RESUMO
Serine proteases are important environmental contributors of enterovirus biocontrol. However, the structural features of molecular interaction accounting for the susceptibility of enteroviruses to proteases remains unexplained. Here, we describe the molecular mechanisms involved in the recruitment of serine proteases to viral capsids. Among the virus types used, coxsackievirus A9 (CVA9), but not CVB5 and echovirus 11 (E11), was inactivated by Subtilisin A in a host-independent manner, while Bovine Pancreatic Trypsin (BPT) only reduced CVA9 infectivity in a host-dependent manner. Predictive interaction models of each protease with capsid protomers indicate the main targets as internal disordered protein (IDP) segments exposed either on the 5-fold vertex (DE loop VP1) or at the 5/2-fold intersection (C-terminal end VP1) of viral capsids. We further show that a functional binding protease/capsid depends on both the strength and the evolution over time of protease-VP1 complexes, and lastly on the local adaptation of proteases on surrounding viral regions. Finally, we predicted three residues on CVA9 capsid that trigger cleavage by Subtilisin A, one of which may act as a sensor residue contributing to enzyme recognition on the DE loop. Overall, this study describes an important biological mechanism involved in enteroviruses biocontrol.
Assuntos
Proteínas do Capsídeo , Capsídeo , Serina Proteases , Capsídeo/metabolismo , Serina Proteases/metabolismo , Serina Proteases/química , Serina Proteases/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Humanos , Enterovirus/enzimologia , Enterovirus/fisiologia , Animais , Enterovirus Humano B/fisiologia , Enterovirus Humano B/enzimologiaRESUMO
Enteroviruses cause a wide range of disorders with varying presentations and severities, and some enteroviruses have emerged as serious public health concerns. These include Coxsackievirus B3 (CVB3), an active causative agent of viral myocarditis, and Coxsackievirus B4 (CVB4), which may accelerate the progression of type 1 diabetes. The 3C proteases from CVB3 and CVB4 play important roles in the propagation of these viruses. In this study, the 3C proteases from CVB3 and CVB4 were expressed in Escherichia coli and purified by affinity chromatography and gel-filtration chromatography. The crystals of the CVB3 and CVB4 3C proteases diffracted to 2.10 and 2.01â Å resolution, respectively. The crystal structures were solved by the molecular-replacement method and contained a typical chymotrypsin-like fold and a conserved His40-Glu71-Cys147 catalytic triad. Comparison with the structures of 3C proteases from other enteroviruses revealed high similarity with minor differences, which will guide the design of 3C-targeting inhibitors with broad-spectrum properties.
Assuntos
Proteases Virais 3C , Sequência de Aminoácidos , Cisteína Endopeptidases , Enterovirus Humano B , Modelos Moleculares , Proteínas Virais , Proteases Virais 3C/química , Cristalografia por Raios X , Enterovirus Humano B/enzimologia , Enterovirus Humano B/química , Enterovirus Humano B/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Domínio Catalítico , Humanos , Conformação Proteica , Clonagem MolecularRESUMO
Enteroviruses, including Coxsackievirus B3 (CVB3), are pervasive pathogens that cause significant disease, including cardiomyopathies. Unfortunately, no treatments or vaccines are available for infected individuals. We identified the host polyamine pathway as a potential drug target, as inhibiting polyamine biosynthesis significantly reduces enterovirus replication in vitro and in vivo. Here, we show that CVB3 is sensitive to polyamine depletion through the polyamine analog diethylnorspermidine (DENSpm), which enhances polyamine catabolism through induction of polyamine acetylation. We demonstrate that CVB3 acquires resistance to DENSpm via mutation of the 2A protease, which enhances proteolytic activity in the presence of DENSpm. Resistance to DENSpm occurred via mutation of a non-catalytic site mutation and results in decreased fitness. These data demonstrate that potential for targeting polyamine catabolism as an antiviral target as well as highlight a potential mechanism of resistance.
Assuntos
Antivirais/farmacologia , Cisteína Endopeptidases/genética , Enterovirus Humano B/efeitos dos fármacos , Poliaminas/farmacologia , Proteínas Virais/genética , Antivirais/química , Cisteína Endopeptidases/metabolismo , Farmacorresistência Viral , Enterovirus Humano B/enzimologia , Enterovirus Humano B/metabolismo , Enterovirus Humano B/fisiologia , Infecções por Enterovirus/virologia , Humanos , Mutação , Poliaminas/química , Poliaminas/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Picornaviral RNA-dependent RNA polymerases (RdRPs) have low replication fidelity that is essential for viral fitness and evolution. Their global fold consists of the classical "cupped right hand" structure with palm, fingers, and thumb domains, and these RdRPs also possess a unique contact between the fingers and thumb domains. This interaction restricts movements of the fingers, and RdRPs use a subtle conformational change within the palm domain to close their active sites for catalysis. We have previously shown that this core RdRP structure and mechanism provide a platform for polymerases to fine-tune replication rates and fidelity to optimize virus fitness. Here, we further elucidated the structural basis for differences in replication rates and fidelity among different viruses by generating chimeric RdRPs from poliovirus and coxsackievirus B3. We designed these chimeric polymerases by exchanging the fingers, pinky finger, or thumb domains. The results of biochemical, rapid-quench, and stopped-flow assays revealed that differences in biochemical activity map to individual modular domains of this polymerase. We found that the pinky finger subdomain is a major regulator of initiation and that the palm domain is the major determinant of catalytic rate and nucleotide discrimination. We further noted that thumb domain interactions with product RNA regulate translocation and that the palm and thumb domains coordinately control elongation complex stability. Several RdRP chimeras supported the growth of infectious poliovirus, providing insights into enterovirus species-specific protein-protein interactions required for virus replication.
Assuntos
Enterovirus Humano B , Poliovirus , RNA Viral , RNA Polimerase Dependente de RNA , Proteínas Virais , Enterovirus Humano B/enzimologia , Enterovirus Humano B/genética , Células HeLa , Humanos , Poliovirus/enzimologia , Poliovirus/genética , Domínios Proteicos , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
AIMS: Coxsackievirus B3 (CVB3) is known to be an important cause of myocarditis and dilated cardiomyopathy. Enterovirus-2C (E2C) is a viral RNA helicase. It inhibits host protein synthesis. Based on these facts, we hypothesize that the inhibition of 2C may suppress virus replication and prevent enterovirus-mediated cardiomyopathy. METHODS AND RESULTS: We generated a chemically modified enterovirus-2C inhibitor (E2CI). From the in vitro assay, E2CI was showed strong antiviral effects. For in vivo testing, mice were treated with E2CI intraperitoneally injected daily for three consecutive days at a dose of 8mg/kg per day, after CVB3 post-infection (p.i) (CVB3 + E2CI, n = 33). For the infected controls (CVB3 only, n = 35), mice were injected with PBS (phosphate buffered saline) in a DBA/2 strain to establish chronic myocarditis. The four-week survival rate of E2CI-treated mice was significantly higher than that of controls (92% vs. 71%; p < 0.05). Virus titers and myocardial damage were significantly reduced in the E2CI treated group. In addition, echocardiography indicated that E2CI administration dramatically maintained mouse heart function compared to control at day 28 p.i chronic stage (LVIDD, 3.1 ± 0.08 vs. 3.9 ± 0.09, p < 0.01; LVDS, 2.0 ± 0.07 vs. 2.5 ± 0.07, p < 0.001; FS, 34.8 ± 1.6% vs. 28.5 ± 1.5%; EF, 67. 9 ± 2.9% vs. 54.7 ± 4.7%, p < 0.05; CVB3 + E2CI, n = 6 vs. CVB3, n = 4). Moreover, E2CI is effectively worked in human iPS (induced pluripotent stem cell) derived cardiomyocytes. CONCLUSION: Enterovirus-2C inhibitor (E2CI) was significantly reduced viral replication, chronic myocardium damage, and CVB3-induced mortality in DBA/2 mice. These results suggested that E2CI is a novel therapeutic agent for the treatment of enterovirus-mediated diseases.
Assuntos
Antivirais/farmacologia , Infecções por Coxsackievirus/tratamento farmacológico , Enterovirus Humano B/enzimologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miocardite/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , RNA Helicases/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/uso terapêutico , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/prevenção & controle , Doença Crônica , Infecções por Coxsackievirus/complicações , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/fisiologia , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas/virologia , Luciferases de Renilla/análise , Masculino , Camundongos , Camundongos Endogâmicos DBA , Miocardite/etiologia , Miocardite/virologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/virologia , Oxidiazóis/farmacologia , Oxidiazóis/uso terapêutico , Oxazóis/farmacologia , Oxazóis/uso terapêutico , Proteínas Recombinantes de Fusão/metabolismo , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/prevenção & controle , Replicação Viral/efeitos dos fármacosRESUMO
Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed terminal amine isotopic labeling of substrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3Cpros) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3Cproin vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3Cpro-targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3Cpro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3Cpro substrates in vivo, we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner.IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection. Although several host protein targets have been identified, the entire list of proteins that are targeted is not known. In this study, we used a novel unbiased proteomics approach to identify â¼100 novel host targets of the enterovirus 3C protease, thus providing further insights into the network of cellular pathways that are modulated to promote virus infection.
Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Cisteína Endopeptidases/metabolismo , Enterovirus Humano B/enzimologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Poliovirus/enzimologia , Proteínas Virais/metabolismo , Proteases Virais 3C , Células HeLa , Humanos , Marcação por Isótopo/métodos , Especificidade por Substrato/fisiologiaRESUMO
Enteroviruses (EVs), such as the Coxsackie B-viruses (CVBs), are common human pathogens, which can cause severe diseases including meningitis, myocarditis and neonatal sepsis. EVs encode two proteases (2Apro and 3Cpro), which perform the proteolytic cleavage of the CVB polyprotein and also cleave host cell proteins to facilitate viral replication. The 2Apro cause direct damage to the infected heart and tools to investigate 2Apro and 3Cpro expression may contribute new knowledge on virus-induced pathologies. Here, we developed new antibodies to CVB-encoded 2Apro and 3Cpro; Two monoclonal 2Apro antibodies and one 3Cpro antibody were produced. Using cells infected with selected viruses belonging to the EV A, B and C species and immunocytochemistry, we demonstrate that the 3Cpro antibody detects all of the EV species B (EV-B) viruses tested and that the 2Apro antibody detects all EV-B viruses apart from Echovirus 9. We furthermore show that the new antibodies work in Western blotting, immunocyto- and immunohistochemistry, and flow cytometry to detect CVBs. Confocal microscopy demonstrated the expression kinetics of 2Apro and 3Cpro, and revealed a preferential cytosolic localization of the proteases in CVB3 infected cells. In summary, the new antibodies detect proteases that belong to EV species B in cells and tissue using multiple applications.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Cisteína Endopeptidases/imunologia , Enterovirus Humano B/imunologia , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/imunologia , Proteínas Virais/imunologia , Proteases Virais 3C , Animais , Antígenos Virais/genética , Células Cultivadas , Clonagem Molecular , Cisteína Endopeptidases/genética , Enterovirus Humano B/enzimologia , Enterovirus Humano B/genética , Infecções por Enterovirus/virologia , Expressão Gênica , Células HeLa , Humanos , Imuno-Histoquímica , Camundongos , Sorogrupo , Proteínas Virais/genéticaRESUMO
Nuclear factor of activated T cells 5 (NFAT5)/Tonicity enhancer binding protein (TonEBP) is a transcription factor induced by hypertonic stress in the kidney. However, the function of NFAT5 in other organs has rarely been studied, even though it is ubiquitously expressed. Indeed, although NFAT5 was reported to be critical for heart development and function, its role in infectious heart diseases has remained obscure. In this study, we aimed to understand the mechanism by which NFAT5 interferes with infection of Coxsackievirus B3 (CVB3), a major cause of viral myocarditis. Our initial results demonstrated that although the mRNA level of NFAT5 remained constant during CVB3 infection, NFAT5 protein level decreased because the protein was cleaved. Bioinformatic prediction and verification of the predicted site by site-directed mutagenesis experiments determined that the NFAT5 protein was cleaved by CVB3 protease 2A at Glycine 503. Such cleavage led to the inactivation of NFAT5, and the 70-kDa N-terminal cleavage product (p70-NFAT5) exerted a dominant negative effect on the full-length NFAT5 protein. We further showed that elevated expression of NFAT5 to counteract viral protease cleavage, especially overexpression of a non-cleavable mutant of NFAT5, significantly inhibited CVB3 replication. Ectopic expression of NFAT5 resulted in elevated expression of inducible nitric oxide synthase (iNOS), a factor reported to inhibit CVB3 replication. The necessity of iNOS for the anti-CVB3 effect of NFAT5 was supported by the observation that inhibition of iNOS blocked the anti-CVB3 effect of NFAT5. In a murine model of viral myocarditis, we observed that treatment with hypertonic saline or mannitol solution upregulated NFAT5 and iNOS expression, inhibited CVB3 replication and reduced tissue damage in the heart. Taken together, our data demonstrate that the anti-CVB3 activity of NFAT5 is impaired during CVB3 infection due to 2A-mediated cleavage of NFAT5. Thus induction of NFAT5 by hypertonic agents may be a promising strategy for the development of anti-CVB3 therapeutics.
Assuntos
Infecções por Coxsackievirus/virologia , Cisteína Endopeptidases/metabolismo , Enterovirus Humano B/enzimologia , Miocardite/virologia , Miócitos Cardíacos/virologia , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/patologia , Enterovirus Humano B/imunologia , Enterovirus Humano B/fisiologia , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos A , Mutação , Miocardite/imunologia , Miocardite/metabolismo , Miocardite/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteólise , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Fatores de Transcrição/genética , Replicação ViralRESUMO
Favipiravir (T-705) is a broad-spectrum antiviral agent that has been approved in Japan for the treatment of influenza virus infections. T-705 also inhibits the replication of various RNA viruses, including chikungunya virus (CHIKV). We demonstrated earlier that the K291R mutation in the F1 motif of the RNA-dependent RNA polymerase (RdRp) of CHIKV is responsible for low-level resistance to T-705. Interestingly, this lysine is highly conserved in the RdRp of positive-sense single-stranded RNA (+ssRNA) viruses. To obtain insights into the unique broad-spectrum antiviral activity of T-705, we explored the role of this lysine using another +ssRNA virus, namely, coxsackievirus B3 (CVB3). Introduction of the corresponding K-to-R substitution in the CVB3 RdRp (K159R) resulted in a nonviable virus. Replication competence of the K159R variant was restored by spontaneous acquisition of an A239G substitution in the RdRp. A mutagenesis analysis at position K159 identified the K159M variant as the only other viable variant which had also acquired the A239G substitution. The K159 substitutions markedly decreased the processivity of the purified viral RdRp, which was restored by the introduction of the A239G mutation. The K159R A239G and K159M A239G variants proved, surprisingly, more susceptible than the wild-type virus to T-705 and exhibited lower fidelity in polymerase assays. Furthermore, the K159R A239G variant was found to be highly attenuated in mice. We thus demonstrate that the conserved lysine in the F1 motif of the RdRp of +ssRNA viruses is involved in the broad-spectrum antiviral activity of T-705 and that it is a key amino acid for the proper functioning of the enzyme.IMPORTANCE In this study, we report the key role of a highly conserved lysine residue of the viral polymerase in the broad-spectrum antiviral activity of favipiravir (T-705) against positive-sense single-stranded RNA viruses. Substitutions of this conserved lysine have a major negative impact on the functionality of the RdRp. Furthermore, we show that this lysine is involved in the fidelity of the RdRp and that the RdRp fidelity influences the sensitivity of the virus for the antiviral efficacy of T-705. Consequently, these results provide insights into the mechanism of the antiviral activity of T-705 and may lay the basis for the design of novel chemical scaffolds that may be endowed with a more potent broad-spectrum antiviral activity than that of T-705.
Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/genética , Lisina/metabolismo , Pirazinas/farmacologia , RNA Polimerase Dependente de RNA/química , Amidas/administração & dosagem , Motivos de Aminoácidos , Animais , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/genética , Chlorocebus aethiops , Farmacorresistência Viral/genética , Enterovirus Humano B/enzimologia , Japão , Lisina/genética , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Mutagênese , Mutação , Pirazinas/administração & dosagem , RNA Polimerase Dependente de RNA/genética , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Viral proteases are proteolytic enzymes that orchestrate the assembly of viral components during the viral life cycle and proliferation. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis are presented of protease 3C, the main protease of an emerging enterovirus, coxsackievirus B3, that is responsible for many cases of viral myocarditis. Polycrystalline protein precipitates suitable for X-ray powder diffraction (XRPD) measurements were produced in the presence of 22-28%(w/v) PEG 4000, 0.1â M Tris-HCl, 0.2â M MgCl2 in a pH range from 7.0 to 8.5. A polymorph of monoclinic symmetry (space group C2, unit-cell parameters a = 77.9, b = 65.7, c = 40.6â Å, ß = 115.9°) was identified via XRPD. These results are the first step towards the complete structural determination of the molecule via XRPD and a parallel demonstration of the accuracy of the method.
Assuntos
Cisteína Endopeptidases/química , Enterovirus Humano B/química , Proteínas Virais/química , Proteases Virais 3C , Sequência de Aminoácidos , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Enterovirus Humano B/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Difração de Raios XRESUMO
The RNA-dependent RNA polymerases from positive-strand RNA viruses, such as picornaviruses and flaviviruses, close their active sites for catalysis via a unique NTP-induced conformational change in the palm domain. Combined with a fully prepositioned templating nucleotide, this mechanism is error-prone and results in a distribution of random mutations in the viral progeny often described as a quasi-species. Here we examine the extent to which noncognate NTPs competitively inhibit single-cycle elongation by coxsackievirus B3 3D(pol), a polymerase that generates three to four mutations per 10 kb of RNA synthesized during viral infection. Using an RNA with a templating guanosine combined with 2-aminopurine fluorescence as a reporter for elongation, we find that the cognate CTP has a Km of 24 µM and the three noncognate nucleotides competitively inhibit the reaction with Kic values of 500 µM for GTP, 1300 µM for ATP, and 3000 µM for UTP. Unexpectedly, ATP also acted as an uncompetitive inhibitor with a Kiu of 1800 µM, resulting in allosteric modulation of 3D(pol) that slowed the polymerase elongation rate ≈4-fold. ATP uncompetitive inhibition required the ß- and γ-phosphates, and its extent was significantly diminished in two previously characterized low-fidelity polymerases. This led to further mutational analysis and the identification of a putative allosteric binding site below the NTP entry channel at the interface of conserved motifs A and D, although cocrystallization failed to reveal any density for bound ATP in this pocket. The potential role of an ATP allosteric effect during the virus life cycle is discussed.
Assuntos
Trifosfato de Adenosina/farmacologia , Enterovirus Humano B/enzimologia , Inibidores Enzimáticos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sequência de Bases , Domínio Catalítico , Sequências Repetidas Invertidas , Cinética , Modelos Moleculares , RNA/genética , RNA/metabolismo , RNA Polimerase Dependente de RNA/químicaRESUMO
Positive strand RNA viruses replicate via a virally encoded RNA-dependent RNA polymerase (RdRP) that uses a unique palm domain active site closure mechanism to establish the canonical two-metal geometry needed for catalysis. This mechanism allows these viruses to evolutionarily fine-tune their replication fidelity to create an appropriate distribution of genetic variants known as a quasispecies. Prior work has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background. In the work presented here, we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel where it may act as a nucleotide sensor to trigger active site closure. Crystallography, stopped-flow kinetics, quench-flow reactions, and infectious virus studies were used to characterize 15 engineered mutations in coxsackievirus B3 polymerase. Mutations that interfere with the transport of the metal A Mg(2+) ion into the active site had only minor effects on RdRP function, but the stacking interaction between Phe(364) and Pro(357), which is absolutely conserved in enteroviral polymerases, was found to be critical for processive elongation and virus growth. Mutating Phe(364) to tryptophan resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice. The data further illustrate the importance of the palm domain movement for RdRP active site closure and demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis.
Assuntos
Enterovirus Humano B/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Replicação Viral , Sequência de Aminoácidos , Biocatálise , Cristalização , Enterovirus Humano B/fisiologia , Cinética , Modelos Moleculares , Conformação Proteica , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Homologia de Sequência de AminoácidosRESUMO
Human coxsackievirus B3 (CVB3) 3C protease plays an essential role in the viral replication of CVB3, which is a non-enveloped and positive single-stranded RNA virus belonging to Picornaviridae family, causing acute viral myocarditis mainly in children. During optimization based on SAR studies of benserazide (3), which was reported as a novel anti-CVB3 3C(pro) agent from a screening of compound libraries, the 2,3,4-trihydroxybenzyl moiety of 3 was identified as a key pharmacophore for inhibitory activity against CVB3 3C(pro). Further optimization was performed by the introduction of various aryl-alkyl substituted hydrazide moieties instead of the serine moiety of 3. Among the optimized compounds, 11Q, a 4-hydroxyphenylpentanehydrazide derivative, showed the most potent inhibitory activity (IC50 = 0.07 µM). Enzyme kinetics studies indicated that 11Q exhibited a mixed inhibitory mechanism of action. The antiviral activity against CVB3 was confirmed using the further derived analogue (14b) with more cell permeable valeryl ester group at the 2,3,4-trihydroxy moiety.
Assuntos
Enterovirus Humano B/efeitos dos fármacos , Hidrazinas/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Antivirais/química , Antivirais/farmacologia , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Criança , Cisteína Endopeptidases , Enterovirus Humano B/enzimologia , Humanos , Hidrazinas/química , Permeabilidade , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Bibliotecas de Moléculas Pequenas , Relação Estrutura-AtividadeRESUMO
To determine whether 2A protease of the enterovirus genus with type I internal ribosome entry site (IRES) effect on the viral replication of type II IRES, coxsackievirus B3(CVB3)-encoded protease 2A and encephalomyocarditis virus (EMCV) IRES (Type II)-dependent or cap-dependent report gene were transiently co-expressed in eukaryotic cells. We found that CVB3 2A protease not only inhibited translation of cap-dependent reporter genes through the cleavage of eIF4GI, but also conferred high EMCV IRES-dependent translation ability and promoted EMCV replication. Moreover, deletions of short motif (aa13-18 RVVNRH, aa65-70 KNKHYP, or aa88-93 PRRYQSH) resembling the nuclear localization signals (NLS) or COOH-terminal acidic amino acid motif (aa133-147 DIRDLLWLEDDAMEQ) of CVB3 2A protease decreased both its EMCV IRES-dependent translation efficiency and destroy its cleavage on eukaryotic initiation factor 4G (eIF4G) I. Our results may provide better understanding into more effective interventions and treatments for co-infection of viral diseases.
Assuntos
Infecções por Cardiovirus/virologia , Cisteína Endopeptidases/metabolismo , Vírus da Encefalomiocardite/fisiologia , Enterovirus Humano B/enzimologia , Infecções por Enterovirus/virologia , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Vírus da Encefalomiocardite/genética , Enterovirus Humano B/genética , Humanos , Biossíntese de Proteínas , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Coxsackievirus B3 is the main cause of human viral myocarditis and cardiomyopathy. Virally encoded Coxsackievirus 3C protease (3C(pro)) plays an essential role in viral proliferation. Here, benserazide was discovered as a novel inhibitor from a drug library screen targeting Coxsackievirus 3C(pro) using a FRET-based enzyme assay. Benserazide, whose chemical structure has no electrophilic functional groups, was characterized as a non-competitive inhibitor by enzyme kinetic studies. A molecular docking study with benserazide and its analogs indicated that a novel putative allosteric binding site was involved. Specifically, a 2,3,4-trihydroxybenzyl moiety was determined to be a key pharmacophore for the enzyme's inhibitory activity. We suggest that the putative allosteric binding site may be a novel target for future therapeutic strategies.
Assuntos
Benserazida/farmacologia , Enterovirus Humano B/enzimologia , Inibidores de Proteases/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Sítio Alostérico , Cisteína Endopeptidases/genética , Transferência Ressonante de Energia de Fluorescência , Simulação de Acoplamento Molecular , Proteínas Virais/genéticaRESUMO
We have previously demonstrated that infection by coxsackievirus B3 (CVB3), a positive-stranded RNA enterovirus, results in the accumulation of insoluble ubiquitin-protein aggregates, which resembles the common feature of neurodegenerative diseases. The importance of protein aggregation in viral pathogenesis has been recognized; however, the underlying regulatory mechanisms remain ill-defined. Transactive response DNA-binding protein-43 (TDP-43) is an RNA-binding protein that has an essential role in regulating RNA metabolism at multiple levels. Cleavage and cytoplasmic aggregation of TDP-43 serves as a major molecular marker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration and contributes significantly to disease progression. In this study, we reported that TDP-43 is translocated from the nucleus to the cytoplasm during CVB3 infection through the activity of viral protease 2A, followed by the cleavage mediated by viral protease 3C. Cytoplasmic translocation of TDP-43 is accompanied by reduced solubility and increased formation of protein aggregates. The cleavage takes place at amino-acid 327 between glutamine and alanine, resulting in the generation of an N- and C-terminal cleavage fragment of ~35 and ~8 kDa, respectively. The C-terminal product of TDP-43 is unstable and quickly degraded through the proteasome degradation pathway, whereas the N-terminal truncation of TDP-43 acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Lastly, we demonstrated that knockdown of TDP-43 results in an increase in viral titers, suggesting a protective role for TDP-43 in CVB3 infection. Taken together, our findings suggest a novel model by which cytoplasmic redistribution and cleavage of TDP-43 as a consequence of CVB3 infection disrupts the solubility and transcriptional activity of TDP-43. Our results also reveal a mechanism evolved by enteroviruses to support efficient viral infection.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endopeptidases/metabolismo , Enterovirus Humano B/enzimologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Enterovirus Humano B/fisiologia , Células HeLa , Humanos , Masculino , Camundongos , Microscopia de Fluorescência , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Replicação ViralRESUMO
UNLABELLED: Picornavirus infection involves a dynamic interplay of host and viral protein interactions that modulates cellular processes to facilitate virus infection and evade host antiviral defenses. Here, using a proteomics-based approach known as TAILS to identify protease-generated neo-N-terminal peptides, we identify a novel target of the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoproteinM(hnRNP M), a nucleocytoplasmic shuttling RNA-binding protein that is primarily known for its role in pre-mRNA splicing. hnRNPMis cleaved in vitro by poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in poliovirus- and CVB3-infected HeLa cells and in the hearts of CVB3-infected mice. hnRNPMrelocalizes from the nucleus to the cytoplasm during poliovirus infection. Finally, depletion of hnRNPMusing small interfering RNA knockdown approaches decreases poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus internal ribosome entry site translation and viral RNA stability. We propose that cleavage of and subverting the function of hnRNPMis a general strategy utilized by picornaviruses to facilitate viral infection. IMPORTANCE: Enteroviruses, a member of the picornavirus family, are RNA viruses that cause a range of diseases, including respiratory ailments, dilated cardiomyopathy, and paralysis. Although enteroviruses have been studied for several decades, the molecular basis of infection and the pathogenic mechanisms leading to disease are still poorly understood. Here, we identify hnRNPMas a novel target of a viral proteinase. We demonstrate that the virus subverts the function of hnRNPMand redirects it to a step in the viral life cycle. We propose that cleavage of hnRNPMis a general strategy that picornaviruses use to facilitate infection.
Assuntos
Cisteína Endopeptidases/metabolismo , Enterovirus Humano B/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Interações Hospedeiro-Patógeno , Poliovirus/fisiologia , Proteínas Virais/metabolismo , Proteases Virais 3C , Animais , Enterovirus Humano B/enzimologia , Infecções por Enterovirus/patologia , Infecções por Enterovirus/virologia , Células HeLa , Coração/virologia , Humanos , Camundongos , Miocárdio/patologia , Poliovirus/enzimologia , ProteóliseRESUMO
The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.
Assuntos
Antivirais/química , Cardiovirus/enzimologia , Enterovirus Humano B/enzimologia , Poliovirus/enzimologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Animais , Sítios de Ligação , Chlorocebus aethiops , Células HeLa , Humanos , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
UNLABELLED: Viral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity. IMPORTANCE: Positive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects on in vitro nucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth in vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.
Assuntos
Enterovirus Humano B/enzimologia , Enterovirus Humano B/fisiologia , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Replicação Viral , Cristalografia por Raios X , Análise Mutacional de DNA , Enterovirus Humano B/genética , Humanos , Cinética , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/genéticaRESUMO
BACKGROUND: Coxsackievirus B3 (CVB3) is a common cause of myocarditis and dilated cardiomyopathy. CVB3 3C protease (3CP) cleaves the viral polyprotein during replication. We tested whether a water soluble 3CP inhibitor (3CPI) had antiviral effects in a chronic myocarditis model. METHODS: Chronic myocarditis was established using DBA/2 strain mice. Starting on post-infection (p.i) day 3, CVB3-infected mice (n=41) were treated with 3CPI by daily intraperitoneal (i.p.) injection at a concentration of 50 µM (1.7 mg/kg/day) per day for 3 consecutive days. Additional mice (n=49) were injected with PBS as a control. RESULTS: The 5-week survival rate was significantly higher with 3CPI treatment (82.3% versus 47.9%; P<0.05). Organ virus titers at day 3 and 7 and myocardial damage were significantly lower in 3CPI-treated mice. Echocardiography at day 31 indicated strong protection of heart function by 3CPI (FS, 51.2±1.5 versus 26.1±1.5%; P<0.001). Hemodynamic measurements indicated that 3CPI treatment markedly reduced CVB3-induced LV dysfunction on day 31 (dP/dTmax, 5302±352 versus 4103±408 mmHg/s, P<0.05; dP/dTmin, -3798±212 versus -2814±206 mmHg/s, P<0.01). CONCLUSIONS: Water soluble 3CPI was delivered through i.p. injection after CVB3 infection. This agent preserved heart function and decreased organ viral titers and myocardial damage. Soluble 3CPI may be beneficial in the treatment of cardiomyopathy associated with enterovirus infection.