaluate the HAdVs detection rates. The genotype of HAdVs positive samples was achieved by the sequencing of the PCR products. HAdVs were detected in 64% (65/102) of positive wastewater samples. A substantial increase in the frequencies of HAdVs was observed at the exit of the two wastewater treatment techniques studied. The typing of HAdVs species F showed the occurrence of only HAdVs type 41. This data acquired for the first time in Tunisia showed high persistence and survival of HAdVs in the two biological wastewater treatment techniques experienced, and mainly highlighted the poor virological quality of the treated wastewater intended for recycling, agriculture reuse, and discharges into the natural receiving environments. Consequently, tertiary wastewater treatment appeared necessary in this case to decrease the load of enteric viruses flowing in the water environment.
Assuntos
Adenovírus Humanos/química , Enterovirus/imunologia , Águas Residuárias/virologia , Adenovírus Humanos/genética , Adenovírus Humanos/isolamento & purificação , Cidades , Enterovirus/química , Genótipo , Hospitais , Humanos , Reação em Cadeia da Polimerase/métodos , Tunísia , Microbiologia da ÁguaRESUMO
AIM: To assess the potential of a viability dye and an enzymatic reverse transcription quantitative PCR (RT-qPCR) pretreatment to discriminate between infectious and noninfectious enteric viruses. METHODS AND RESULTS: Enterovirus (EntV), norovirus (NoV) GII.4 and hepatitis A virus (HAV) were inactivated at 95°C for 10 min, and four methods were used to compare the efficiency of inactivation: (i) cell culture plaque assay for HAV and EntV, (ii) RT-qPCR alone, (iii) RT-qPCR assay preceded by RNase treatment, and (iv) pretreatment with a viability dye (reagent D (RD)) followed by RT-qPCR. In addition, heat-inactivated NoV was treated with RD coupled with surfactants to increase the efficiency of the viability dye. No treatment was able to completely discriminate infectious from noninfectious viruses. RD-RT-qPCR reduced more efficiently the detection of noninfectious viruses with little to no removal observed with RNase. RD-RT-qPCR method was the closest to cell culture assay. The combination of surfactants and RD did not show relevant improvements on the removal of inactivated viruses signal compared with viability RT-qPCR, with the exception of Triton X-100. CONCLUSION: The use of surfactant/RD-RT-qPCR, although not being able to completely remove the signal from noninfectious viral particles, yielded a better estimation of viral infectivity. SIGNIFICANCE AND IMPACT OF THE STUDY: Surfactant/RD-RT-qPCR may be an advantageous tool for a better detection of infectious viruses with potential significant impact in the risk assessment of the presence of enteric viruses.
Assuntos
Enterovirus/química , Vírus da Hepatite A/química , Norovirus/química , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Caliciviridae/virologia , Enterovirus/genética , Enterovirus/crescimento & desenvolvimento , Enterovirus/isolamento & purificação , Infecções por Enterovirus/virologia , Hepatite A/virologia , Vírus da Hepatite A/genética , Vírus da Hepatite A/crescimento & desenvolvimento , Vírus da Hepatite A/fisiologia , Temperatura Alta , Humanos , Norovirus/genética , Norovirus/crescimento & desenvolvimento , Norovirus/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/química , Inativação de VírusRESUMO
Biocompatible gold nanoclusters can be utilized as contrast agents in virus imaging. The labeling of viruses can be achieved noncovalently but site-specifically by linking the cluster to the hydrophobic pocket of a virus via a lipid-like pocket factor. We have estimated the binding affinities of three different pocket factors of echovirus 1 (EV1) in molecular dynamics simulations combined with non-equilibrium free-energy calculations. We have also studied the effects on binding affinities with a pocket factor linked to the Au102pMBA44 nanocluster in different protonation states. Although the absolute binding affinities are over-estimated for all the systems, the trend is in agreement with recent experiments.3 Our results suggest that the natural pocket factor (palmitic acid) can be replaced by molecules pleconaril (drug) and its derivative Kirtan1 that have higher estimated binding affinities. Our results also suggest that including the gold nanocluster does not decrease the affinity of the pocket factor to the virus, but the affinity is sensitive to the protonation state of the nanocluster, i.e., to pH conditions. The methodology introduced in this work helps in the design of optimal strategies for gold-virus bioconjugation for virus detection and manipulation.
Assuntos
Capsídeo/metabolismo , Enterovirus/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Antivirais/metabolismo , Sítios de Ligação , Capsídeo/química , Enterovirus/química , Ouro/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Dinâmica Molecular , Oxidiazóis/metabolismo , Oxazóis , Ácido Palmítico/metabolismo , TermodinâmicaRESUMO
INTRODUCTION: Since drinking water can be a vehicle for the transmission of pathogens, the detection of enteric viruses in these water samples is essential to establish the appropriate measures to control and prevent associated diseases. OBJECTIVE: To analyze the results obtained for enteric viruses in water samples for human consumption received at the Colombian Instituto Nacional de Salud and establish their association with the data on water quality in Colombian municipalities. MATERIALS AND METHODS: We conducted a descriptive-retrospective analysis of the results obtained in the detection of rotavirus, enterovirus, hepatitis A virus and adenovirus in water samples received for complementary studies of enteric hepatitis, acute diarrheal disease and foodborne diseases. Data were correlated with the results of water quality surveillance determined by the national human consumption water quality index (IRCA). RESULTS: Of the 288 samples processed from 102 Colombian municipalities, 50.7% were positive for viruses: 26.73% for hepatitis A virus, 20.48% for enterovirus and rotavirus and 18.05% for adenovirus. Viruses were detected in 48.26% of non-treated water samples and in 45.83% of treated water samples. The IRCA index showed no correlation with the presence of viruses. CONCLUSIONS: The presence of viruses in water represents a public health risk and, therefore, the prevention of virus transmission through water requires appropriate policies to reinforce water supply systems and improve epidemiological surveillance.
Assuntos
Adenoviridae/patogenicidade , Infecções por Enterovirus/microbiologia , Enterovirus/química , Vírus/imunologia , Microbiologia da Água , Adenoviridae/química , Colômbia , Enterovirus/imunologia , Enterovirus/metabolismo , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/metabolismo , Humanos , Estudos Retrospectivos , Vírus/metabolismo , Abastecimento de ÁguaRESUMO
Human enteroviruses (EVs) comprise >100 different types. Research suggests a non-chance association between EV infections and type 1 diabetes. Immunohistochemical studies with the anti-EV antibody 5D-8.1 have shown that the EV capsid antigen is present in pancreatic islet cells of diabetic subjects. When it was noticed that 5D-8.1 may cross-react with human proteins, doubt was casted on the significance of the above histopathologic findings. To address this issue, properties of EV antibodies 5D-8.1 and 9D5 have been investigated using peptide microarrays, peptide substitution scanning, immunofluorescence of EV-infected cells, EV neutralization assays, bioinformatics analysis. Evidence indicates that the two antibodies bind to distinct non-neutralizing linear epitopes in VP1 and are specific for a vast spectrum of EV types (not for other human viruses). However, their epitopes may align with a few human proteins at low expected values. When tested by immunofluorescence, high concentrations of 5D-8.1 yelded faint cytoplasmic staining in uninfected cells. At reduced concentrations, both antibodies produced dotted staining only in the cytoplasm of infected cells and recognized both acute and persistent EV infection. Thus, the two monoclonals represent distinct and independent probes for hunting EVs in tissues of patients with diabetes or other endocrine conditions.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Diabetes Mellitus/fisiopatologia , Enterovirus/imunologia , Sequência de Aminoácidos , Enterovirus/química , Epitopos/química , Epitopos/imunologia , Humanos , Homologia de Sequência de AminoácidosRESUMO
UNLABELLED: The NF-κB signaling network, which is an ancient signaling pathway, plays a pivotal role in innate immunity and constitutes a first line of defense against invading pathogens, including viruses. However, numerous viruses possess evolved strategies to antagonize the activation of the NF-κB signaling pathway. Our previous study demonstrated that the nonstructural protein 2C of enterovirus 71 (EV71), which is the major pathogen of hand, foot, and mouth disease, inhibits tumor necrosis factor alpha (TNF-α)-mediated activation of NF-κB by suppressing IκB kinase ß (IKKß) phosphorylation. Nevertheless, the mechanism underlying the inhibition of IKKß phosphorylation by EV71 2C remains largely elusive. We demonstrate that EV71 2C interacts with all isoforms of the protein phosphatase 1 (PP1) catalytic subunit (the PP1α, PP1ß, and PP1γ isoforms) through PP1-docking motifs. EV71 2C has no influence on the subcellular localization of PP1. In addition, the PP1-binding-deficient EV71 2C mutant 3E3L nearly completely lost the ability to suppress IKKß phosphorylation and NF-κB activation was markedly restored in the mutant, thereby indicating that PP1 binding is efficient for EV71 2C-mediated inhibition of IKKß phosphorylation and NF-κB activation. We further demonstrate that 2C forms a complex with PP1 and IKKß to dephosphorylate IKKß. Notably, we reveal that other human enteroviruses, including poliovirus (PV), coxsackie A virus 16 (CVA16), and coxsackie B virus 3 (CVB3), use 2C proteins to recruit PP1, leading to the inhibition of IKKß phosphorylation. Our findings indicate that enteroviruses exploit a novel mechanism to inhibit IKKß phosphorylation by recruiting PP1 and IKKß to form a complex through 2C proteins, which ultimately results in the inhibition of the NF-κB signaling pathway. IMPORTANCE: The innate antiviral immunity system performs an essential function in recognizing and eliminating invading viruses. Enteroviruses include a number of important human pathogens, including poliovirus (PV), EV71, and coxsackieviruses (CVs). As 2C is the most conserved and complex nonstructural protein of enteroviruses, its biological function is largely unclear, whereas the 2A and 3C proteinases of enteroviruses are well characterized. We reveal that EV71 2C forms a complex with PP1 and IKKß to maintain IKKß in an unphosphorylated and inactive state, resulting in the inactivation of the TNF-α-mediated NF-κB signaling pathway. We provide evidence that the 2C proteins of the enteroviruses PV, CVA16, and CVB3 suppress IKKß phosphorylation through the same mechanism involving PP1. We demonstrate that enteroviruses exploit a novel mechanism involving PP1 to regulate innate antiviral immunity, and our findings may be particularly important for understanding the pathogenicity of enteroviruses.
Assuntos
Proteínas de Transporte/metabolismo , Enterovirus/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas de Transporte/genética , Enterovirus/química , Enterovirus/genética , Enterovirus Humano A/química , Enterovirus Humano A/metabolismo , Enterovirus Humano B/metabolismo , Células HeLa , Humanos , Fosforilação , Poliovirus/química , Poliovirus/metabolismo , Ligação Proteica , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
Enterovirus 71 (EV71) plays an important role in hand-foot-and-mouth disease. In this study, a series of diarylhydrazide analogues was synthesized, and the systematic exploration of SAR led to potent enterovirus inhibitors, of which compound 15 exhibits significant improvements in inhibition potency with an EC50 value of 0.02 µM against EV71. It is very interesting that this class of diarylhydrazides exhibits activities against a series of human enteroviruses at the picomolar level, including EV71 and Coxsackieviruses B1 (CVB1), CVB2, CVB3, CVB4, CVB5, and CVB6 (EC50 as low as 0.5 nM). Compared with the reference antienterovirus drug 1 (enviroxime) and known inhibitor 5 (WIN 51711), the four highly selective compounds 15, 27, 41 and 47 inhibited EV71 replication with EC50 values of 0.17-0.02 µM and SI values in a range of 978.4-12338. A preliminary mechanistic study indicated that VP1 might be the target site for this type of compound.
Assuntos
Antivirais/farmacologia , Enterovirus/efeitos dos fármacos , Hidrazinas/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Chlorocebus aethiops , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Enterovirus/química , Hidrazinas/síntese química , Hidrazinas/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Células Vero , Proteínas Estruturais Virais/químicaRESUMO
Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin-magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.
Assuntos
Culinária/métodos , Crassostrea/virologia , Enterovirus/fisiologia , Doenças Transmitidas por Alimentos/virologia , Frutos do Mar/virologia , Inativação de Vírus , Animais , Enterovirus/química , Temperatura Alta , Humanos , Norovirus/química , Norovirus/fisiologiaRESUMO
Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16), as the main agents causing hand, foot and mouth disease (HFMD), have become a serious public health concern in the Asia-Pacific region. Recently, various neutralizing B cell epitopes of EV71 were identified as targets for promising vaccine candidates. Structural studies of Picornaviridae indicated that potent immunodominant epitopes typically lie in the hypervariable loop of capsid surfaces. However, cross-neutralizing antibodies and cross-protection between EV71 and CVA16 have not been observed. Therefore, we speculated that divergent sequences of the two viruses are key epitopes for inducing protective neutralizing responses. In this study, we selected 10 divergent epitope candidates based on alignment of the EV71 and CVA16 P1 amino acid sequences using the Multalin interface page, and these epitopes are conserved among all subgenotypes of EV71. Simultaneously, by utilizing the norovirus P particle as a novel vaccine delivery carrier, we identified the 71-6 epitope (amino acid 176-190 of VP3) as a conformational neutralizing epitope against EV71 in an in vitro micro-neutralization assay as well as an in vivo protection assay in mice. Altogether, these results indicated that the incorporation of the 71-6 epitope into the norovirus P domain can provide a promising candidate for an effective synthetic peptide-based vaccine against EV71.