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1.
J Med Chem ; 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38687966

RESUMO

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.

3.
Chemosphere ; 352: 141307, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38307338

RESUMO

The present study compares the effect of using different solvents on the electrochemical properties of the reduced TiO2 nanotubes (TiO2-rNTs) layered Ti/TiO2-rNTs/SnO2-Sb/PbO2 anodes. The electrodes are prepared using three different solvent-based precursors: (i) isopropanol, (ii) ethylene glycol and citric acid (Pechini method), and (iii) 2-hydroxyethylammonium acetate (2HEAA) ionic liquid (IL) via the thermal decomposition route. The decomposition mechanism of precursor solutions was explored using the thermogravimetric (TGA) analysis. Further, the physicochemical properties of the electrodes are examined using Field emission Scanning Electron microscopy (FE-SEM), X-ray diffraction spectroscopy (XRD), and X-ray photoelectron emission spectroscopy (XPS). The results revealed that solvents with higher viscosity and slower decomposition rates support better film uniformity and higher stability of the electrode. The TiO2 -rNTs bottom layer and PbO2 top layer helped obtain higher film stability, increased working potential window (2.2 V vs. SHE) of the electrode, and the repeatability of the results. The performance of different electrodes based on the precursor solution is found as IL â‰« Pechini > Isopropanol. 4-chlorophenol (4-CP) is used as a model pollutant to test the performance of IL-Ti/TiO2-rNTs/SnO2-Sb/PbO2 anode in an anodic oxidation (AO) coupled electro-Fenton (EF) treatment. Further, the reliability of the electrode is evaluated by mineralizing other persistent organic pollutants (POPs) like tetracyclin, phenol, 2-chlorophenol (2-CP), and 2,4-dichlorophenol (2,4-DCP). Under the optimized conditions, the proposed system was able to mineralize the tetracyclin, phenol, 2-CP, 2,4-DCP, and 4-CP up to 78.91, 82.07, 74.96, 78.78, and 69.3 %, respectively. Moreover, the degradation mechanism of chlorophenols is proposed.


Assuntos
Óxidos , Poluentes Químicos da Água , Óxidos/química , Poluentes Orgânicos Persistentes , 2-Propanol , Reprodutibilidade dos Testes , Titânio/química , Oxirredução , Fenóis , Fenol/química , Eletrodos , Solventes , Poluentes Químicos da Água/química
4.
Chemosphere ; 336: 139225, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37356583

RESUMO

The influence of anode materials on the electrochemical treatment of tannery wastewater (TWW) was evaluated using Pt, Ti/RuO2-IrO2 (DSA), Ti/SnO2-Sb, Ti/PbO2, and Ti/SnO2-Sb/PbO2 electrodes. The comparison of the degradation mechanism of these electrodes in the electro-Fenton (EF) treatment was evaluated. The Ti/SnO2-Sb/PbO2 anode was efficient, with high electrocatalytic activity, stability, and reproducibility of the degradation results. Further, the study was extended to define the ability of sequential EF and electrocoagulation (EC) processes to clean TWW. The EC treatment was conducted using Al electrodes, and the performance of the combined treatment was evaluated by the removal of chemical oxygen demand (COD), turbidity, total suspended solids (TSS), sulfide, and Cr removal. The role of chlorides and sulfate salts during both treatments was evaluated by monitoring the concentration changes of these anions during the whole treatment using ion chromatography (IC). A sequential 1.5 h EF and 1 h EC treatment were applied to achieve a satisfactory degradation of (81.2 ± 3.9)% COD, >98% Cr, >99% turbidity, TSS, and sulfide removal. Additionally, the combined treatment was found to be more efficient towards the COD removal, achieving about 22.5% higher COD removal consuming almost the same amount of electrical energy.


Assuntos
Poluentes Químicos da Água , Purificação da Água , Oxirredução , Reprodutibilidade dos Testes , Eletrocoagulação , Águas Residuárias , Eletrodos , Purificação da Água/métodos , Poluentes Químicos da Água/química , Titânio/química
5.
Proc Natl Acad Sci U S A ; 120(16): e2214997120, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-37043537

RESUMO

While somatic variants of TRAF7 (Tumor necrosis factor receptor-associated factor 7) underlie anterior skull-base meningiomas, here we report the inherited mutations of TRAF7 that cause congenital heart defects. We show that TRAF7 mutants operate in a dominant manner, inhibiting protein function via heterodimerization with wild-type protein. Further, the shared genetics of the two disparate pathologies can be traced to the common origin of forebrain meninges and cardiac outflow tract from the TRAF7-expressing neural crest. Somatic and inherited mutations disrupt TRAF7-IFT57 interactions leading to cilia degradation. TRAF7-mutant meningioma primary cultures lack cilia, and TRAF7 knockdown causes cardiac, craniofacial, and ciliary defects in Xenopus and zebrafish, suggesting a mechanistic convergence for TRAF7-driven meningiomas and developmental heart defects.


Assuntos
Cardiopatias Congênitas , Neoplasias Meníngeas , Meningioma , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cardiopatias Congênitas/genética , Neoplasias Meníngeas/genética , Meningioma/genética , Meningioma/patologia , Mutação , Crânio/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Humanos , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral
6.
Front Vet Sci ; 9: 1028077, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36387381

RESUMO

The foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) is a papain like protease that cleaves the viral polyprotein and several host factors affecting host cell translation and induction of innate immunity. Introduction of Lpro mutations ablating catalytic activity is not tolerated by the virus, however, complete coding sequence deletion or introduction of targeted amino acid substitutions can render viable progeny. In proof-of-concept studies, we have previously identified and characterized FMDV Lpro mutants that are attenuated in cell culture and in animals, while retaining their capacity for inducing a strong adaptive immunity. By using molecular modeling, we have now identified a His residue (H138), that resides outside the substrate binding and catalytic domain, and is highly conserved across serotypes. Mutation of H138 renders possible FMDV variants of reduced virulence in vitro and in vivo. Kinetics studies showed that FMDV A12-LH138L mutant replicates similarly to FMDV A12-wild type (WT) virus in cells that do not offer immune selective pressure, but attenuation is observed upon infection of primary or low passage porcine epithelial cells. Western blot analysis on protein extracts from these cells, revealed that while processing of translation initiation factor eIF-4G was slightly delayed, no degradation of innate sensors or effector molecules such as NF-κB or G3BP2 was observed, and higher levels of interferon (IFN) and IFN-stimulated genes (ISGs) were induced after infection with A12-LH138L as compared to WT FMDV. Consistent with the results in porcine cells, inoculation of swine with this mutant resulted in a mild, or in some cases, no clinical disease but induction of a strong serological adaptive immune response. These results further support previous evidence that Lpro is a reliable target to derive numerous viable FMDV strains that alone or in combination could be exploited for the development of novel FMD vaccine platforms.

7.
Antiviral Res ; 208: 105429, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36208677

RESUMO

Vero cells are widely used for antiviral tests and virology research for SARS-CoV-2 as well as viruses from various other families. However, Vero cells generally express high levels of multi-drug resistance 1 (MDR1) or Pgp protein, the efflux transporter of foreign substances including many antiviral compounds, affecting the antiviral activity as well as interpretation of data. To address this, a Pgp gene knockout VeroE6 cell line (VeroE6-Pgp-KO) was generated using CRISPR-CAS9 technology. These cells no longer expressed the Pgp protein as indicated by flow cytometry analysis following staining with a Pgp-specific monoclonal antibody. They also showed significantly reduced efflux transporter activity in the calcein acetoxymethyl ester (calcein AM) assay. The VeroE6-Pgp-KO cells and the parental VeroE6 cells were each infected with SARS-CoV-2 to test antiviral activities of remdesivir and nirmatrelvir, two known Pgp substrates, in the presence or absence of a Pgp inhibitor. The compounds showed antiviral activities in VeroE6-Pgp-KO cells similar to that observed in the presence of the Pgp inhibitor. Thus, the newly established VeroE6-Pgp-KO cell line adds a new in vitro virus infection system for SARS-CoV-2 and possibly other viruses to test antiviral therapies without a need to control the Pgp activity. Removal of the Pgp inhibitor for antiviral assays will lead to less data variation and prevent failed assays.


Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Humanos , Chlorocebus aethiops , Animais , SARS-CoV-2/genética , Antivirais/farmacologia , Técnicas de Inativação de Genes , Células Vero , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular
8.
Nat Med ; 27(12): 2165-2175, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34887573

RESUMO

Intracranial aneurysm (IA) rupture leads to subarachnoid hemorrhage, a sudden-onset disease that often causes death or severe disability. Although genome-wide association studies have identified common genetic variants that increase IA risk moderately, the contribution of variants with large effect remains poorly defined. Using whole-exome sequencing, we identified significant enrichment of rare, deleterious mutations in PPIL4, encoding peptidyl-prolyl cis-trans isomerase-like 4, in both familial and index IA cases. Ppil4 depletion in vertebrate models causes intracerebral hemorrhage, defects in cerebrovascular morphology and impaired Wnt signaling. Wild-type, but not IA-mutant, PPIL4 potentiates Wnt signaling by binding JMJD6, a known angiogenesis regulator and Wnt activator. These findings identify a novel PPIL4-dependent Wnt signaling mechanism involved in brain-specific angiogenesis and maintenance of cerebrovascular integrity and implicate PPIL4 gene mutations in the pathogenesis of IA.


Assuntos
Encéfalo/irrigação sanguínea , Ciclofilinas/genética , Aneurisma Intracraniano/genética , Neovascularização Patológica/genética , Proteínas de Ligação a RNA/genética , Ciclofilinas/fisiologia , Humanos , Mutação , Proteínas de Ligação a RNA/fisiologia , Sequenciamento do Exoma , Via de Sinalização Wnt/fisiologia
9.
Science ; 374(6575): 1586-1593, 2021 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-34726479

RESUMO

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.


Assuntos
Tratamento Farmacológico da COVID-19 , Lactamas/farmacologia , Lactamas/uso terapêutico , Leucina/farmacologia , Leucina/uso terapêutico , Nitrilas/farmacologia , Nitrilas/uso terapêutico , Prolina/farmacologia , Prolina/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , Inibidores de Protease Viral/farmacologia , Inibidores de Protease Viral/uso terapêutico , Administração Oral , Animais , COVID-19/virologia , Ensaios Clínicos Fase I como Assunto , Coronavirus/efeitos dos fármacos , Modelos Animais de Doenças , Quimioterapia Combinada , Humanos , Lactamas/administração & dosagem , Lactamas/farmacocinética , Leucina/administração & dosagem , Leucina/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Nitrilas/administração & dosagem , Nitrilas/farmacocinética , Prolina/administração & dosagem , Prolina/farmacocinética , Ensaios Clínicos Controlados Aleatórios como Assunto , Ritonavir/administração & dosagem , Ritonavir/uso terapêutico , SARS-CoV-2/fisiologia , Inibidores de Protease Viral/administração & dosagem , Inibidores de Protease Viral/farmacocinética , Replicação Viral/efeitos dos fármacos
10.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32581111

RESUMO

Many RNA viruses encode a proof-reading deficient, low-fidelity RNA-dependent polymerase (RdRp), which generates genetically diverse populations that can adapt to changing environments and thwart antiviral therapies. 3Dpol, the RdRp of the foot-and-mouth disease virus (FMDV), is responsible for replication of viral genomes. The 3Dpol N terminus encodes a nuclear localization signal (NLS) sequence,MRKTKLAPT, important for import of the protein to host nucleus. Previous studies showed that substitutions at residues 18 and 20 of the NLS are defective in proper incorporation of nucleotides and RNA binding. Here, we use a systematic alanine scanning mutagenesis approach to understand the role of individual residues of the NLS in nuclear localization and nucleotide incorporation activities of 3Dpol We identify two residues of 3Dpol NLS, T19 and L21, that are important for the maintenance of enzyme fidelity. The 3Dpol NLS alanine substitutions of T19 and L21 results in aberrant incorporation of nucleoside analogs, conferring a low fidelity phenotype of the enzyme. A molecular dynamics simulation of RNA- and mutagen (RTP)-bound 3Dpol revealed that the T19 residue participates in a hydrogen bond network, including D165 in motif F and R416 at the C terminus of the FMDV 3Dpol and RNA template-primer. Based on these findings and previous studies, we conclude that at least the first six residues of theMRKTKLAPT sequence motif play a vital role in the maintenance of faithful RNA synthesis activity (fidelity) of FMDV 3Dpol, suggesting that the role of the NLS motif in similar viral polymerases needs to be revisited.IMPORTANCE In this study, we employed genetic and molecular dynamics approaches to analyze the role of individual amino acids of the FMDV 3Dpol nuclear localization signal (NLS). The NLS residues were mutated to alanine using a type A full-genome cDNA clone, and the virus progeny was analyzed for defects in growth and in competition with the parental virus. We identified two mutants in 3Dpol, T19A and L21A, that exhibited high rate of mutation, were sensitive to nucleotide analogs, and displayed reduced replicative fitness compared to the parental virus. Using molecular dynamics simulation, we demonstrated that residues T19 and L21 played a role in the structural configuration of the interaction network at the 3Dpol palm subdomain. Cumulatively, our data suggest that the T19 and L21 3Dpol amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.


Assuntos
Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/fisiologia , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Genoma Viral , Simulação de Dinâmica Molecular , Mutagênese , Mutação , Sinais de Localização Nuclear/química , Nucleotídeos , Conformação Proteica , RNA Viral , Replicação Viral
11.
PLoS One ; 14(4): e0210847, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31022193

RESUMO

Foot-and-mouth disease (FMD) is a highly contagious viral disease that severely impacts global food security and is one of the greatest constraints on international trade of animal products. Extensive viral population diversity and rapid, continuous mutation of circulating FMD viruses (FMDVs) pose significant obstacles to the control and ultimate eradication of this important transboundary pathogen. The current study investigated mechanisms contributing to within-host evolution of FMDV in a natural host species (cattle). Specifically, vaccinated and non-vaccinated cattle were infected with FMDV under controlled, experimental conditions and subsequently sampled for up to 35 days to monitor viral genomic changes as related to phases of disease and experimental cohorts. Consensus-level genomic changes across the entire FMDV coding region were characterized through three previously defined stages of infection: early, transitional, and persistent. The overall conclusion was that viral evolution occurred via a combination of two mechanisms: emergence of full-genomic minority haplotypes from within the inoculum super-swarm, and concurrent continuous point mutations. Phylogenetic analysis indicated that individuals were infected with multiple distinct haplogroups that were pre-existent within the ancestral inoculum used to infect all animals. Multiple shifts of dominant viral haplotype took place during the early and transitional phases of infection, whereas few shifts occurred during persistent infection. Overall, this work suggests that the establishment of the carrier state is not associated with specific viral genomic characteristics. These insights into FMDV population dynamics have important implications for virus sampling methodology and molecular epidemiology.


Assuntos
Portador Sadio/veterinária , Evolução Molecular , Vírus da Febre Aftosa/genética , Febre Aftosa/virologia , Genoma Viral/genética , Animais , Proteínas do Capsídeo/genética , Portador Sadio/imunologia , Portador Sadio/virologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/virologia , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Vírus da Febre Aftosa/imunologia , Vírus da Febre Aftosa/isolamento & purificação , Haplótipos , Estudos Longitudinais , Mutação , Filogenia , RNA Viral/genética , Vacinas Virais/administração & dosagem
12.
J Vet Diagn Invest ; 30(5): 699-707, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29916768

RESUMO

The highly contagious foot-and-mouth disease virus (FMDV) afflicts cloven-hoofed animals, resulting in significant costs because of loss of trade and recovery from disease. We developed a sensitive, specific, and rapid competitive ELISA (cELISA) to detect serum antibodies to FMDV. The cELISA utilized a monoclonal blocking antibody specific for a highly conserved FMDV nonstructural 3B epitope, a recombinant mutant FMDV 3ABC coating protein, and optimized format variables including serum incubation for 90 min at 20-25°C. Samples from 16 animals experimentally infected with one FMDV serotype (A, O, Asia, or SAT-1) demonstrated early detection capacity beginning 7 d post-inoculation. All samples from 55 vesicular stomatitis virus antibody-positive cattle and 44 samples from cloven-hoofed animals affected by non-FMD vesicular diseases were negative in the cELISA, demonstrating 100% analytical specificity. The diagnostic sensitivity was 100% against sera from 128 cattle infected with isolates of all FMDV serotypes, emphasizing serotype-agnostic results. Diagnostic specificities of U.S. cattle ( n = 1135) and swine ( n = 207) sera were 99.4% and 100%, respectively. High repeatability and reproducibility were demonstrated with 3.1% coefficient of variation in percent inhibition data and 100% agreement using 2 kit lots and 400 negative control serum samples, with no difference between bench and biosafety cabinet operation. Negative results from vaccinated, uninfected cattle, pig, and sheep sera confirmed the DIVA (differentiate infected from vaccinated animals) capability. This rapid (<3 h), select agent-free assay with high sensitivity and specificity, DIVA capability, and room temperature processing capability will serve as a useful tool in FMDV surveillance, emergency preparedness, response, and outbreak recovery programs.


Assuntos
Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática/veterinária , Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/diagnóstico , Proteínas não Estruturais Virais/isolamento & purificação , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/virologia , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/imunologia , Febre Aftosa/virologia , Ovinos , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/virologia , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia
13.
Front Microbiol ; 9: 485, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29616004

RESUMO

Viral genomes have evolved to maximize their potential of overcoming host defense mechanisms and to induce a variety of disease syndromes. Structurally, a genome of a virus consists of coding and noncoding regions, and both have been shown to contribute to initiation and progression of disease. Accumulated work in picornaviruses has stressed out the importance of the noncoding RNAs, or untranslated 5'- and 3'-regions (UTRs), in both replication and translation of viral genomes. Unsurprisingly, defects in these processes have been reported to cause viral attenuation and affect viral pathogenicity. However, substantial evidence suggests that these untranslated RNAs may influence the outcome of the host innate immune response. This review discusses the involvement of 5'- and 3'-terminus UTRs in induction and regulation of host immunity and its consequences for viral life cycle and virulence.

14.
Virology ; 512: 132-143, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28961454

RESUMO

The S fragment of the FMDV 5' UTR is predicted to fold into a long stem-loop structure and it has been implicated in virus-host protein interactions. In this study, we report the minimal S fragment sequence required for virus viability and show a direct correlation between the extent of the S fragment deletion mutations and attenuated phenotypes. Furthermore, we provide novel insight into the role of the S fragment in modulating the host innate immune response. Importantly, in an FMDV mouse model system, all animals survive the inoculation with the live A24 FMDV-S4 mutant, containing a 164 nucleotide deletion in the upper S fragment loop, at a dose 1000 higher than the one causing lethality by parental A24 FMDV, indicating that the A24 FMDV-S4 virus is highly attenuated in vivo. Additionally, mice exposed to high doses of live A24 FMDV-S4 virus are fully protected when challenged with parental A24 FMDV virus.


Assuntos
Regiões 5' não Traduzidas/genética , Vírus da Febre Aftosa/fisiologia , Imunidade Inata/fisiologia , Replicação Viral/fisiologia , Animais , Bovinos , Linhagem Celular , Cricetinae , Vírus da Febre Aftosa/genética , RNA Viral/genética , Deleção de Sequência , Replicação Viral/genética
15.
J Virol ; 91(15)2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28515297

RESUMO

Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3Dpol) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3Dpol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237FHF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237ILF) and W237LLF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3Dpol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3Dpol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237FHF substitution or W237ILF and W237LLF mutations were highly attenuated in animals. Our study shows that obtaining 3Dpol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.


Assuntos
Antígenos Virais/genética , Antígenos Virais/metabolismo , Vírus da Febre Aftosa/enzimologia , Vírus da Febre Aftosa/patogenicidade , Engenharia de Proteínas , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Substituição de Aminoácidos , Animais , Antivirais , Células Cultivadas , Cricetinae , Análise Mutacional de DNA , Modelos Animais de Doenças , Farmacorresistência Viral , Fluoruracila/farmacologia , Febre Aftosa/patologia , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/crescimento & desenvolvimento , Camundongos , Mutagênese Sítio-Dirigida , Mutação Puntual , Ribavirina/farmacologia , Suínos , Triptofano/genética , Triptofano/metabolismo , Ensaio de Placa Viral
16.
Virus Res ; 223: 181-9, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27497620

RESUMO

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The viral genome encodes for more than 150 genes, and only a select few of these genes have been studied in some detail. Here we report the characterization of open reading frame Ep152R that has a predicted complement control module/SCR domain. This domain is found in Vaccinia virus proteins that are involved in blocking the immune response during viral infection. A recombinant ASFV harboring a HA tagged version of the Ep152R protein was developed (ASFV-G-Ep152R-HA) and used to demonstrate that Ep152R is an early virus protein. Attempts to construct recombinant viruses having a deleted Ep152R gene were consistently unsuccessful indicating that Ep152R is an essential gene. Interestingly, analysis of host-protein interactions for Ep152R using a yeast two-hybrid screen, identified BAG6, a protein previously identified as being required for ASFV replication. Furthermore, fluorescent microscopy analysis confirms that Ep152R-BAG6 interaction actually occurs in cells infected with ASFV.


Assuntos
Vírus da Febre Suína Africana/fisiologia , Febre Suína Africana/metabolismo , Febre Suína Africana/virologia , Genes Essenciais , Chaperonas Moleculares/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Sequência Conservada , Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Macrófagos/virologia , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Fases de Leitura Aberta , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas/métodos , Transporte Proteico , Deleção de Sequência , Suínos , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/química , Replicação Viral
17.
Virology ; 495: 136-47, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27209448

RESUMO

Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co(2+) affinity columns. Electron microscopy and biochemical assays showed that the 6xHis FMDVs readily assembled into antigen: adjuvant complexes in solution, by conjugating with Ni(2+)-chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). Animals Immunized with the inactivated 6xHis-FMDV:MPLA:NiNLP vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone. Induction of anti-6xHis and anti-FMDV neutralizing antibodies in the immunized animals could be exploited in the differentiation of vaccinated from infected animals needed for the improvement of FMD control measures. The novel marker vaccine/nanolipid technology described here has broad applications for the development of distinctive and effective immune responses to other pathogens of importance.


Assuntos
Adjuvantes Imunológicos , Vírus da Febre Aftosa/imunologia , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Íons , Metais , Nanopartículas , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Vírus da Febre Aftosa/genética , Ordem dos Genes , Genoma Viral , Lipoproteínas/imunologia , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Virais/genética
18.
Virology ; 492: 108-17, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26914509

RESUMO

A companion study reported Jumonji-C domain containing protein 6 (JMJD6) is involved in an integrin- and HS-independent pathway of FMDV infection in CHO cells. JMJD6 localization was investigated in animal tissues from cattle infected with either wild type A24-FMDV (A24-WT) or mutant FMDV (JMJD6-FMDV) carrying E95K/S96L and RGD to KGE mutations in VP1. Additionally, pathogenesis of mutant JMJD6-FMDV was investigated in cattle through aerosol and intraepithelial lingual (IEL) inoculation. Interestingly, JMJD6-FMDV pathogenesis was equivalent to A24-WT administered by IEL route. In contrast, JMJD6-FMDV aerosol-infected cattle did not manifest signs of FMD and animals showed no detectable viremia. Immunofluorescent microscopy of post-mortem tissue revealed JMJD6-FMDV exclusively co-localized with JMJD6(+) cells while A24-WT was occasionally found in JMJD6(+) cells. In vitro, chemical uptake inhibitors demonstrated JMJD6-FMDV entered cells via clathrin-coated pit endocytosis. In vivo, JMJD6-FMDV exhibited preference for JMJD6(+) cells, but availability of this alternative receptor likely depends on route of inoculation.


Assuntos
Doenças dos Bovinos/prevenção & controle , Vírus da Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Histona Desmetilases com o Domínio Jumonji/imunologia , Vacinação , Administração por Inalação , Animais , Células CHO , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , Linhagem Celular , Vesículas Revestidas por Clatrina/metabolismo , Cricetulus , Endocitose , Células Epiteliais/virologia , Febre Aftosa/imunologia , Febre Aftosa/patologia , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/metabolismo , Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Integrinas/genética , Integrinas/metabolismo , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Mutagênese , Mutação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Língua
19.
Virology ; 492: 38-52, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26896934

RESUMO

Foot-and-mouth disease virus (FMDV) utilizes four integrins (αvß1, αvß3, αvß6, and αvß8) as its primary cell receptor. During cell culture propagation, FMDV frequently adapts to use heparan sulfate (HS), and rarely utilizes an unidentified third receptor. Capsid mutations acquired by a soluble integrin resistant FMDV cause (i) adaptation to CHO-677 cells (ii) increased affinity to membrane-bound Jumonji C-domain containing protein 6 (JMJD6) (iii) induced JMJD6 re-localization from the cell surface and cytoplasm to the nucleus. Interestingly, pre-treatment of cells with N- and C-terminal JMJD6 antibodies or by simultaneous incubation of mutant virus with soluble JMJD6 (but not by treatment with HS or αvß6) impaired virus infectivity in cultured cells. JMJD6 and mutant virus co-purified by reciprocal co-immunoprecipitation. Molecular docking predictions suggested JMJD6 C-terminus interacts with mutated VP1 capsid protein. We conclude when specific VP1 mutations are displayed, JMJD6 contributes to FMDV infectivity and may be a previously unidentified FMDV receptor.


Assuntos
Proteínas do Capsídeo/química , Vírus da Febre Aftosa/genética , Integrinas/química , Histona Desmetilases com o Domínio Jumonji/química , Receptores Virais/química , Sequência de Aminoácidos , Animais , Células CHO , Células COS , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Bovinos , Chlorocebus aethiops , Cricetulus , Cristalografia por Raios X , Células Epiteliais/virologia , Vírus da Febre Aftosa/metabolismo , Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Integrinas/genética , Integrinas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Rim/virologia , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
20.
Virol J ; 12: 224, 2015 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-26695943

RESUMO

BACKGROUND: The nuclear protein Src-associated protein of 68 kDa in mitosis (Sam68) is known to bind RNA and be involved in cellular processes triggered in response to environmental stresses, including virus infection. Interestingly, Sam68 is a multi-functional protein implicated in the life cycle of retroviruses and picornaviruses and is also considered a marker of virus-induced stress granules (SGs). Recently, we demonstrated the partial redistribution of Sam68 to the cytoplasm in FMDV infected cells, its interaction with viral protease 3C(pro), and found a significant reduction in viral titers as consequence of Sam68-specific siRNA knockdowns. Despite of that, details of how it benefits FMDV remains to be elucidated. METHODS: Sam68 cytoplasmic localization was examined by immunofluorescent microscopy, counterstaining with antibodies against Sam68, a viral capsid protein and markers of SGs. The relevance of RAAA motifs in the IRES was investigated using electromobility shift assays with Sam68 protein and parental and mutant FMDV RNAs. In addition, full genome WT and mutant or G-luc replicon RNAs were tested following transfection in mammalian cells. The impact of Sam68 depletion to virus protein and RNA synthesis was investigated in a cell-free system. Lastly, through co-immunoprecipitation, structural modeling, and subcellular fractionation, viral protein interactions with Sam68 were explored. RESULTS: FMDV-induced cytoplasmic redistribution of Sam68 resulted in it temporarily co-localizing with SG marker: TIA-1. Mutations that disrupted FMDV IRES RAAA motifs, with putative affinity to Sam68 in domain 3 and 4 cause a reduction on the formation of ribonucleoprotein complexes with this protein and resulted in non-viable progeny viruses and replication-impaired replicons. Furthermore, depletion of Sam68 in cell-free extracts greatly diminished FMDV RNA replication, which was restored by addition of recombinant Sam68. The results here demonstrated that Sam68 specifically co-precipitates with both FMDV 3D(pol) and 3C(pro) consistent with early observations of FMDV 3C(pro)-induced cleavage of Sam68. CONCLUSION: We have found that Sam68 is a specific binding partner for FMDV non-structural proteins 3C(pro) and 3D(pol) and showed that mutations at RAAA motifs in IRES domains 3 and 4 cause a decrease in Sam68 affinity to these RNA elements and rendered the mutant RNA non-viable. Interestingly, in FMDV infected cells re-localized Sam68 was transiently detected along with SG markers in the cytoplasm. These results support the importance of Sam68 as a host factor co-opted by FMDV during infection and demonstrate that Sam68 interact with both, FMDV RNA motifs in the IRES and viral non-structural proteins 3C(pro) and 3D(pol).


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos Virais/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Vírus da Febre Aftosa/fisiologia , Febre Aftosa/patologia , Febre Aftosa/virologia , Interações Hospedeiro-Patógeno , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Proteases Virais 3C , Animais , Antígenos Virais/química , Linhagem Celular , Cisteína Endopeptidases/química , Citoplasma/química , Análise Mutacional de DNA , Imunoprecipitação , Sítios Internos de Entrada Ribossomal , Microscopia de Fluorescência , Modelos Moleculares , Ligação Proteica , Conformação Proteica , RNA Viral/genética , RNA Viral/metabolismo , Proteínas não Estruturais Virais/química , Proteínas Virais/química
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