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1.
Commun Biol ; 4(1): 841, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230602

RESUMO

Characterizing protein-protein interactions (PPIs) is an effective method to help explore protein function. Here, through integrating a newly identified split human Rhinovirus 3 C (HRV 3 C) protease, super-folder GFP (sfGFP), and ClpXP-SsrA protein degradation machinery, we developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to explore protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. We firstly identified a highly efficient split HRV 3 C protease with high re-assembly ability and then incorporated it into the SPEC method. The SPEC method could convert the cellular protein-protein interaction to quantitative fluorescence signals through a split HRV 3 C protease-mediated proteolytic reaction with high efficiency and broad temperature adaptability. Using SPEC method, we explored the interactions among effectors of representative type I-E and I-F CRISPR/Cas complexes, which combining with subsequent studies of Cas3 mutations conferred further understanding of the functions and structures of CRISPR/Cas complexes.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mapas de Interação de Proteínas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Endopeptidase Clp/genética , Enterovirus/enzimologia , Enterovirus/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteólise , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
Expert Opin Ther Targets ; 25(6): 479-489, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34253126

RESUMO

Introduction: Enteroviruses are common viruses causing a huge number of acute and chronic infections and producing towering economic costs. Similarly, coronaviruses cause seasonal mild infections, epidemics, and even pandemics and can lead to severe respiratory symptoms. It is important to develop broadly acting antiviral molecules to efficiently tackle the infections caused by thes.Areas covered: This review illuminates the differences and similarities between enteroviruses and coronaviruses and examines the most appealing therapeutic targets to combat both virus groups. Publications of both virus groups and deposited structures discovered through PubMed to March 2021 for viral proteases have been evaluated.Expert opinion: The main protease of coronaviruses and enteroviruses share similarities in their structure and function. These proteases process their viral polyproteins and thus drugs that bind to the active site have potential to target both virus groups. It is important to develop drugs that target more evolutionarily conserved processes and proteins. Moreover, it is a wise strategy to concentrate on processes that are similar between several virus families.


Assuntos
Antivirais/farmacologia , Coronavirus/fisiologia , Enterovirus/fisiologia , Animais , Coronavirus/efeitos dos fármacos , Coronavirus/enzimologia , Cisteína Endopeptidases/metabolismo , Enterovirus/efeitos dos fármacos , Enterovirus/enzimologia , Humanos , Especificidade por Substrato
3.
mBio ; 12(2)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33849973

RESUMO

Enteroviruses (EV) deploy two proteases that mediate viral polyprotein cleavage and host cell manipulation. Here, we report that EV 2A proteases cleave all three members of the YTHDF protein family, cytosolic N 6-methyladenosine (m6A) "readers" that regulate target mRNA fate. YTHDF protein cleavage occurs very early during infection, before viral translation is detected or cytopathogenic effects are observed. Preemptive YTHDF protein depletion enhanced viral translation and replication but only in cells with restrained viral translation, signs of inefficient 2A protease activity, and protective innate host immune responses. This effect corresponded with repression of interferon (IFN)-stimulated gene (ISG) induction, while type I/III IFN production was not significantly altered. Moreover, YTHDF3 depletion impaired JAK/STAT signaling in cells treated with type I, but not type II, IFN. YTHDF3 depletion's stimulatory effect on viral dynamics was dampened by JAK/STAT blockade and enhanced by type I IFN pretreatment of cells. We propose that EV 2A proteases cleave YTHDF proteins to antagonize ISG induction in infected cells.IMPORTANCE It is believed that ∼7,000 messenger RNAs (mRNAs) are subject to N 6-methyladenosine modification. The biological significance of this remains mysterious. The YTHDF m6A readers are three related proteins with high affinity for m6A-modified mRNA, yet their biological functions remain obscure. We discovered that polio/enteroviruses elicit very early proteolysis of YTHDF1 to 3 in infected cells. Our research demonstrates that YTHDF3 acts as a positive regulator of antiviral JAK/STAT signaling in response to positive single-strand RNA virus infection, enabling type I interferon (IFN)-mediated gene regulatory programs to unfurl in infected cells. Our observation of viral degradation of the YTHDF proteins demonstrates that they are key response modifiers in the innate antiviral immune response.


Assuntos
Enterovirus/genética , Interferon Tipo I/metabolismo , Janus Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Virais/metabolismo , Linhagem Celular , Enterovirus/enzimologia , Células HeLa , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Janus Quinases/genética , Proteólise , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Fatores de Transcrição STAT/genética , Proteínas Virais/genética
4.
Proc Natl Acad Sci U S A ; 117(24): 13499-13508, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32467165

RESUMO

The existence of multiple serotypes renders vaccine development challenging for most viruses in the Enterovirus genus. An alternative and potentially more viable strategy for control of these viruses is to develop broad-spectrum antivirals by targeting highly conserved proteins that are indispensable for the virus life cycle, such as the 3C protease. Previously, two single-chain antibody fragments, YDF and GGVV, were reported to effectively inhibit human rhinovirus 14 proliferation. Here, we found that both single-chain antibody fragments target sites on the 3C protease that are distinct from its known drug site (peptidase active site) and possess different mechanisms of inhibition. YDF does not block the active site but instead noncompetitively inhibits 3C peptidase activity through an allosteric effect that is rarely seen for antibody protease inhibitors. Meanwhile, GGVV antagonizes the less-explored regulatory function of 3C in genome replication. The interaction between 3C and the viral genome 5' noncoding region has been reported to be important for enterovirus genome replication. Here, the interface between human rhinovirus 14 3C and its 5' noncoding region was probed by hydrogen-deuterium exchange coupled mass spectrometry and found to partially overlap with the interface between GGVV and 3C. Consistently, prebinding of GGVV completely abolishes interaction between human rhinovirus 14 3C and its 5' noncoding region. The epitopes of YDF and GGVV, therefore, represent two additional sites of therapeutic vulnerability in rhinovirus. Importantly, the GGVV epitope appears to be conserved across many enteroviruses, suggesting that it is a promising target for pan-enterovirus inhibitor screening and design.


Assuntos
Antivirais/farmacologia , Cisteína Endopeptidases/química , Enterovirus/efeitos dos fármacos , Anticorpos de Cadeia Única/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Regiões 5' não Traduzidas , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Antivirais/química , Antivirais/metabolismo , Cisteína Endopeptidases/metabolismo , Enterovirus/enzimologia , Epitopos , Genoma Viral , RNA Viral/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Proteínas Virais/metabolismo
5.
Nucleic Acids Res ; 48(10): 5591-5602, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32286652

RESUMO

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1-1000 s duration, of which the short-lived ones (1-10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.


Assuntos
Vírus de RNA/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Temperatura , Replicação Viral , Bacteriófago phi 6/enzimologia , Enterovirus/enzimologia , Ativação Enzimática , Cinética , Microscopia , Poliovirus/enzimologia
6.
J Med Chem ; 63(9): 4562-4578, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32045235

RESUMO

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease-inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the α-ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus.


Assuntos
Antivirais/farmacologia , Coronavirus/efeitos dos fármacos , Enterovirus/efeitos dos fármacos , Lactamas/farmacologia , Peptidomiméticos/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/síntese química , Antivirais/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Chlorocebus aethiops , Coronavirus/enzimologia , Proteases 3C de Coronavírus , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Desenho de Fármacos , Enterovirus/enzimologia , Humanos , Lactamas/síntese química , Lactamas/metabolismo , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Inibidores de Proteases/síntese química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Ligação Proteica , Células Vero , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteínas Virais/metabolismo
7.
Viruses ; 12(1)2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936473

RESUMO

BACKGROUND: Enteroviruses are a group of common non-enveloped RNA viruses that cause symptoms ranging from mild respiratory infections to paralysis. Due to the abundance of enterovirus infections it is hard to distinguish between on-going and previous infections using immunological assays unless the IgM fraction is studied. METHODS: In this study we show using Indirect ELISA and capture IgM ELISA that an IgG antibody response against the nonstructural enteroviral proteins 2A and 3C can be used to distinguish between IgM positive (n = 22) and IgM negative (n = 20) human patients with 83% accuracy and a diagnostic odds ratio of 30. Using a mouse model, we establish that the antibody response to the proteases is short-lived compared to the antibody response to the structural proteins in. As such, the protease antibody response serves as a potential marker for an acute infection. CONCLUSIONS: Antibody responses against enterovirus proteases are shorter-lived than against structural proteins and can differentiate between IgM positive and negative patients, and therefore they are a potential marker for acute infections.


Assuntos
Anticorpos Antivirais/sangue , Enterovirus/enzimologia , Enterovirus/imunologia , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Peptídeo Hidrolases/imunologia , Doença Aguda , Adulto , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Biomarcadores/sangue , Cisteína Endopeptidases/imunologia , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Lactente , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo Hidrolases/classificação , Proteínas Virais/imunologia
8.
Biol Chem ; 400(3): 405-415, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30521472

RESUMO

Proteases are crucial for regulating biological processes in organisms through hydrolysis of peptide bonds. Recombinant proteases have moreover become important tools in biotechnological, and biomedical research and as therapeutics. We have developed a label-free high-throughput method for quantitative assessment of proteolytic activity in Escherichia coli. The screening method is based on co-expression of a protease of interest and a reporter complex. This reporter consists of an aggregation-prone peptide fused to a fluorescent protein via a linker that contains the corresponding substrate sequence. Cleavage of the substrate rescues the fluorescent protein from aggregation, resulting in increased fluorescence that correlates to proteolytic activity, which can be monitored using flow cytometry. In one round of flow-cytometric cell sorting, we isolated an efficiently cleaved tobacco etch virus (TEV) substrate from a 1:100 000 background of non-cleavable sequences, with around 6000-fold enrichment. We then engineered the 3C protease from coxsackievirus B3 (CVB3 3Cpro) towards improved proteolytic activity on the substrate LEVLFQ↓GP. We isolated highly proteolytic active variants from a randomly mutated CVB3 3Cpro library with up to 4-fold increase in activity. The method enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for protease substrate profiling, as well as directed evolution of proteases.


Assuntos
Cisteína Endopeptidases/metabolismo , Enterovirus/enzimologia , Citometria de Fluxo , Fluorescência , Proteínas Virais/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Engenharia de Proteínas
9.
Angew Chem Int Ed Engl ; 57(39): 12702-12706, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30118570

RESUMO

Analogous to reversible post-translational protein modifications, the ability to attach and subsequently remove modifications on proteins would be valuable for protein and biological research. Although bioorthogonal functionalities have been developed to conjugate or cleave protein modifications, they are introduced into proteins on separate residues and often with bulky side chains, limiting their use to one type of control and primarily protein surface. Here we achieved dual control on one residue by genetically encoding S-propargyl-cysteine (SprC), which has bioorthogonal alkyne and propargyl groups in a compact structure, permitting usage in protein interior in addition to surface. We demonstrated its incorporation at the dimer interface of glutathione transferase for in vivo crosslinking via thiol-yne click chemistry, and at the active site of human rhinovirus 3C protease for masking and then turning on enzyme activity via Pd-cleavage of SprC into Cys. In addition, we installed biotin onto EGFP via Sonogashira coupling of SprC and then tracelessly removed it via Pd cleavage. SprC is small in size, commercially available, nontoxic, and allows for bond building and breaking on a single residue. Genetically encoded SprC will be valuable for chemically controlling proteins with an essential Cys and for reversible protein modifications.


Assuntos
Cisteína Endopeptidases/metabolismo , Cisteína/química , Proteínas de Fluorescência Verde/química , Proteínas Virais/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Biotina/química , Catálise , Domínio Catalítico , Química Click , Cisteína/metabolismo , Cisteína Endopeptidases/química , Enterovirus/enzimologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Methanosarcina/metabolismo , Mutagênese Sítio-Dirigida , Paládio/química , Pargilina/química , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Proteínas Virais/química
10.
J Med Chem ; 61(3): 1218-1230, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29328649

RESUMO

Lead structure discovery mainly focuses on the identification of noncovalently binding ligands. Covalent linkage, however, is an essential binding mechanism for a multitude of successfully marketed drugs, although discovered by serendipity in most cases. We present a concept for the design of fragments covalently binding to proteases. Covalent linkage enables fragment binding unrelated to affinity to shallow protein binding sites and at the same time allows differentiated targeted hit verification and binding location verification through mass spectrometry. We describe a systematic and rational computational approach for the identification of covalently binding fragments from compound collections inhibiting enteroviral 3C protease, a target with high therapeutic potential. By implementing reactive groups potentially forming covalent bonds as a chemical feature in our 3D pharmacophore methodology, covalent binders were discovered by high-throughput virtual screening. We present careful experimental validation of the virtual hits using enzymatic assays and mass spectrometry unraveling a novel, previously unknown irreversible inhibition of the 3C protease by phenylthiomethyl ketone-based fragments. Subsequent synthetic optimization through fragment growing and reactivity analysis against catalytic and noncatalytic cysteines revealed specific irreversible 3C protease inhibition.


Assuntos
Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Enterovirus/enzimologia , Cetonas/química , Cetonas/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Domínio Catalítico , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/metabolismo , Desenho de Fármacos , Ensaios de Triagem em Larga Escala , Cetonas/metabolismo , Modelos Moleculares , Relação Estrutura-Atividade , Especificidade por Substrato , Proteínas Virais/química
11.
Virol Sin ; 32(6): 548-552, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29164396

RESUMO

The RNA-dependent RNA polymerases (RdRPs) encoded by RNA viruses represent a unique class of nucleic acid polymerases. Unlike other classes of single-subunit polymerases, viral RdRPs have evolved a unique conformational change in their palm domain to close the active site during catalysis. The hallmark of this conformational change is the backbone shift of the polymerase motif A from an "open" state to a "closed" state, allowing two universally conserved aspartic acid residues to orient toward each other for divalent metal binding and catalysis. The "closed" motif A conformation was only observed upon the binding of correct NTP in RdRP catalytic complexes or under rare conditions such as induced by a bound lutetium ion or a bound glutamate molecule. By solving the crystal structure of the catalytic elongation complex of the coxsackievirus RdRP, we in this work observed for the first time the "closed" motif A conformation in the absence of an NTP substrate or other conformational-change-inducing factors. This observation emphasizes the intrinsic dynamic features of viral RdRP motif A, and solidifies the structural basis for how this important structural element participates in catalytic events of the RdRPs.


Assuntos
Enterovirus/enzimologia , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Cristalografia por Raios X , Modelos Moleculares
12.
Artigo em Inglês | MEDLINE | ID: mdl-28461310

RESUMO

Hand-foot-and-mouth disease (HFMD), caused by enterovirus, is a threat to public health worldwide. To date, enterovirus 71 (EV71) has been one of the major causative agents of HFMD in the Pacific-Asia region, and outbreaks with EV71 cause millions of infections. However, no drug is currently available for clinical therapeutics. In our previous works, we developed a set of protease inhibitors (PIs) targeting the EV71 3C protease (3Cpro). Among these are NK-1.8k and NK-1.9k, which have various active groups and high potencies and selectivities. In the study described here, we determined the structures of the PI NK-1.8k in complex with wild-type (WT) and drug-resistant EV71 3Cpro Comparison of these structures with the structure of unliganded EV71 3Cpro and its complex with AG7088 indicated that the mutation of N69 to a serine residue destabilized the S2 pocket. Thus, the mutation influenced the cleavage activity of EV71 3Cpro and the inhibitory activity of NK-1.8k in an in vitro protease assay and highlighted that site 69 is an additional key site for PI design. More information for the optimization of the P1' to P4 groups of PIs was also obtained from these structures. Together with the results of our previous works, these in-depth results elucidate the inhibitory mechanism of PIs and shed light to develop PIs for the clinical treatment of infections caused by EV71 and other enteroviruses.


Assuntos
Antivirais/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Enterovirus/enzimologia , Inibidores de Proteases/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Antivirais/química , Doença de Mão, Pé e Boca/enzimologia , Doença de Mão, Pé e Boca/metabolismo , Isoxazóis/química , Isoxazóis/metabolismo , Mutação , Fenilalanina/análogos & derivados , Inibidores de Proteases/química , Estrutura Terciária de Proteína , Pirrolidinonas/química , Pirrolidinonas/metabolismo , Valina/análogos & derivados
13.
J Virol ; 91(14)2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28490584

RESUMO

Enteroviruses (EVs) are implicated in a wide range of diseases in humans and animals. In this study, a novel enterovirus (enterovirus species G [EVG]) (EVG 08/NC_USA/2015) was isolated from a diagnostic sample from a neonatal pig diarrhea case and identified by using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582-nucleotide insertion, flanked by 3Cpro cleavage sites at the 5' and 3' ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54 to 68% amino acid identity to torovirus (ToV) papain-like protease (PLP) (ToV-PLP). Structural homology modeling predicts that this protease adopts a fold and a catalytic site characteristic of minimal PLP catalytic domains. This structure is similar to those of core catalytic domains of the foot-and-mouth disease virus leader protease and coronavirus PLPs, which act as deubiquitinating and deISGylating (interferon [IFN]-stimulated gene 15 [ISG15]-removing) enzymes on host cell substrates. Importantly, the recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities and demonstrated the ability to suppress IFN-ß expression. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus. Compared to the wild-type virus, the ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event.IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we identified a special case of cross-order recombination between enterovirus G (order Picornavirales) and torovirus (order Nidovirales). This naturally occurring recombination event may have broad implications for other picornaviral and/or nidoviral species. Importantly, we demonstrated that the exogenous ToV-PLP gene that was inserted into the EVG genome encodes a deubiquitinase/deISGylase and potentially suppresses host cellular innate immune responses. Our results provide insights into how a gain of function through genetic recombination, in particular cross-order recombination, may improve the ability of a virus to evade host immunity.


Assuntos
Enzimas Desubiquitinantes/genética , Enterovirus/enzimologia , Enterovirus/genética , Fezes/virologia , Mutagênese Insercional , Torovirus/enzimologia , Torovirus/genética , Animais , Animais Recém-Nascidos , Diarreia/veterinária , Enterovirus/isolamento & purificação , Metagenômica , RNA Viral/genética , Recombinação Genética , Análise de Sequência de DNA , Suínos , Doenças dos Suínos/virologia , Estados Unidos
14.
Biophys Chem ; 219: 9-16, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27668727

RESUMO

Hand foot and mouth disease (HFMD) epidemic has occurred in many countries. Coxsackievirus A16 (CV-A16) and Enterovirus A71 (EV-A71) are the main causes of HFMD. Up to now, there are no anti-HFMD drugs available. Rupintrivir, a broad-spectrum inhibitor, is a drug candidate for HFMD treatment, while other HFMD inhibitors designed from several studies have a relatively low efficiency. Therefore, in this work we aim to study the binding mechanisms of rupintrivir and a peptidic α,ß-unsaturated ethyl ester (SG85) against both CV-A16 and EV-A71 3C proteases (3Cpro) using all-atoms molecular dynamics simulation. The obtained results indicate that SG85 shows a stronger binding affinity than rupintrivir against CV-A16. Both inhibitors exhibit a comparable affinity against EV-A71 3Cpro. The molecular information of the binding of the two inhibitors to the proteases will be elucidated. Thus, it is implied that these two compounds may be used as leads for further anti-HFMD drug design and development.


Assuntos
Enterovirus Humano A/enzimologia , Enterovirus/enzimologia , Simulação de Dinâmica Molecular , Proteínas Virais/antagonistas & inibidores , Cisteína Endopeptidases , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Doença de Mão, Pé e Boca/virologia , Humanos , Técnicas In Vitro , Isoxazóis/farmacologia , Oligopeptídeos/farmacologia , Peptídeos/farmacologia , Fenilalanina/análogos & derivados , Ligação Proteica , Pirrolidinonas/farmacologia , Valina/análogos & derivados
15.
Nucleic Acids Res ; 44(14): 6883-95, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27317698

RESUMO

Genetic recombination in single-strand, positive-sense RNA viruses is a poorly understand mechanism responsible for generating extensive genetic change and novel phenotypes. By moving a critical cis-acting replication element (CRE) from the polyprotein coding region to the 3' non-coding region we have further developed a cell-based assay (the 3'CRE-REP assay) to yield recombinants throughout the non-structural coding region of poliovirus from dually transfected cells. We have additionally developed a defined biochemical assay in which the only protein present is the poliovirus RNA dependent RNA polymerase (RdRp), which recapitulates the strand transfer events of the recombination process. We have used both assays to investigate the role of the polymerase fidelity and nucleotide turnover rates in recombination. Our results, of both poliovirus intertypic and intratypic recombination in the CRE-REP assay and using a range of polymerase variants in the biochemical assay, demonstrate that RdRp fidelity is a fundamental determinant of recombination frequency. High fidelity polymerases exhibit reduced recombination and low fidelity polymerases exhibit increased recombination in both assays. These studies provide the basis for the analysis of poliovirus recombination throughout the non-structural region of the virus genome and provide a defined biochemical assay to further dissect this important evolutionary process.


Assuntos
Enterovirus/enzimologia , Enterovirus/genética , RNA Polimerase Dependente de RNA/metabolismo , Recombinação Genética , Animais , Sequência de Bases , Bioensaio , Replicação do DNA , DNA Intergênico/genética , Genoma Viral , Células HeLa , Humanos , Camundongos , Mutação/genética , Nucleotídeos/metabolismo , Poliovirus/genética , RNA Polimerase Dependente de RNA/genética , Moldes Genéticos , Replicação Viral
16.
Rev Med Virol ; 26(4): 251-67, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27145174

RESUMO

Enteroviruses are common human pathogens, and infections are particularly frequent in children. Severe infections can lead to a variety of diseases, including poliomyelitis, aseptic meningitis, myocarditis and neonatal sepsis. Enterovirus infections have also been implicated in asthmatic exacerbations and type 1 diabetes. The large disease spectrum of the closely related enteroviruses may be partially, but not fully, explained by differences in tissue tropism. The molecular mechanisms by which enteroviruses cause disease are poorly understood, but there is increasing evidence that the two enteroviral proteases, 2A(pro) and 3C(pro) , are important mediators of pathology. These proteases perform the post-translational proteolytic processing of the viral polyprotein, but they also cleave several host-cell proteins in order to promote the production of new virus particles, as well as to evade the cellular antiviral immune responses. Enterovirus-associated processing of cellular proteins may also contribute to pathology, as elegantly demonstrated by the 2A(pro) -mediated cleavage of dystrophin in cardiomyocytes contributing to Coxsackievirus-induced cardiomyopathy. It is likely that improved tools to identify targets for these proteases will reveal additional host protein substrates that can be linked to specific enterovirus-associated diseases. Here, we discuss the function of the enteroviral proteases in the virus replication cycle and review the current knowledge regarding how these proteases modulate the infected cell in order to favour virus replication, including ways to avoid detection by the immune system. We also highlight new possibilities for the identification of protease-specific cellular targets and thereby a way to discover novel mechanisms contributing to disease. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Cisteína Endopeptidases/metabolismo , Enterovirus/enzimologia , Enterovirus/fisiologia , Interações Hospedeiro-Patógeno , Replicação Viral , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Enterovirus/patogenicidade , Humanos , Evasão da Resposta Imune , Conformação Proteica , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
17.
J Gen Virol ; 97(6): 1368-1380, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26935471

RESUMO

Type I IFNs play an important role in the immune response to enterovirus infections. Their importance is underscored by observations showing that many enteroviruses including coxsackie B viruses (CVBs) have developed strategies to block type I IFN production. Recent studies have highlighted a role for the type III IFNs (also called IFNλs) in reducing permissiveness to infections with enteric viruses including coxsackievirus. However, whether or not CVBs have measures to evade the effects of type III IFNs remains unknown. By combining virus infection studies and different modes of administrating the dsRNA mimic poly I : C, we discovered that CVBs target both TLR3- and MDA5/RIG-I-mediated type III IFN expression. Consistent with this, the cellular protein expression levels of the signal transduction proteins TRIF and IPS1 were reduced and no hyperphosphorylation of IRF-3 was observed following infection with the virus. Notably, decreased expression of full-length TRIF and IPS1 and the appearance of cleavage products was observed upon both CVB3 infection and in cellular protein extracts incubated with recombinant 2Apro, indicating an important role for the viral protease in subverting the cellular immune system. Collectively, our study reveals that CVBs block the expression of type III IFNs, and that this is achieved by a similar mechanism as the virus uses to block type I IFN production. We also demonstrate that the virus blocks several intracellular viral recognition pathways of importance for both type I and III IFN production. The simultaneous targeting of numerous arms of the host immune response may be required for successful viral replication and dissemination.


Assuntos
Cisteína Endopeptidases/metabolismo , Enterovirus/imunologia , Enterovirus/patogenicidade , Evasão da Resposta Imune , Imunidade Inata , Interleucinas/antagonistas & inibidores , Proteínas Virais/metabolismo , Enterovirus/enzimologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferons , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo
18.
PLoS One ; 10(10): e0141383, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26509685

RESUMO

Unc93b is an endoplasmic reticulum (ER)-resident transmembrane protein that serves to bind and traffic toll-like receptors (TLRs) from the ER to their appropriate subcellular locations for ligand sensing. Because of its role in TLR trafficking, Unc93b is necessary for an effective innate immune response to coxsackievirus B3 (CVB), a positive-sense single stranded RNA virus belonging to the enterovirus family. Here, we show that Unc93b is cleaved by a CVB-encoded cysteine protease (3Cpro) during viral replication. Further, we define a role for Unc93b in the induction of apoptotic cell death and show that expression of wild-type Unc93b, but not a mutant incapable of binding TLRs or exiting the ER (H412R), induces apoptosis. Furthermore, we show that cellular caspases activated during apoptosis directly cleave Unc93b. Interestingly, we show that the 3Cpro- and caspase-mediated cleavage of Unc93b both occur within ten amino acids in the distal N-terminus of Unc93b. Mechanistically, neither caspase-mediated nor 3Cpro-mediated cleavage of Unc93b altered its trafficking function, inhibited its role in facilitating TLR3 or TLR8 signaling, or altered its apoptosis-inducing effects. Taken together, our studies show that Unc93b is targeted by both viral- and host cell-specific proteases and identify a function of Unc93b in the induction of apoptotic cell death.


Assuntos
Apoptose/genética , Enterovirus/enzimologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Peptídeo Hidrolases/metabolismo , Caspases/metabolismo , Linhagem Celular , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Infecções por Enterovirus/genética , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/virologia , Expressão Gênica , Humanos , Mutação , Transporte Proteico , Proteólise , Transdução de Sinais , Receptores Toll-Like/metabolismo , Proteínas Virais/metabolismo
19.
J Virol ; 89(21): 11069-79, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26311873

RESUMO

UNLABELLED: Enteroviruses proteolyze nuclear pore complex (NPC) proteins (Nups) during infection, leading to disruption of host nuclear transport pathways and alterations in nuclear permeability. To better understand how enteroviruses exert these effects on nuclear transport, the mechanisms and consequences of Nup98 proteolysis were examined. The results indicate that Nup98 is rapidly targeted for degradation following enterovirus infection and that this is mediated by the enterovirus 2A protease (2A(pro)). Incubation of bacterially expressed or in vitro-translated Nup98 with 2A(pro) results in proteolytic cleavage at multiple sites in vitro, indicating that 2A(pro) cleaves Nup98 directly. Site-directed mutagenesis of putative cleavage sites identified Gly374 and Gly552 as the sites of 2A(pro) proteolysis in Nup98 in vitro and in infected cells. Indirect immunofluorescence assays using an antibody that recognizes the N terminus of Nup98 revealed that proteolysis releases the N-terminal FG-rich region from the NPC. In contrast, similar analyses using an antibody to the C terminus indicated that this region is retained at the nuclear rim. Nup88, a core NPC component that serves as a docking site for Nup98, also remains at the NPC in infected cells. These findings support a model whereby the selective removal of Nup FG repeat domains leads to increased NPC permeability and inhibition of certain transport pathways, while retention of structural domains maintains the overall NPC structure and leaves other transport pathways unaffected. IMPORTANCE: Enteroviruses are dependent upon host nuclear RNA binding proteins for efficient replication. This study examines the mechanisms responsible for alterations in nuclear transport in enterovirus-infected cells that lead to the cytoplasmic accumulation of these proteins. The results demonstrate that the enterovirus 2A protease directly cleaves the nuclear pore complex (NPC) protein, Nup98, at amino acid positions G374 and G552 both in vitro and in infected cells. Cleavage at these positions results in the selective removal of the FG-containing N terminus of Nup98 from the NPC, while the C terminus remains associated. Nup88, a core component of the NPC that serves as a docking site for the C terminus of Nup98, remains associated with the NPC in infected cells. These findings help to explain the alterations in permeability and nuclear transport in enterovirus-infected cells and how NPCs remain functional for certain trafficking pathways despite significant alterations to their compositions.


Assuntos
Transporte Ativo do Núcleo Celular/genética , Cisteína Endopeptidases/metabolismo , Enterovirus/enzimologia , Modelos Genéticos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas Virais/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Mutagênese , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína
20.
PLoS Pathog ; 11(7): e1005067, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26218680

RESUMO

RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3'-to-5' unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.


Assuntos
Infecções por Enterovirus/metabolismo , Enterovirus/enzimologia , Chaperonas Moleculares/metabolismo , RNA Helicases/metabolismo , RNA Viral/genética , Proteínas não Estruturais Virais/metabolismo , Trifosfato de Adenosina/metabolismo , Humanos , Replicação Viral/fisiologia
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