RESUMO
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
The nucleic acid polymerase-catalyzed nucleotidyl transfer reaction associated with polymerase active site closure is a key step in the nucleotide addition cycle (NAC). Two proton transfer events can occur in such a nucleotidyl transfer: deprotonation of the priming nucleotide 3'-hydroxyl nucleophile and protonation of the pyrophosphate (PPi) leaving group. In viral RNA-dependent RNA polymerases (RdRPs), whether and how active site residues participate in this two-proton transfer reaction remained to be clarified. Here we report a 2.5 Šresolution crystal structure of enterovirus 71 (EV71) RdRP in a catalytically closed pre-chemistry conformation, with a proposed proton donor candidate K360 in close contact with the NTP γ-phosphate. Enzymology data reveal that K360 mutations not only reduce RdRP catalytic efficiency but also alter pH dependency profiles in both elongation and pre-elongation synthesis modes. Interestingly, mutations at R174, an RdRP-invariant residue in motif F, had similar effects with additional impact on the Michaelis constant of NTP (KM,NTP). However, direct participation in protonation was not evident for K360 or R174. Our data suggest that both K360 and R174 participate in nucleotidyl transfer, while their possible roles in acid-base or positional catalysis are discussed in comparison with other classes of nucleic acid polymerases.
Assuntos
Enterovirus Humano A , RNA Polimerase Dependente de RNA , Catálise , Modelos Moleculares , Ácidos Nucleicos , Nucleotídeos/química , Nucleotidiltransferases , Prótons , RNA Viral , RNA Polimerase Dependente de RNA/química , Enterovirus Humano A/enzimologiaRESUMO
Several viruses have been proven to inhibit the formation of RNA processing bodies (P-bodies); however, knowledge regarding whether enterovirus blocks P-body formation remains unclear, and the detailed molecular mechanisms and functions of picornavirus regulation of P-bodies are limited. Here, we show the crucial role of 2A protease in inhibiting P-bodies to promote viral replication during enterovirus 71 infection. Moreover, we found that the activity of 2A protease is essential to inhibit P-body formation, which was proven by the result that infection with EV71-2AC110S, a 2A protease activity-inactivated recombinant virus, failed to block the formation of P-bodies. Furthermore, we show that DDX6, a scaffolding protein of P-bodies, interacted with viral RNA to facilitate viral replication rather than viral translation, by using a Renilla luciferase mRNA reporter system and nascent RNA capture assay. Altogether, our data first demonstrate that the 2A protease of enterovirus inhibits P-body formation to facilitate viral RNA synthesis by recruiting the P-body components to viral RNA. IMPORTANCE Processing bodies (P-bodies) are constitutively present in eukaryotic cells and play an important role in the mRNA cycle, including regulation of gene expression and mRNA degradation. The P-body is the structure that viruses manipulate to facilitate their survival. Here, we show that the 2A protease alone was efficient to block P-body formation during enterovirus 71 infection, and its activity is essential. When the assembly of P-bodies was blocked by 2A protease, DDX6 and 4E-T, which were required for P-body formation, bound to viral RNA to facilitate viral RNA synthesis. We propose a model revealing that EV71 manipulates P-body formation to generate an environment that is conducive to viral replication by facilitating viral RNA synthesis: 2A protease blocked P-body assembly to make it possible for virus to take advantage of P-body components.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Enterovirus Humano A/metabolismo , Peptídeo Hidrolases/metabolismo , RNA Viral/biossíntese , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/ultraestrutura , RNA Helicases DEAD-box/metabolismo , Enterovirus Humano A/enzimologia , Enterovirus Humano A/fisiologia , Células HeLa , Humanos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ribonucleoproteínas/metabolismo , Replicação ViralRESUMO
With CA16, enterovirus-71 is the causative agent of hand foot and mouth disease (HFMD) which occurs mostly in children under 5 years-old and responsible of several outbreaks since a decade. Most of the time, HFMD is a mild disease but can progress to severe complications such as meningitis, brain stem encephalitis, acute flaccid paralysis (AFP) and even death; EV71 has been identified in all severe cases. Therefore, it is actually one of the most public health issues that threatens children's life. [Formula: see text] is a protease which plays important functions in EV71 infection. To date, a lot of [Formula: see text] inhibitors have been tested but none of them has been approved yet. Therefore, a drug screening is still an utmost importance in order to treat and/or prevent EV71 infections. This work highlights the EV71 life cycle, [Formula: see text] functions and [Formula: see text] inhibitors recently screened. It permits to well understand all mechanisms about [Formula: see text] and consequently allow further development of drugs targeting [Formula: see text]. Thus, this review is helpful for screening of more new [Formula: see text] inhibitors or for designing analogues of well known [Formula: see text] inhibitors in order to improve its antiviral activity.
Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Enterovirus Humano A/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Doença de Mão, Pé e Boca/tratamento farmacológico , RNA Viral/antagonistas & inibidores , Animais , Antivirais/isolamento & purificação , Criança , Avaliação Pré-Clínica de Medicamentos/tendências , Enterovirus Humano A/enzimologia , Inibidores Enzimáticos/isolamento & purificação , Doença de Mão, Pé e Boca/complicações , Doença de Mão, Pé e Boca/virologia , Humanos , Camundongos , Inibidores da Síntese de Ácido Nucleico/farmacologia , FilogeniaRESUMO
Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot and mouth disease (HFMD) which seriously threatened the safety and lives of infants and young children. However, there are no licensed direct antiviral agents to cure the HFMD. In this study, a series of quinoline formamide analogues as effective enterovirus inhibitors were developed, subsequent systematic structure-activity relationship (SAR) studies demonstrated that these quinoline formamide analogues exhibited good potency to treat EV-A71 infection. As described, the most efficient EV-A71 inhibitor 6i showed good anti-EV-A71 activity (EC50 = 1.238 µM) in RD cells. Furthermore, compound 6i could effectively prevent death of virus infected mice at dose of 6 mg/kg. When combined with emetine (0.1 mg/kg), this treatment could completely prevent the clinical symptoms and death of virus infected mice. Mechanism study indicated that compound 6i inhibited EV-A71 via targeting 2C helicase, thus impeding RNA remodeling and metabolism. Taken together, these data indicated that 6i is a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.
Assuntos
Antivirais/farmacologia , Dibucaína/farmacologia , Enterovirus Humano A/efeitos dos fármacos , Infecções por Enterovirus/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , RNA Helicases/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/química , Dibucaína/síntese química , Dibucaína/química , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Enterovirus Humano A/enzimologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Camundongos Endogâmicos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , RNA Helicases/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/metabolismoRESUMO
We describe here the design, synthesis, and evaluation of a macrocyclic peptidomimetic as a potent agent targeting enterovirus A71 (EV71). The compound has a 15-membered macrocyclic ring in a defined conformation. Yamaguchi esterification reaction was used to close the 15-membered macrocycle instead of the typical Ru-catalyzed ring-closing olefin metathesis reaction. The crystallographic characterization of the complex between this compound and its target, 3C protease from EV71, validated the design and paved the way for the generation of a new series of anti-EV71 agents.
Assuntos
Antivirais/síntese química , Desenho de Fármacos , Compostos Macrocíclicos/química , Proteases Virais 3C/química , Proteases Virais 3C/metabolismo , Animais , Antivirais/sangue , Antivirais/metabolismo , Antivirais/farmacologia , Sítios de Ligação , Catálise , Domínio Catalítico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Estabilidade de Medicamentos , Enterovirus Humano A/efeitos dos fármacos , Enterovirus Humano A/enzimologia , Esterificação , Humanos , Compostos Macrocíclicos/sangue , Compostos Macrocíclicos/metabolismo , Compostos Macrocíclicos/farmacologia , Camundongos , Simulação de Dinâmica Molecular , Rutênio/químicaRESUMO
Each polymerase nucleotide addition cycle is associated with two primary conformational changes of the catalytic complex: the pre-chemistry active site closure and post-chemistry translocation. While active site closure is well interpreted by numerous crystallographic snapshots, translocation intermediates are rarely captured. Here we report three types of intermediate structures in an RNA-dependent RNA polymerase (RdRP). The first two types, captured in forward and reverse translocation events, both highlight the role of RdRP-unique motif G in restricting the RNA template movement, corresponding to the rate-limiting step in translocation. By mutating two critical residues in motif G, we obtain the third type of intermediates that may mimic the transition state of this rate-limiting step, demonstrating a previously unidentified movement of the template strand. We propose that a similar strategy may be utilized by other classes of nucleic acid polymerases to ensure templating nucleotide positioning for efficient catalysis through restricting interactions with template RNA.
Assuntos
RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Transporte Biológico Ativo , Domínio Catalítico , Cristalografia por Raios X , Enterovirus Humano A/enzimologia , Enterovirus Humano A/genética , Genes Virais , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Conformação Proteica , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , Elongação da Transcrição Genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Human rhinoviruses (HRVs) are the predominant infectious agents for the common cold worldwide. The HRV-C species cause severe illnesses in children and are closely related to acute exacerbations of asthma. 3C protease, a highly conserved enzyme, cleaves the viral polyprotein during replication and assists the virus in escaping the host immune system. These key roles make 3C protease an important drug target. A few structures of 3Cs complexed with an irreversible inhibitor rupintrivir have been determined. These structures shed light on the determinants of drug specificity. Here we describe the structures of HRV-C15 3C in free and inhibitor-bound forms. The volume-decreased S1' subsite and half-closed S2 subsite, which were thought to be unique features of enterovirus A 3C proteases, appear in the HRV-C 3C protease. Rupintrivir assumes an "intermediate" conformation in the complex, which might open up additional avenues for the design of potent antiviral inhibitors. Analysis of the features of the three-dimensional structures and the amino acid sequences of 3C proteases suggest new applications for existing drugs.
Assuntos
Proteases Virais 3C/antagonistas & inibidores , Proteases Virais 3C/química , Antivirais/química , Desenho de Fármacos , Enterovirus Humano A/efeitos dos fármacos , Isoxazóis/química , Fenilalanina/análogos & derivados , Pirrolidinonas/química , Valina/análogos & derivados , Cristalografia por Raios X , Enterovirus Humano A/enzimologia , Isoxazóis/farmacologia , Modelos Moleculares , Fenilalanina/química , Fenilalanina/farmacologia , Estrutura Terciária de Proteína , Pirrolidinonas/farmacologia , Análise de Sequência de DNA , Valina/química , Valina/farmacologiaRESUMO
Enteroviruses, which may cause neurological complications, have become a public health threat worldwide in recent years. Interactions between cellular proteins and enteroviral proteins could interfere with cellular biological processes to facilitate viral replication in infected cells. Enteroviral RNA-dependent RNA polymerase (RdRP), known as 3D protein, mainly functions as a replicase for viral RNA synthesis in infected cells. However, the 3D protein encoded by enterovirus A71 (EV-A71) could also interact with several cellular proteins to regulate cellular events and responses during infection. To globally investigate the functions of the EV-A71 3D protein in regulating biological processes in host cells, we performed immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify host proteins that may associate with the 3D protein. We found that the 3D protein interacts with factors involved in translation-related biological processes, including ribosomal proteins. In addition, polysome profiling analysis showed that the 3D protein cosediments with small and large subunits of ribosomes. We further discovered that the EV-A71 3D protein could enhance EV-A71 internal ribosome entry site (IRES)-dependent translation as well as cap-dependent translation. Collectively, this research demonstrated that the RNA polymerase encoded by EV-A71 could join a functional ribosomal complex and positively regulate viral and host translation.
Assuntos
Enterovirus Humano A/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Linhagem Celular , Cromatografia Líquida , Células HEK293 , Células HeLa , Humanos , Sítios Internos de Entrada Ribossomal , Biossíntese de Proteínas , Espectrometria de Massas em Tandem , Proteínas Virais/metabolismoRESUMO
Virus-encoded proteases play diverse roles in the efficient replication of enterovirus 71 (EV71), which is the causative agent of human hand, foot, and mouth disease (HFMD). However, it is unclear how host proteases affect viral proliferation. Here, we designed activity-based probes (ABPs) based on an inhibitor of the main EV71 protease (3Cpro), which is responsible for the hydrolysis of the EV71 polyprotein, and successfully identified host candidates that bind to the ABPs. Among the candidates, the host cysteine protease autophagy-related protein 4 homolog B (ATG4B), a key component of the autophagy machinery, was demonstrated to hydrolytically process the substrate of EV71 3Cpro and had activity comparable to that of the viral protease. Genetic disruption of ATG4B confirmed that the enzyme is indispensable for viral proliferation in vivo Our results not only further the understanding of host-virus interactions in EV71 biology but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.IMPORTANCE Enterovirus 71 (EV71), one of the major pathogens of human HFMD, has caused outbreaks worldwide. How EV71 efficiently assesses its life cycle with elaborate interactions with multiple host factors remains to be elucidated. In this work, we deconvoluted that the host ATG4B protein processes the viral polyprotein with its cysteine protease activity and helps EV71 replicate through a chemical biology strategy. Our results not only further the understanding of the EV71 life cycle but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proliferação de Células/fisiologia , Cisteína Endopeptidases/metabolismo , Enterovirus Humano A/metabolismo , Proteases Virais 3C , Proteínas Relacionadas à Autofagia/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cisteína Endopeptidases/genética , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Enterovirus Humano A/efeitos dos fármacos , Enterovirus Humano A/enzimologia , Enterovirus Humano A/crescimento & desenvolvimento , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno/fisiologia , Modelos Moleculares , Conformação Proteica , Proteoma , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Targeted covalent inhibitors (TCIs) have attracted growing attention from the pharmaceutical industry in recent decades because they have potential advantages in terms of efficacy, selectivity, and safety. TCIs have recently evolved into a new version with reversibility that can be systematically modulated. This feature may diminish the risk of haptenization and help optimize the drug-target residence time as needed. The enteroviral 3C protease (3Cpro) is a valuable therapeutic target, but the development of 3Cpro inhibitors is far from satisfactory. Therefore, we aimed to apply a reversible TCI approach to the design of novel 3Cpro inhibitors. The introduction of various substituents onto the α-carbon of classical Michael acceptors yielded inhibitors bearing several classes of warheads. Using steady-state kinetics and biomolecular mass spectrometry, we confirmed the mode of reversible covalent inhibition and elucidated the mechanism by which the potency and reversibility were affected by electronic and steric factors. This research produced several potent inhibitors with good selectivity and suitable reversibility; moreover, it validated the reversible TCI approach in the field of viral infection, suggesting broader applications in the design of reversible covalent inhibitors for other proteases.
Assuntos
Acrilamidas/química , Antivirais/química , Cianoacrilatos/química , Enterovirus Humano A/enzimologia , Inibidores Enzimáticos/química , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Acrilamidas/síntese química , Antivirais/síntese química , Cianoacrilatos/síntese química , Cisteína Endopeptidases , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Estrutura MolecularRESUMO
BACKGROUND: Enterovirus 71 (EV71) is the main pathogen of hand-foot-mouth disease (HFMD) and sometimes causes several neurological complications. However, the underlying mechanism of the host response to the virus infection remains unclear. OBJECTIVE: To reveal the cell-specific transcriptional response of cultured RD cells following infection with EV71, and better understand the molecular mechanisms of virus-host interactions. METHODS: The RD cells were infected with or without EV71 for 24 h, and then transcriptome sequencing and qRT-PCR were performed to analyze the transcriptome difference of functional genes. RESULTS: More than 15000 genes were identified in transcriptome sequencing. In comparison with uninfected RD cells, 329 DEGs were identified in cells infected with EV71. GO and KEGG pathway enrichment analysis showed that most of the DEGs were related to DNA binding, transcriptional regulation, immune response and inflammatory response, apoptosis inducing factors and enriched in JAK-STAT and MAPK signaling pathways. TXNIP (thioredoxin-interacting protein) gene was further demonstrated to play an important role participating in cellular apoptosis induced by EV71, and the apoptosis and death mediated by TXNIP during EV71 infection was triggered by viral 2A protease (2Apro), not 3C protease (3Cpro). CONCLUSION: Our study demonstrated that RD cells have a significant response to EV71 infection, including immune response and apoptosis. 2Apro might be a key inducer relative to the cellular apoptosis and death mediated by TXNIP during EV71 infection. These data would contribute to preferably understand the process at the molecular level and provide theoretical foundation for diagnosis and treatment of EV71-related diseases.
Assuntos
Apoptose , Proteínas de Transporte/genética , Cisteína Endopeptidases/genética , Infecções por Enterovirus/genética , Transcriptoma , Proteínas Virais/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Enterovirus Humano A/enzimologia , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Proteínas Virais/metabolismoRESUMO
Enteroviruses are members of Pichornaviridae family consisting of human enterovirus group A, B, C, and D as well as nonhuman enteroviruses. Hand, foot, and mouth disease (HFMD) is a serious disease which is usually seen in the Asia-Pacific region in children. Enterovirus 71 and coxsackievirus A16 are two important viruses responsible for HFMD which are members of group A enterovirus. IFN α and ß are two cytokines, which have a major activity in the innate immune system against viral infections. Most of the viruses have some weapons against these cytokines. EV71 has two main proteases called 2A and 3C, which are important for polyprotein processing and virus maturation. Several studies have indicated that they have a significant effect on different cellular pathways such as interferon production and signaling pathway. The aim of this study was to investigate the latest findings about the interaction of 2A and 3C protease of EV71 and IFN production/signaling pathway and their inhibitory effects on this pathway.
Assuntos
Cisteína Endopeptidases/metabolismo , Enterovirus Humano A/patogenicidade , Doença de Mão, Pé e Boca/virologia , Evasão da Resposta Imune , Fatores Imunológicos/antagonistas & inibidores , Interferon Tipo I/antagonistas & inibidores , Proteínas Virais/metabolismo , Proteases Virais 3C , Ásia/epidemiologia , Enterovirus Humano A/enzimologia , Doença de Mão, Pé e Boca/epidemiologia , HumanosRESUMO
Enterovirus A71 (EV-A71) has emerged as a major pathogen causing hand, foot, and mouth disease, as well as neurological disorders. The host immune response affects the outcomes of EV-A71 infection, leading to either resolution or disease progression. However, the mechanisms of how the mammalian innate immune system detects EV-A71 infection to elicit antiviral immunity remain elusive. Here, we report that the Toll-like receptor 3 (TLR3) is a key viral RNA sensor for sensing EV-A71 infection to trigger antiviral immunity. Expression of TLR3 in HEK293 cells enabled the cells to sense EV-A71 infection, leading to type I, IFN-mediated antiviral immunity. Viral double-stranded RNA derived from EV-A71 infection was a key ligand for TLR3 detection. Silencing of TLR3 in mouse and human primary immune cells impaired the activation of IFN-ß upon EV-A71 infection, thus reinforcing the importance of the TLR3 pathway in defending against EV-A71 infection. Our results further demonstrated that TLR3 was a target of EV-A71 infection. EV-A71 protease 2A was implicated in the downregulation of TLR3. Together, our results not only demonstrate the importance of the TLR3 pathway in response to EV-A71 infection, but also reveal the involvement of EV-A71 protease 2A in subverting TLR3-mediated antiviral defenses.
Assuntos
Cisteína Endopeptidases/imunologia , Enterovirus Humano A/imunologia , RNA Viral/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Células Cultivadas , Regulação para Baixo , Enterovirus Humano A/enzimologia , Inativação Gênica , Células HEK293 , Humanos , Imunidade Inata , Interferon beta/imunologia , Camundongos , Camundongos Endogâmicos C57BL , RNA de Cadeia Dupla/imunologia , Receptor 3 Toll-Like/genéticaRESUMO
A recently reported potent inhibitor of enterovirus 71 3C protease, ( R)-1, was found to have stability and potential toxicity issues due to the presence of a cyanohydrin moiety. Modifying the labile cyanohydrin moiety, by serendipity, led to the discovery of 4-iminooxazolidin-2-one-based inhibitors 4e and 4g with potent inhibitory activity and significantly improved stability. In vivo pharmacokinetic studies of 4e also demonstrated high plasma exposure and moderate half-life. These compounds have shown potential of becoming anti-EV71 drug candidates.
Assuntos
Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Enterovirus Humano A/enzimologia , Nitrilas/química , Oxazóis/química , Oxazóis/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Animais , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Oxazóis/metabolismo , Conformação Proteica , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
A three-dimensional quantitative structure-activity relationships model of enterovirus A71 3C protease inhibitors was constructed in this study. The protein-ligand interaction fingerprint was analyzed to generate a pharmacophore model. A predictive and reliable three-dimensional quantitative structure-activity relationships model was built based on the Flexible Alignment of AutoGPA. Moreover, three novel compounds (I-III) were designed and evaluated for their biochemical activity against 3C protease and anti-enterovirus A71 activity in vitro. III exhibited excellent inhibitory activity (IC50 = 0.031 ± 0.005 µM, EC50 = 0.036 ± 0.007 µM). Thus, this study provides a useful quantitative structure-activity relationships model to develop potent inhibitors for enterovirus A71 3C protease.
Assuntos
Enterovirus Humano A/enzimologia , Inibidores de Proteases/química , Relação Quantitativa Estrutura-Atividade , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Amidas/química , Amidas/metabolismo , Amidas/farmacologia , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Desenho de Fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Proteínas Virais/metabolismoRESUMO
Target-guided screening of fragments (TGSOF) was developed and employed in the identification of EV-A71 3C protease (3Cpro) inhibitors. We identified 4-acetylpyridine and 3-acetylpyridine as effective P3 fragments of an inhibitor and obtained the corresponding irreversible inhibitors 12c and 12fvia this method. Furthermore, based on 12c and 12f, we have obtained reversible inhibitors 17c and 17f. These results demonstrated that TGSOF is a useful strategy for identifying suitable fragments in developing leads in drug discovery.
Assuntos
Descoberta de Drogas , Enterovirus Humano A/enzimologia , Inibidores de Proteases/farmacologia , Piridinas/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Estrutura Molecular , Inibidores de Proteases/química , Piridinas/química , Relação Estrutura-Atividade , Proteínas Virais/metabolismoRESUMO
Enterovirus D68 (EV-D68) has been increasingly associated with severe respiratory illness and neurological complications in children worldwide. However, no vaccine is currently available to prevent EV-D68 infection. In the present study, we investigated the possibility of developing a virus-like particle (VLP)-based EV-D68 vaccine. We found that co-expression of the P1 precursor and 3CD protease of EV-D68 in Pichia pastoris yeast resulted in the generation of EV-D68 VLPs, which were composed of processed VP0, VP1, and VP3 capsid proteins and were visualized as ~30 nm spherical particles. Mice immunized with these VLPs produced serum antibodies capable of specifically neutralizing EV-D68 infections in vitro. The in vivo protective efficacy of the EV-D68 VLP candidate vaccine was assessed in two challenge experiments. The first challenge experiment showed that neonatal mice born to the VLP-immunized dams were fully protected from lethal EV-D68 infection, whereas in the second experiment, passive transfer of anti-VLP sera was found to confer complete protection in the recipient mice. Collectively, these results demonstrate the proof-of-concept for VLP-based broadly effective EV-D68 vaccines.
Assuntos
Anticorpos Neutralizantes/sangue , Enterovirus Humano A/imunologia , Infecções por Enterovirus/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Antivirais/sangue , Anticorpos Antivirais/uso terapêutico , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Modelos Animais de Doenças , Enterovirus Humano A/enzimologia , Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/imunologia , Imunização Passiva , Camundongos , Pichia/genética , Vacinação , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologiaRESUMO
Enterovirus A71 (EV-A71) is a positive-strand RNA virus that causes hand-foot-mouth disease and neurological complications in children and infants. Although the underlying mechanisms remain to be further defined, impaired immunity is thought to play an important role. The host zinc-finger antiviral protein (ZAP), an IFN-stimulated gene product, has been reported to specifically inhibit the replication of certain viruses. However, whether ZAP restricts the infection of enteroviruses remains unknown. Here, we report that EV-A71 infection upregulates ZAP mRNA in RD and HeLa cells. Moreover, ZAP overexpression rendered 293 T cells resistant to EV-A71 infection, whereas siRNA-mediated depletion of endogenous ZAP enhanced EV-A71 infection. The EV-A71 infection stimulated site-specific proteolysis of two ZAP isoforms, leading to the accumulation of a 40 kDa N-terminal ZAP fragment in virus-infected cells. We further revealed that the 3C protease (3Cpro) of EV-A71 mediates ZAP cleavage, which requires protease activity. Furthermore, ZAP variants with single amino acid substitutions at Gln-369 were resistant to 3Cpro cleavage, implying that Gln-369 is the sole cleavage site in ZAP. Moreover, although ZAP overexpression inhibited EV-A71 replication, the cleaved fragments did not show this effect. Our results indicate that an equilibrium between ZAP and enterovirus 3Cpro controls viral infection. The findings in this study suggest that viral 3Cpro mediated ZAP cleavage may represent a mechanism to escape host antiviral responses.
Assuntos
Cisteína Endopeptidases/metabolismo , Enterovirus Humano A/enzimologia , Interações Hospedeiro-Patógeno , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Proteases Virais 3C , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Cisteína Endopeptidases/genética , Enterovirus Humano A/genética , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Células Musculares/metabolismo , Células Musculares/virologia , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Células Sf9/imunologia , Células Sf9/virologia , Transdução de Sinais , Spodoptera , Proteínas Virais/genéticaRESUMO
Enterovirus 71 (EV71) is a major pathogen of hand, foot, and mouth disease (HFMD). To date, no antiviral drug has been approved to treat EV71 infection. Due to the essential role that EV71 3 C protease (3Cpro) plays in the viral life cycle, it is generally considered as a highly appealing target for antiviral drug development. In this study, we present a transgene-encoded biosensor that can accurately, sensitively and quantitatively report the proteolytic activity of EV71 3Cpro. This biosensor is based on the catalyzed activity of a pro-interleukin (IL)-1ß-enterovirus 3Cpro cleavage site-Gaussia Luciferase (GLuc) fusion protein that we named i-3CS-GLuc. GLuc enzyme is inactive in the fusion protein because of aggregation caused by pro-IL-1ß. However, the 3Cpro of EV71 and other enteroviruses, such as coxsackievirus A9 (CVA9), coxsackievirus B3 (CVB3), and poliovirus can recognize and process the canonical enterovirus 3Cpro cleavage site between pro-IL-1ß and GLuc, thereby releasing and activating GLuc and resulting in increased luciferase activity. The high sensitivity, ease of use, and applicability as a transgene in cell-based assays of i-3CS-GLuc biosensor make it a powerful tool for studying viral protease proteolytic events in living cells and for achieving high-throughput screening of antiviral agents.