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1.
Methods Mol Biol ; 2203: 205-221, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833214

RESUMO

We have developed a screening system using the yeast Saccharomyces cerevisiae to identify eukaryotic genes involved in the replication of mammalian viruses. Yeast come with various advantages, but in the context of coronavirus research and the system outlined here, they are simple and easy to work with and can be used at biosafety level 2. The system involves inducible expression of individual viral proteins and identification of detrimental phenotypes in the yeast. Yeast knockout and overexpression libraries can then be used for genome-wide screening of host proteins that provide a suppressor phenotype. From the yeast hits, a narrowed list of candidate genes can be produced to investigate for roles in viral replication. Since the system only requires expression of viral proteins, it can be used for any current or emerging virus, regardless of biocontainment requirements and ability to culture the virus. In this chapter, we will outline the protocols that can be used to take advantage of S. cerevisiae as a tool to advance understanding of how viruses interact with eukaryotic cells.


Assuntos
Coronavirus/fisiologia , Coronavirus/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Saccharomyces cerevisiae/genética , Plasmídeos , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Replicação Viral
2.
J Vet Sci ; 21(2): e24, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32233132

RESUMO

The pandemic of avian influenza viruses (AIVs) in Asia has caused enormous economic loss in poultry industry and human health threat, especially clade 2.3.4.4 H5 and H7 subtypes in recent years. The endemic chicken H6 virus in Taiwan has also brought about human and dog infections. Since wild waterfowls is the major AIV reservoir, it is important to monitor the diversified subtypes in wildfowl flocks in early stage to prevent viral reassortment and transmission. To develop a more efficient and sensitive approach is a key issue in epidemic control. In this study, we integrate multiplex reverse transcription recombinase polymerase amplification (RT-RPA) and capillary electrophoresis (CE) for high-throughput detection and differentiation of AIVs in wild waterfowls in Taiwan. Four viral genes were detected simultaneously, including nucleoprotein (NP) gene of all AIVs, hemagglutinin (HA) gene of clade 2.3.4.4 H5, H6 and H7 subtypes. The detection limit of the developed detection system could achieve as low as one copy number for each of the four viral gene targets. Sixty wild waterfowl field samples were tested and all of the four gene signals were unambiguously identified within 6 h, including the initial sample processing and the final CE data analysis. The results indicated that multiplex RT-RPA combined with CE was an excellent alternative for instant simultaneous AIV detection and subtype differentiation. The high efficiency and sensitivity of the proposed method could greatly assist in wild bird monitoring and epidemic control of poultry.


Assuntos
Eletroforese Capilar/veterinária , Vírus da Influenza A/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Proteínas Virais/isolamento & purificação , Animais , Anseriformes , Eletroforese Capilar/métodos , Genes Virais , Ensaios de Triagem em Larga Escala/veterinária , Influenza Aviária/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Taiwan
3.
J Virol ; 94(10)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132237

RESUMO

For cell entry, vaccinia virus requires fusion with the host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that the viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, we performed experimental evolutionary analyses by repeatedly passaging vaccinia virus in HeLa cells overexpressing the A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that they had accumulated a unique G9R open reading frame (ORF) mutation, resulting in a single His44Tyr amino acid change. We engineered a recombinant vaccinia virus to express the G9H44Y mutant protein, and it readily infected HeLa-A56/K2 cells. Moreover, similar to the ΔA56 virus, the G9H44Y mutant virus on HeLa cells had a cell fusion phenotype, indicating that G9H44Y-mediated membrane fusion was less prone to inhibition by A56/K2. Coimmunoprecipitation experiments demonstrated that the G9H44Y protein bound to A56/K2 at neutral pH, suggesting that the H44Y mutation did not eliminate the binding of G9 to A56/K2. Interestingly, upon acid treatment to inactivate A56/K2-mediated fusion inhibition, the G9H44Y mutant virus induced robust cell-cell fusion at pH 6, unlike the pH 4.7 required for control and revertant vaccinia viruses. Thus, A56/K2 fusion suppression mainly targets the G9 protein. Moreover, the G9H44Y mutant protein escapes A56/K2-mediated membrane fusion inhibition most likely because it mimics an acid-induced intermediate conformation more prone to membrane fusion.IMPORTANCE It remains unclear how the multiprotein entry fusion complex of vaccinia virus mediates membrane fusion. Moreover, vaccinia virus contains fusion suppressor proteins to prevent the aberrant activation of this multiprotein complex. Here, we used experimental evolution to identify adaptive mutant viruses that overcome membrane fusion inhibition mediated by the A56/K2 protein complex. We show that the H44Y mutation of the G9 protein is sufficient to overcome A56/K2-mediated membrane fusion inhibition. Treatment of virus-infected cells at different pHs indicated that the H44Y mutation lowers the threshold of fusion inhibition by A56/K2. Our study provides evidence that A56/K2 inhibits the viral fusion complex via the latter's G9 subcomponent. Although the G9H44Y mutant protein still binds to A56/K2 at neutral pH, it is less dependent on low pH for fusion activation, implying that it may adopt a subtle conformational change that mimics a structural intermediate induced by low pH.


Assuntos
Fusão de Membrana , Mutação , Vírus Vaccinia/genética , Vírus Vaccinia/isolamento & purificação , Proteínas Virais/genética , Fusão Celular , Membrana Celular , Evolução Molecular , Regulação Viral da Expressão Gênica , Genes Virais/genética , Genoma Viral , Células HeLa , Humanos , Proteínas Recombinantes , Vírus Vaccinia/crescimento & desenvolvimento , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/isolamento & purificação , Proteínas Virais/isolamento & purificação , Internalização do Vírus
4.
Biochim Biophys Acta Proteins Proteom ; 1868(6): 140406, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32135196

RESUMO

Phage virion protein (PVP) identification plays key role in elucidating relationships between phages and hosts. Moreover, PVP identification can facilitate the design of related biochemical entities. Recently, several machine learning approaches have emerged for this purpose and have shown their potential capacities. In this study, the proposed PVP identifiers are systemically reviewed, and the related algorithms and tools are comprehensively analyzed. We summarized the common framework of these PVP identifiers and constructed our own novel identifiers based upon the framework. Furthermore, we focus on a performance comparison of all PVP identifiers by using a training dataset and an independent dataset. Highlighting the pros and cons of these identifiers demonstrates that g-gap DPC (dipeptide composition) features are capable of representing characteristics of PVPs. Moreover, SVM (support vector machine) is proven to be the more effective classifier to distinguish PVPs and non-PVPs.


Assuntos
Bacteriófagos/metabolismo , Biologia Computacional/métodos , Aprendizado de Máquina , Proteínas Virais/isolamento & purificação , Vírion/metabolismo , Algoritmos , Máquina de Vetores de Suporte
5.
Biosens Bioelectron ; 154: 112068, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32056963

RESUMO

Here we report the development of a high throughput, all-solution phase, and isothermal detection system for African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with a fluorescence-based point-of-care (POC) system for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 h. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 h of incubation, resulting in an improved detection limit of 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact and simple ASFV detection, intended for low resource settings.


Assuntos
Vírus da Febre Suína Africana/isolamento & purificação , Febre Suína Africana/diagnóstico , Técnicas Biossensoriais , Proteínas Virais/isolamento & purificação , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/patogenicidade , Animais , Sistemas CRISPR-Cas/genética , DNA de Cadeia Simples/química , Fluorescência , Sistemas Automatizados de Assistência Junto ao Leito , Suínos , Proteínas Virais/química
6.
Sensors (Basel) ; 20(3)2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32028629

RESUMO

Since the norovirus is the main cause of acute gastroenteritis all over the world, its fast detection is crucial in medical diagnostics. In this work, a rapid, sensitive, and selective optical fiber biosensor for the detection of norovirus virus-like particles (VLPs) is reported. The sensor is based on highly sensitive long-period fiber gratings (LPFGs) coated with antibodies against the main coat protein of the norovirus. Several modification methods were verified to obtain reliable immobilization of protein receptors on the LPFG surface. We were able to detect 1 ng/mL norovirus VLPs in a 40-min assay in a label-free manner. Thanks to the application of an optical fiber as the sensor, there is a possibility to increase the user's safety by separating the measurement point from the signal processing setup. Moreover, our sensor is small and light, and the proposed assay is straightforward. The designed LPFG-based biosensor could be applied in both fast norovirus detection and in vaccine testing.


Assuntos
Anticorpos/isolamento & purificação , Técnicas Biossensoriais , Gastroenterite/genética , Norovirus/isolamento & purificação , Gastroenterite/diagnóstico , Gastroenterite/imunologia , Gastroenterite/virologia , Humanos , Norovirus/patogenicidade , Proteínas Virais/imunologia , Proteínas Virais/isolamento & purificação
7.
J Virol ; 94(8)2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32024777

RESUMO

Malignant catarrhal fever (MCF) is a rare but frequently lethal disease of certain cloven-hoofed animals. At least 10 different viruses, all members of the Macavirus genus in the subfamily Gammaherpesvirinae, are known as causative agents of MCF. Among these, ovine herpesvirus 2 (OvHV-2) is the most frequent and economically most important MCF agent. Phenotypically, MCF is characterized by severe lymphocytic arteritis-periarteritis, which leads to the accumulation of activated lymphocytes accompanied by apoptosis and necrosis in a broad range of tissues. However, a viral factor that might be responsible for tissue damage has not yet been identified. We have studied a seemingly intergenic locus on the OvHV-2 genome, which was previously shown to be transcriptionally highly active in MCF-affected tissue. We identified by 5' and 3' rapid amplification of cDNA ends (RACE) a conserved, double-spliced transcript that encoded a 9.9-kDa hydrophobic protein. The newly detected gene, Ov8.25, and its splicing pattern were conserved among OvHV-2 strains of different origins. Upon transient expression of synthetic variants of this gene in various cell types, including bovine lymphocytes, the protein (pOv8.25) was shown to target mitochondria, followed by caspase-dependent apoptosis and necrosis. Notably, a deletion mutant of the same protein lost these abilities. Finally, we detected pOv8.25 in brain-infiltrating lymphocytes of cattle with MCF. Thus, the cell death-causing properties of pOv8.25 in affected cells may be involved in the emergence of typical MCF-associated apoptosis and necrosis. Thus, we have identified a novel OvHV-2 protein, which might contribute to the phenotype of MCF-related lesions.IMPORTANCE Ovine herpesvirus 2 (OvHV-2) circulates among sheep without causing disease. However, upon transmission to cattle, the same virus instigates a frequently lethal disease, malignant catarrhal fever (MCF). While the cause of death and pathogenesis of tissue lesions are still poorly understood, MCF is characterized by the accumulation of lymphocytes in various tissues, associated with vasculitis and cell death. As infectious virus is hardly present in these lesions, the cause of cell death cannot be explained simply by viral replication. The significance of our research is in identifying and characterizing a previously overlooked gene of OvHV-2 (Ov8.25), which is highly expressed in animals with MCF. Its encoded protein targets mitochondria, causing apoptosis and necrosis, thus contributing to an understanding of the source and nature of cell death. As the corresponding genetic locus is also active in the context of MCF due to a different macavirus, we may have detected a common denominator of the disease phenotype.


Assuntos
Apoptose , Gammaherpesvirinae/genética , Gammaherpesvirinae/metabolismo , Mitocôndrias/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Doenças do Gato/virologia , Gatos , Bovinos , Linhagem Celular , Chlorocebus aethiops , Linfócitos , Febre Catarral Maligna/patologia , Febre Catarral Maligna/virologia , Mitocôndrias/patologia , Necrose/virologia , Alinhamento de Sequência , Ovinos , Doenças dos Ovinos/virologia , Células Vero , Proteínas Virais/isolamento & purificação
8.
Protein Pept Lett ; 27(4): 259-264, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30968770

RESUMO

Phage Virion Proteins (PVP) are essential materials of bacteriophage, which participate in a series of biological processes. Accurate identification of phage virion proteins is helpful to understand the mechanism of interaction between the phage and its host bacteria. Since experimental method is labor intensive and time-consuming, in the past few years, many computational approaches have been proposed to identify phage virion proteins. In order to facilitate researchers to select appropriate methods, it is necessary to give a comprehensive review and comparison on existing computational methods on identifying phage virion proteins. In this review, we summarized the existing computational methods for identifying phage virion proteins and also assessed their performances on an independent dataset. Finally, challenges and future perspectives for identifying phage virion proteins were presented. Taken together, we hope that this review could provide clues to researches on the study of phage virion proteins.


Assuntos
Bacteriófagos/genética , Proteínas Virais/genética , Vírion/genética , Biologia Computacional , Proteínas Virais/isolamento & purificação
9.
J Immunol Methods ; 478: 112712, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31783022

RESUMO

Upper respiratory tract infections are the world's most common infectious disease. The etiologic agents behind upper respiratory tract infections (URTIs) are, in fact, a diverse set of pathogens such as influenza, parainfluenza, adenovirus, rhinovirus, and others. More than 200 pathogens are known to be involved. Differential diagnosis of viral infections is sometimes complicated by their diversity or similarity of clinical presentation. This work is devoted to the development of a method which enables simultaneous detection of six common viral URTI pathogens: IAV; IBV; RSV; hAdV; hPIV2; and hPIV3. Antibody microarray technology is utilized to accomplish the analysis. In preparation for protein microchip creation, we produced, characterized, and selected approximately 50 monoclonal antibodies; for each of the aforementioned pathogens, an optimal monoclonal antibody pair was selected. A protein microchip was created, and its core working conditions were optimized. With a balance between convenience and maximal assay sensitivity in mind, a one-step analysis approach was developed for accomplishing the ELISA-like "sandwich" interaction on the manufactured microchip (antibody microarray). Reference viral strains were used to establish the lower limits of detection (LoD) for the assay. For IAV, the LoD was 0.25 ng/ml total viral protein. For other viruses, the LoD ranged from 1 to 2 ng/ml total protein. These sensitivity limits are slightly better than those of standard ELISA, but inferior to those of PCR. Overall, we believe that the developed microchip is a good alternative to existing methods, allowing relatively quick (overnight), inexpensive, simultaneous screening of several pathogens. The design of the antibody microarray is conducive to further development, and the panel of analyzed pathogens can be expanded to include approximately 50 members.


Assuntos
Antígenos Virais/isolamento & purificação , Análise Serial de Proteínas/métodos , Infecções Respiratórias/diagnóstico , Proteínas Virais/isolamento & purificação , Viroses/diagnóstico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/imunologia , Linhagem Celular , DNA Viral/isolamento & purificação , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Hibridomas , Limite de Detecção , Camundongos , Reação em Cadeia da Polimerase , RNA Viral/isolamento & purificação , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia , Proteínas Virais/imunologia , Viroses/imunologia , Viroses/virologia
10.
Int J Pharm ; 573: 118850, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31759993

RESUMO

Cpl-1, an endolysin derived from Cp-1 phage has been found to be effective in a number of in-vitro and in-vivo pneumococcal infection models. However its lower bioavailability under in-vivo conditions limits its applicability as therapeutic agent. In this study, Cpl-1 loaded chitosan nanoparticles were set up in order to develop a novel therapeutic delivery system to counter antibiotic resistant S. pneumoniae infections. Interactions of chitosan and Cpl-1 were studied by in-silico docking analysis. Chitosan nanoparticles and Cpl-1 loaded chitosan nanoparticles were prepared by using ionic gelation method and the process was optimized by varying chitosan:TPP ratio, pH, stirring time, stirring rate and Cpl-1 concentration. Chitosan nanoparticles and Cpl-1 loaded chitosan nanoparticles were characterized to ascertain successful formation of nanoparticles and entrapment of Cpl-1 into nanoparticles. Chitosan nanoparticles and Cpl-1 loaded nanoparticles were also evaluated for nanoparticle yield, entrapment efficiency, in-vitro release, stability, structural integrity of Cpl-1, in-vitro bioassay, swelling studies, in-vitro biodegradation and heamolysis studies. Mucoadhesion behavior of chitosan nanoparticles and Cpl-1 loaded nanoparticles was explored using mucous glycoprotein assay and ex-vivo mucoadhesion assay, both preparations exhibited their mucoadhesive nature. Cellular cytotoxicity and immune stimulation studies revealed biocompatible nature of nanoparticles. The results of this study confirm that chitosan nanoparticles are a promising biocompatible candidate for Cpl-1 delivery with a significant potential to increase bioavailability of enzyme that in turn can increase its in-vivo half life to treat S. pneumoniae infections.


Assuntos
Portadores de Fármacos/química , Composição de Medicamentos/métodos , Endopeptidases/administração & dosagem , Nanopartículas/química , Pneumonia Pneumocócica/tratamento farmacológico , Proteínas Virais/administração & dosagem , Células A549 , Administração Intranasal , Animais , Bacteriófagos/enzimologia , Disponibilidade Biológica , Quitosana/química , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/farmacocinética , Avaliação Pré-Clínica de Medicamentos , Liberação Controlada de Fármacos , Endopeptidases/química , Endopeptidases/isolamento & purificação , Endopeptidases/farmacocinética , Estudos de Viabilidade , Meia-Vida , Humanos , Masculino , Teste de Materiais , Camundongos , Simulação de Acoplamento Molecular , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/microbiologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacocinética , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pneumoniae/virologia , Proteínas Virais/química , Proteínas Virais/isolamento & purificação , Proteínas Virais/farmacocinética
11.
Int J Biol Macromol ; 147: 980-989, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31715241

RESUMO

Bacteriophages (phages), or bacterial viruses, have recently received increasing attention, especially considering pan-drug-resistant bacteria, and studies on lytic bacteriophage proteins would help develop antibiotic candidates to treat these bacterial infections. We previously isolated and sequenced a Streptomyces avermitilis bacteriophage, phiSASD1. This study aimed to clone and express ORF40 and ORF19, previously predicted as endolysin (termed LytSD) and holin (termed HolSD), two crucial phage proteins involved in host lysis. The yield of LytSD was 17.2 mg per liter of culture, and the optimal lysis conditions were investigated. When applied exogenously, LytSD lysed 7/18 of the tested bacterial strains, including S. avermitilis, Bacillus subtilis, Staphylococcus aureus, Sarcina lutea, and Enterococcus faecalis. As regards HolSD, it resulted in growth inhibition of several tested strains and abrupt lysis of E. coli BL21 (DE3) pLysS; furthermore, it complemented the defective λ S allele of non-suppressing E. coli strains to produce phage plaques. Together, these results indicate the function of ORF40 and ORF19 of phage phiSASD1 and their potentials as novel antibiotics to inhibit or lyse pathogens.


Assuntos
Antibacterianos , Clonagem Molecular , Endopeptidases , Siphoviridae , Streptomyces/virologia , Proteínas Virais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Endopeptidases/farmacologia , Fases de Leitura Aberta , Siphoviridae/enzimologia , Siphoviridae/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/farmacologia
12.
J Enzyme Inhib Med Chem ; 35(1): 145-151, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31724441

RESUMO

There were severe panics caused by Severe Acute Respiratory Syndrome (SARS) and Middle-East Respiratory Syndrome-Coronavirus. Therefore, researches targeting these viruses have been required. Coronaviruses (CoVs) have been rising targets of some flavonoids. The antiviral activity of some flavonoids against CoVs is presumed directly caused by inhibiting 3C-like protease (3CLpro). Here, we applied a flavonoid library to systematically probe inhibitory compounds against SARS-CoV 3CLpro. Herbacetin, rhoifolin and pectolinarin were found to efficiently block the enzymatic activity of SARS-CoV 3CLpro. The interaction of the three flavonoids was confirmed using a tryptophan-based fluorescence method, too. An induced-fit docking analysis indicated that S1, S2 and S3' sites are involved in binding with flavonoids. The comparison with previous studies showed that Triton X-100 played a critical role in objecting false positive or overestimated inhibitory activity of flavonoids. With the systematic analysis, the three flavonoids are suggested to be templates to design functionally improved inhibitors.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Vírus da SARS/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Flavonoides/síntese química , Flavonoides/química , Humanos , Estrutura Molecular , Vírus da SARS/enzimologia , Relação Estrutura-Atividade , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
13.
Proc Natl Acad Sci U S A ; 116(49): 24738-24747, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31740606

RESUMO

Here, we report on the discovery in Caenorhabditis nematodes of multiple vertically transmitted RNAs coding for putative RNA-dependent RNA polymerases. Their sequences share similarity to distinct RNA viruses, including bunyaviruses, narnaviruses, and sobemoviruses. The sequences are present exclusively as RNA and are not found in DNA form. The RNAs persist in progeny after bleach treatment of adult animals, indicating vertical transmission of the RNAs. We tested one of the infected strains for transmission to an uninfected strain and found that mating of infected animals with uninfected animals resulted in infected progeny. By in situ hybridization, we detected several of these RNAs in the cytoplasm of the male and female germline of the nematode host. The Caenorhabditis hosts were found defective in degrading exogenous double-stranded RNAs, which may explain retention of viral-like RNAs. Strikingly, one strain, QG551, harbored three distinct virus-like RNA elements. Specific patterns of small RNAs complementary to the different viral-like RNAs were observed, suggesting that the different RNAs are differentially recognized by the RNA interference (RNAi) machinery. While vertical transmission of viruses in the family Narnaviridae, which are known as capsidless viruses, has been described in fungi, these observations provide evidence that multicellular animal cells harbor similar viruses.


Assuntos
Caenorhabditis/virologia , Transmissão Vertical de Doença Infecciosa/veterinária , Vírus de RNA/patogenicidade , RNA Viral/genética , Proteínas Virais/genética , Animais , Caenorhabditis/genética , Feminino , Masculino , Estabilidade de RNA , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/isolamento & purificação , Proteínas Virais/isolamento & purificação , Replicação Viral/genética
14.
Curr Microbiol ; 76(12): 1477-1486, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31612259

RESUMO

Expression and secretion of recombinant proteins in the endotoxin-free bacterium, Bacillus subtilis, has been thoroughly studied, but overexpression in the cytoplasm has been limited to only a few proteins. Here, we used the robust IPTG-inducible promoter, Pgrac212, to overexpress human rhinovirus 3C protease (HRV3C) in the cytoplasm of B. subtilis cells. A novel solubility tag, the N-terminal domain of the lysS gene of B. subtilis coding for a lysyl-tRNA synthetase was placed at the N terminus with a cleavage site for the endoprotease HRV3C, followed by His-HRV3C or His-GST-HRV3C. The recombinant protease was purified by using a Ni-NTA column. In this study, the His-HRV3C and His-GST-HRV3C proteases were overexpressed in the cytoplasm of B. subtilis at 11% and 16% of the total cellular proteins, respectively. The specific protease activities were 8065 U/mg for His-HRV3C and 3623 U/mg for His-GST-HRV3C. The purified enzymes were used to cleave two different substrates followed by purification of the two different protein targets, the green fluorescent protein and the beta-galactosidase. In conclusion, the combination of an inducible promoter Pgrac212 and a solubility tag allowed the overexpression of the HRV3C protease in the cytoplasm of B. subtilis. The resulting fusion protein was purified using a nickel column and was active in cleaving target proteins to remove the fusion tags. This study offers an effective method for producing recombinant proteins in the cytoplasm of endotoxin-free bacteria.


Assuntos
Bacillus subtilis/genética , Cisteína Endopeptidases/genética , Citoplasma/metabolismo , Microbiologia Industrial/métodos , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Rhinovirus/enzimologia , Proteínas Virais/genética , Bacillus subtilis/metabolismo , Clonagem Molecular , Cisteína Endopeptidases/isolamento & purificação , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Isopropiltiogalactosídeo/farmacologia , Lisina-tRNA Ligase/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Rhinovirus/genética , Solubilidade , Proteínas Virais/isolamento & purificação , beta-Galactosidase/genética
15.
Viral Immunol ; 32(9): 370-382, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31644382

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) still poses a threat to the swine industry worldwide. Currently, commercial vaccines against PRRSV, which consist of modified live or inactivated virus, reduce symptoms and viremia in immunized pigs, but efficacy against heterologous strains is variable. This has led to the development of subunit vaccines that contain viral antigens that show the highest variability. In this work, a chimeric protein comprising short amino acid sequences from glycoprotein 3 (GP3), glycoprotein 4 (GP4), glycoprotein 5 (GP5), and M (matrix protein) proteins of PRRSV was designed and expressed in Escherichia coli. This protein, designated as PRRSVchim, was purified by immobilized metal affinity chromatography and evaluated. PRRSVchim was identified by immunoglobulin G (IgG) presence in serum samples from PRRSV-positive pigs. Also, the protein probed to be antigenic in immunized mice and piglets and provided some degree of protection against challenge with a PRRSV field isolate. These results show the potential of PRRSVchim protein for both PRRSV diagnostic and immunoprophylaxis.


Assuntos
Antígenos Virais/imunologia , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Epitopos/genética , Escherichia coli , Feminino , Glicoproteínas/administração & dosagem , Glicoproteínas/genética , Glicoproteínas/imunologia , Glicoproteínas/isolamento & purificação , Imunogenicidade da Vacina , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Células RAW 264.7 , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Suínos , Vacinação/veterinária , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificação
16.
Elife ; 82019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31502535

RESUMO

CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because Acrs are challenging to identify, their natural diversity and impact on microbial ecosystems are underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection to isolate ten DNA fragments from human oral and fecal metagenomes that inhibit Streptococcus pyogenes Cas9 (SpyCas9) in Escherichia coli. The most potent Acr from this set, AcrIIA11, was recovered from a Lachnospiraceae phage. We found that AcrIIA11 inhibits SpyCas9 in bacteria and in human cells. AcrIIA11 homologs are distributed across diverse bacteria; many distantly-related homologs inhibit both SpyCas9 and a divergent Cas9 from Treponema denticola. We find that AcrIIA11 antagonizes SpyCas9 using a different mechanism than other previously characterized Type II-A Acrs. Our study highlights the power of functional selection to uncover widespread Cas9 inhibitors within diverse microbiomes.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Microbiota , Proteínas Virais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Fezes/microbiologia , Fezes/virologia , Humanos , Metagenômica , Boca/microbiologia , Boca/virologia , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
17.
Nat Commun ; 10(1): 3048, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296855

RESUMO

Bacteriophages typically hijack the host bacterial transcriptional machinery to regulate their own gene expression and that of the host bacteria. The structural basis for bacteriophage protein-mediated transcription regulation-in particular transcription antitermination-is largely unknown. Here we report the 3.4 Å and 4.0 Å cryo-EM structures of two bacterial transcription elongation complexes (P7-NusA-TEC and P7-TEC) comprising the bacteriophage protein P7, a master host-transcription regulator encoded by bacteriophage Xp10 of the rice pathogen Xanthomonas oryzae pv. Oryzae (Xoo) and discuss the mechanisms by which P7 modulates the host bacterial RNAP. The structures together with biochemical evidence demonstrate that P7 prevents transcription termination by plugging up the RNAP RNA-exit channel and impeding RNA-hairpin formation at the intrinsic terminator. Moreover, P7 inhibits transcription initiation by restraining RNAP-clamp motions. Our study reveals the structural basis for transcription antitermination by phage proteins and provides insights into bacterial transcription regulation.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Fatores de Elongação da Transcrição/metabolismo , Proteínas Virais/metabolismo , Xanthomonas/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Interações entre Hospedeiro e Microrganismos/genética , Oryza/microbiologia , Estrutura Secundária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Regiões Terminadoras Genéticas/genética , Transcrição Genética , Fatores de Elongação da Transcrição/isolamento & purificação , Fatores de Elongação da Transcrição/ultraestrutura , Proteínas Virais/isolamento & purificação , Proteínas Virais/ultraestrutura , Xanthomonas/virologia
18.
Mol Biotechnol ; 61(8): 622-630, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31165966

RESUMO

Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg2+ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm-baculovirus expression vector system (silkworm-BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 µg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg2+, Mn2+, Ca2+, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm-BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale.


Assuntos
DNA Topoisomerases Tipo I/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Vírus Vaccinia , Proteínas Virais/isolamento & purificação , Animais , Baculoviridae/genética , Bombyx/metabolismo , Bombyx/virologia , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Magnésio/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vírus Vaccinia/enzimologia , Vírus Vaccinia/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
19.
Protein Expr Purif ; 161: 78-83, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31051245

RESUMO

Human cytomegalovirus (HCMV), a member of the human herpesvirus family, is a common opportunistic virus causing severe ailments and deaths in people with immature or compromised immune systems. UL23 is a virion protein found in the tegument and is expressed in the cytoplasm in HCMV infected cells. However, UL23 is dispensable for viral replication in cultured cells and little is currently known about its function. In order to further study of UL23, polyclonal antibody of UL23 was prepared. UL23 gene fragment was cloned from HCMV Towne by PCR and ligated into pET28a (+). The recombinant plasmid pET28a (+)-UL23 was transformed into E.coli BL21(DE3) to induce expression of the target protein. Then we efficiently purified the recombinant protein affinity chromatography under unique denaturation conditions. Recombinant UL23 protein was used as immunogen to inoculate New Zealand white rabbits and the sera was collected after the fourth immunization. UL23 Polyclonal antibody was purified from antisera using CNBr-activated Sepharose 4 beads. Our UL23 Polyclonal antibody showed specific reaction with UL23 in ELISA, Western-blot and immunofluorescence. More importantly, UL23 Polyclonal antibody could specifically recognize UL23 protein in HCMV infected cells, which laid a foundation for further study of HCMV UL23.


Assuntos
Anticorpos/análise , Citomegalovirus/metabolismo , Proteínas Virais/análise , Animais , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Imunização , Coelhos , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
20.
Nat Commun ; 10(1): 2104, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068591

RESUMO

Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques.


Assuntos
DNA/metabolismo , Corantes Fluorescentes/química , Imagem Individual de Molécula/métodos , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Endonucleases Flap/química , Endonucleases Flap/isolamento & purificação , Endonucleases Flap/metabolismo , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem , Proteínas Virais/química , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
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