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1.
Gene ; 723: 144134, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589960

RESUMO

Viral kinases are known to undergo autophosphorylation and also phosphorylate viral and host substrates. Viral kinases have been implicated in various diseases and are also known to acquire host kinases for mimicking cellular functions and exhibit virulence. Although substantial analyses have been reported in the literature on diversity of viral kinases, there is a gap in the understanding of sequence and structural similarity among kinases from different classes of viruses. In this study, we performed a comprehensive analysis of protein kinases encoded in viral genomes. Homology search methods have been used to identify kinases from 104,282 viral genomic datasets. Serine/threonine and tyrosine kinases are identified only in 390 viral genomes. Out of seven viral classes that are based on nature of genetic material, only viruses having double-stranded DNA and single-stranded RNA retroviruses are found to encode kinases. The 716 identified protein kinases are classified into 63 subfamilies based on their sequence similarity within each cluster, and sequence signatures have been identified for each subfamily. 11 clusters are well represented with at least 10 members in each of these clusters. Kinases from dsDNA viruses, Phycodnaviridae which infect green algae and Herpesvirales that infect vertebrates including human, form a major group. From our analysis, it has been observed that the protein kinases in viruses belonging to same taxonomic lineages form discrete clusters and the kinases encoded in alphaherpesvirus form host-specific clusters. A comprehensive sequence and structure-based analysis enabled us to identify the conserved residues or motifs in kinase catalytic domain regions across all viral kinases. Conserved sequence regions that are specific to a particular viral kinase cluster and the kinases that show close similarity to eukaryotic kinases were identified by using sequence and three-dimensional structural regions of eukaryotic kinases as reference. The regions specific to each viral kinase cluster can be used as signatures in the future in classifying uncharacterized viral kinases. We note that kinases from giant viruses Marseilleviridae have close similarity to viral oncogenes in the functional regions and in putative substrate binding regions indicating their possible role in cancer.


Assuntos
Proteínas Quinases/química , Proteínas Quinases/genética , Vírus/classificação , Domínio Catalítico , Biologia Computacional/métodos , Bases de Dados de Proteínas , Variação Genética , Fosforilação , Filogenia , Proteínas Quinases/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Vírus/enzimologia , Vírus/patogenicidade
2.
Chemistry ; 25(53): 12303-12307, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31373735

RESUMO

Triplex forming oligonucleotides are used as a tool for gene regulation and in DNA nanotechnology. By incorporating artificial nucleic acids, target affinity and biological stability superior to that of natural DNA may be obtained. This work demonstrates how a chimeric clamp consisting of acyclic (L)-threoninol nucleic acid (aTNA) and DNA can bind DNA and RNA by the formation of a highly stable triplex structure. The (L)-aTNA clamp is released from the target again by the addition of a releasing strand in a strand displacement type of reaction. It is shown that the clamp efficiently inhibits Bsu and T7 RNA polymerase activity and that polymerase activity is reactivated by displacing the clamp. The clamp was successfully applied to the regulation of luciferase expression by reversible binding to the mRNA. When targeting a sequence in the double stranded plasmid, 40 % downregulation of protein expression is achieved.


Assuntos
RNA Polimerases Dirigidas por DNA/química , DNA/química , Ácidos Nucleicos/química , RNA/química , Proteínas Virais/química , Amino Álcoois/química , Butileno Glicóis/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/farmacologia , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia
3.
Dokl Biochem Biophys ; 486(1): 201-205, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367821

RESUMO

Infection of mice with influenza A viruses led to the formation of clones of lymphocytes that specifically recognizes viral domains in the central zone of the NSP protein (amino acid positions 83-119). Computer analysis of the primary structure of the NSP protein showed the presence of T-cell epitopes in the central part of the NSP molecule. The findings indicate that the viral NSP gene is expressed in the infected animals and verify the concept of the bipolar strategy (ambisense strategy) of the influenza A virus genome.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Leucócitos/imunologia , RNA Viral/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Leucócitos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Domínios Proteicos , Proteínas Virais/química , Proteínas Virais/metabolismo
4.
Nat Commun ; 10(1): 2925, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266960

RESUMO

Bacteriophage Q protein engages σ-dependent paused RNA polymerase (RNAP) by binding to a DNA site embedded in late gene promoter and renders RNAP resistant to termination signals. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact Q-engaged arrested complex. The structure reveals key interactions responsible for σ-dependent pause, Q engagement, and Q-mediated transcription antitermination. The structure shows that two Q protomers (QI and QII) bind to a direct-repeat DNA site and contact distinct elements of the RNA exit channel. Notably, QI forms a narrow ring inside the RNA exit channel and renders RNAP resistant to termination signals by prohibiting RNA hairpin formation in the RNA exit channel. Because the RNA exit channel is conserved among all multisubunit RNAPs, it is likely to serve as an important contact site for regulators that modify the elongation properties of RNAP in other organisms, as well.


Assuntos
Bacteriófagos/enzimologia , Códon de Terminação/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Bacteriófagos/química , Bacteriófagos/genética , Códon de Terminação/metabolismo , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Regiões Promotoras Genéticas , Proteínas Virais/genética
5.
Eur J Med Chem ; 178: 64-80, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31176096

RESUMO

Encouraged by our earlier discovery of neuraminidase inhibitors targeting 150-cavity or 430-cavity, herein, to yield more potent inhibitors, we designed, synthesized, and biologically evaluated a series of novel oseltamivir derivatives via modification of C-1 and C5-NH2 of oseltamivir by exploiting 150-cavity and/or 430-cavity. Among the synthesized compounds, compound 15e, the most potent N1-selective inhibitor targeting 150-cavity, showed 1.5 and 1.8 times greater activity than oseltamivir carboxylate (OSC) against N1 (H5N1) and N1 (H5N1-H274Y). In cellular assays, 15e also exhibited greater potency than OSC against H5N1 with EC50 of 0.66 µM. In addition, 15e demonstrated low cytotoxicity in vitro and low acute toxicity in mice. Molecular docking studies provided insights into the high potency of 15e against N1 and N1-H274Y mutant NA. Overall, we envisioned that the significant breakthrough in the discovery of potent group-1-specific neuraminidase inhibitors may lead to further investigation of more potent anti-influenza agents.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Neuraminidase/antagonistas & inibidores , Oseltamivir/análogos & derivados , Oseltamivir/farmacologia , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/química , Antivirais/toxicidade , Domínio Catalítico , Linhagem Celular , Galinhas , Desenho de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Feminino , Influenzavirus A/enzimologia , Influenzavirus B/enzimologia , Masculino , Camundongos , Simulação de Acoplamento Molecular , Neuraminidase/química , Oseltamivir/síntese química , Oseltamivir/toxicidade , Proteínas Virais/química
6.
Anal Bioanal Chem ; 411(19): 4987-4998, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31254054

RESUMO

Influenza infection requires viral escape from early endosomes into the cytosol, which is enabled by an acid-induced irreversible conformational transformation in the viral protein hemagglutinin. Despite the direct relationship between this conformational change and infectivity, label-free methods for characterizing this and other protein conformational changes in biological mixtures are limited. While the chemical reactivity of the protein backbone and side-chain residues is a proxy for protein conformation, coupling this reactivity to quantitative mass spectrometry is a challenge in complex environments. Herein, we evaluate whether electrophilic amidination coupled with pseudo-parallel reaction monitoring is an effective label-free approach to detect the fusion-associated conformational transformation in recombinant hemagglutinin (rHA). We identified rHA peptides that are differentially amidinated between the pre- and post-fusion states, and validated that this difference relies upon the fusion-associated conformational switch. We further demonstrate that we can distinguish the fusion profile in a matrix of digested cellular lysate. This fusion assay can be used to evaluate fusion competence for modified HA. Graphical abstract.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Orthomyxoviridae/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Virais/metabolismo , Amidas/metabolismo , Células HEK293 , Humanos , Limite de Detecção , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Proteínas Virais/química , Proteínas Virais/classificação
7.
Arch Virol ; 164(9): 2327-2332, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31177352

RESUMO

Two distinct genotypes responsible for rabbit hemorrhagic disease (RHD) are reported, GI.1 (RHDV) and GI.2 (RHDV2). Vaccines based on these two genotypes are only partially cross-protective. Hence, knowing which genotype is circulating is important for appropriate control measures. We have investigated 25 field samples isolated between 2015 and 2018 from rabbits with clinical signs of RHD. Only GI.2 (RHDV2) is currently circulating in Tunisia. All Tunisian samples were grouped together with typical genotypic and phenotypic mutations. Therefore, we recommend initiating an extensive preventive vaccination program based on GI.2 vaccines in addition to a regular monitoring of the circulating lagoviruses.


Assuntos
Infecções por Caliciviridae/veterinária , Vírus da Doença Hemorrágica de Coelhos/genética , Vírus da Doença Hemorrágica de Coelhos/isolamento & purificação , Filogenia , Coelhos/virologia , Sequência de Aminoácidos , Animais , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Genótipo , Vírus da Doença Hemorrágica de Coelhos/química , Vírus da Doença Hemorrágica de Coelhos/classificação , Dados de Sequência Molecular , Alinhamento de Sequência , Tunísia/epidemiologia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
8.
Arch Virol ; 164(9): 2285-2295, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31250104

RESUMO

Examination of lumpy skin disease virus (LSDV) isolates from different geographic regions and times revealed that assays developed in our laboratory for differentiating between virulent Israeli viruses and Neethling vaccine virus (NVV) are generally useful in most, if not all, endemic areas in which NVV-based vaccines are used. Recently it was revealed that the LSDV126 gene of field isolates contains a duplicated region of 27 bp (9 aa), while the vaccine viruses have only one copy. Phylogenetic analysis of a 532-bp segment carrying the LSDV126 gene and whole virus genome sequences revealed that LSDV isolates formed two groups: virulent and vaccine viruses. In this analysis, all of the capripox viruses that lack the ability to efficiently infect cattle were found to carry only one copy of the 27-bp fragment, suggesting that the LSDV126 gene plays an important role in the ability of capripox viruses to infect cattle. In silico analysis of potential antigenic sites in LSDV126 revealed that LSDV126 variants with only one copy of the repeat lack a potentially important antigenic epitope, supporting its possible significance in cattle infection. This study provides new information about the nature of the LSDV126 gene and its possible role in the life cycle of LSDV.


Assuntos
Doença Nodular Cutânea/virologia , Vírus da Doença Nodular Cutânea/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Mapeamento de Epitopos , Dosagem de Genes , Doença Nodular Cutânea/diagnóstico , Vírus da Doença Nodular Cutânea/química , Vírus da Doença Nodular Cutânea/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética
9.
Virology ; 533: 21-33, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31078932

RESUMO

Cavally virus (CavV) is a mosquito-borne plus-strand RNA virus in the family Mesoniviridae (order Nidovirales). We present X-ray structures for the CavV 3C-like protease (3CLpro), as a free enzyme and in complex with a peptide aldehyde inhibitor mimicking the P4-to-P1 residues of a natural substrate. The 3CLpro structure (refined to 1.94 Å) shows that the protein forms dimers. The monomers are comprised of N-terminal domains I and II, which adopt a chymotrypsin-like fold, and a C-terminal α-helical domain III. The catalytic Cys-His dyad is assisted by a complex network of interactions involving a water molecule that mediates polar contacts between the catalytic His and a conserved Asp located in the domain II-III junction and is suitably positioned to stabilize the developing positive charge of the catalytic His in the transition state during catalysis. The study also reveals the structural basis for the distinct P2 Asn-specific substrate-binding pocket of mesonivirus 3CLpros.


Assuntos
Culicidae/virologia , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Nidovirales/enzimologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Cristalografia por Raios X , Cisteína Proteases/genética , Nidovirales/química , Nidovirales/genética , Alinhamento de Sequência , Especificidade por Substrato , Proteínas Virais/genética
10.
Molecules ; 24(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067727

RESUMO

A tertiary structure governs, to a great extent, the biological activity of a protein in the living cell and is consequently a central focus of numerous studies aiming to shed light on cellular processes central to human health. Here, we aim to elucidate the structure of the Rift Valley fever virus (RVFV) L protein using a combination of in silico techniques. Due to its large size and multiple domains, elucidation of the tertiary structure of the L protein has so far challenged both dry and wet laboratories. In this work, we leverage complementary perspectives and tools from the computational-molecular-biology and bioinformatics domains for constructing, refining, and evaluating several atomistic structural models of the L protein that are physically realistic. All computed models have very flexible termini of about 200 amino acids each, and a high proportion of helical regions. Properties such as potential energy, radius of gyration, hydrodynamics radius, flexibility coefficient, and solvent-accessible surface are reported. Structural characterization of the L protein enables our laboratories to better understand viral replication and transcription via further studies of L protein-mediated protein-protein interactions. While results presented a focus on the RVFV L protein, the following workflow is a more general modeling protocol for discovering the tertiary structure of multidomain proteins consisting of thousands of amino acids.


Assuntos
Estrutura Terciária de Proteína , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/química , Proteínas Virais/química , Animais , Genoma Viral/genética , Humanos , Conformação Proteica , RNA Viral/química , RNA Viral/genética , Vírus da Febre do Vale do Rift/genética , Proteínas Virais/genética , Replicação Viral/genética
11.
Virol J ; 16(1): 54, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036013

RESUMO

BACKGROUND: The picornaviral 3C protease mediates viral polyprotein maturation and multiple cleavages of host proteins to modulate viral translation and transcription. The 3C protease has been regarded as a valid target due to its structural similarity among different picornaviruses and minimal sequence similarity with host proteins; therefore, the development of potent inhibitors against the 3C protease as an antiviral drug is ongoing. Duck hepatitis A virus (DHAV) belongs to the Picornavidea family and is a major threat to the poultry industry. To date, little is known about the roles of the DHAV 3C protease plays during infection. METHODS: In this study, we compared the full-length DHAV 3C protein sequence with other 3C sequences to obtain an alignment for the construction of a phylogenetic tree. Then, we expressed and purified recombinant DHAV 3C protease in the BL21 expression system using nickel-NTA affinity chromatography. The optimization of the cleavage assay conditions and the kinetic analysis for DHAV 3C protease were done by in vitro cleavage assays with a fluorogenic peptide respectively. The inhibitory activity of rupintrivir against the DHAV 3C protease was further evaluated. The localization of the 3C protease in infected and transfected cells was determined using immunofluorescence and confocal microscopy. RESULTS: Under different expression conditions, the 3C protease was found to be highly expressed after induction with 1 mM IPTG at 16 °C for 10 h. We synthesized a fluorogenic peptide derived from the cleavage site of the DHAV polyprotein and evaluated the protease activity of the DHAV 3C protease for the first time. We used fluorimetric based kinetic analysis to determine kinetic parameters, and Vmax and Km values were determined to be 16.52 nmol/min and 50.78 µM, respectively. Rupintrivir was found to exhibit inhibitory activity against the DHAV 3C protease. Using polyclonal antibody and an indirect immunofluorescence microscopy assay (IFA), it was determined that the DHAV 3C protease was found in the nucleus during infection. In addition, the DHAV 3C protease can enter into the nucleus without the cooperation of viral proteins. CONCLUSIONS: This is the first study to examine the activity of the DHAV 3C protease, and the activity of the DHAV 3C protease is temperature-, pH- and NaCl concentration- dependent. The DHAV 3C protease localizes throughout DHAV-infected cells and can enter into the nucleus in the absence of other viral proteins. The kinetic analysis was calculated, and the Vmax and Km values were 16.52 nmol/min and 50.78 µM, respectively, using the Lineweaver-Burk plot.


Assuntos
Cisteína Endopeptidases/química , Vírus da Hepatite do Pato/enzimologia , Filogenia , Proteínas Virais/química , Vírus da Hepatite do Pato/genética , Concentração de Íons de Hidrogênio , Isoxazóis/farmacologia , Cinética , Pirrolidinonas/farmacologia , Proteínas Recombinantes , Alinhamento de Sequência
12.
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
13.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 340-347, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045563

RESUMO

Ebola virus is an emerging virus that is capable of causing a deadly disease in humans. Replication, transcription and packaging of the viral genome are carried out by the viral nucleocapsid. The nucleocapsid is a complex of the viral nucleoprotein, RNA and several other viral proteins. The nucleoprotein forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins. The 3.1 Šresolution single-particle cryo-electron microscopy structure of the nucleoprotein-RNA helical filament presented here resembles previous structures determined at lower resolution, while providing improved molecular details of protein-protein and protein-RNA interactions. The higher resolution of the structure presented here will facilitate the design and characterization of novel and specific Ebola virus therapeutics targeting the nucleocapsid.


Assuntos
Ebolavirus/química , Nucleocapsídeo/química , Nucleoproteínas/química , RNA Viral/química , Proteínas Virais/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleocapsídeo/ultraestrutura , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
Adv Mater ; 31(23): e1901485, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30977207

RESUMO

Self-assembly guided by biological molecules is a promising approach for fabricating predesigned nanostructures. Protein is one such biomolecule possessing deterministic 3D crystal structure and peptide information, which acts as a good candidate for templating functional nanoparticles (fNPs). However, inadequate coordination efficacy during the establishment of interfacial interactions with fNPs makes it highly challenging to precisely fabricate designed nanostructures and functional materials. Here, a facile and robust strategy is reported for the hierarchical assembly of fNPs into ordered architectures, with unprecedentedly large sizes up to tens of micrometers, using a hollow cylinder-shaped tobacco mosaic virus coat protein (TMV disk). The rational design of the site-specific functional groups on the TMV disk not only demonstrates the powerful capability of directing various discrete fNP assemblies with high controllability but also assists in precise assembly of a TMV monolayer sheet structure for further organizing homogeneous and heterogeneous fNP periodic lattices by varying the types of fNPs. The high precision and adjustability of the pattern fashions of different fNPs unambiguously corroborate the validity of this innovative strategy, which provides a convenient route to design and assemble protein-based hierarchical ordered architectures for use in nanophotonics and nanodevices.


Assuntos
Proteínas do Capsídeo/química , Nanoestruturas/química , Vírus do Mosaico do Tabaco/química , Proteínas Virais/química , Aminoácidos/química , Sítios de Ligação , Nanopartículas/química , Tamanho da Partícula , Ligação Proteica
15.
Math Biosci Eng ; 16(3): 1709-1717, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30947439

RESUMO

The interaction between viral proteins and small molecule compounds is the basis of drug design. Therefore, it is a fundamental challenge to identify viral proteins according to their amino acid sequences in the field of biopharmaceuticals. The traditional prediction methods su er from the data imbalance problem and take too long computation time. To this end, this paper proposes a deep learning framework for virus protein identifying. In the framework, we employ Temporal Convolutional Network(TCN) instead of Recurrent Neural Network(RNN) for feature extraction to improve computation e ciency. We also customize the cost-sensitive loss function of TCN and introduce the misclassification cost of training samples into the weight update of Gradient Boosting Decision Tree(GBDT) to address data imbalance problem. Experiment results show that our framework not only outperforms traditional data imbalance methods but also greatly reduces the computation time with slight performance enhancement.


Assuntos
Produtos Biológicos , Biologia Computacional/métodos , Proteínas Virais/química , Algoritmos , Antivirais/farmacologia , Simulação por Computador , Bases de Dados Factuais , Aprendizado Profundo , Humanos , Internet , Aprendizado de Máquina , Redes Neurais (Computação) , Fatores de Tempo , Viroses/terapia
16.
Analyst ; 144(10): 3389-3397, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-30990481

RESUMO

DNA can be configured into unique high-order structures due to its significantly high programmability, such as a three-way junction-based structure (denoted Y-shaped DNA), for further applications. Herein, we report a label-free fluorescent signal-on biosensor based on the target-driven primer remodeling rolling circle amplification (RCA)-activated multisite-catalytic hairpin assembly (CHA) enabling the concurrent formation of Y-shaped DNA nanotorches (Y-DNTs) for ultrasensitive detection of ochratoxin A (OTA). Two kinds of masterfully-designed probes, termed Complex I and II, were pre-prepared by the combination of a circular template (CT) with an OTA aptamer (S1), a substrate probe (S2) and hairpin probe 1 (HP1), respectively. Target OTA specifically binds to Complex I, resulting in the release of the remnant element in S2 and successive remodeling into a mature primer for RCA by phi29 DNA polymerase, thus a usable primer-CT complex is produced, which actuates primary RCA. Then, numerous Complex II probes can anneal with the first-generation RCA product (RP) with multiple sites to activate the CHA process. With the participation of endonuclease IV (Endo IV) and phi29, HP1 as a pre-primer containing a tetrahydrofuran abasic site mimic (AP site) in Complex II is converted into a mature primer to initiate additional rounds of RCA. So, countless Y-DNTs are formed concurrently containing a G-quadruplex structure that enables the N-methylmesoporphyrin IX (NMM) to be embedded, generating remarkably strong fluorescence signals. The biosensor was demonstrated to enable rapid and accurate highly efficient and selective detection of OTA with an improved detection limit of as low as 0.0002 ng mL-1 and a widened dynamic range of over 4 orders of magnitude. Meanwhile, this method was proven to be capable of being used to analyze actual samples. Therefore, this proposed strategy may be established as a useful and practical platform for the ultrasensitive detection of mycotoxins in food safety testing.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Nanoestruturas/química , Ocratoxinas/análise , Aptâmeros de Nucleotídeos/genética , Fagos Bacilares/enzimologia , Bacteriófago T4/enzimologia , Sequência de Bases , DNA/genética , DNA Ligases/química , DNA Polimerase Dirigida por DNA/química , Desoxirribonuclease IV (Fago T4-Induzido)/química , Fluorescência , Corantes Fluorescentes/química , Contaminação de Alimentos/análise , Quadruplex G , Sequências Repetidas Invertidas , Limite de Detecção , Mesoporfirinas/química , Técnicas de Amplificação de Ácido Nucleico , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Ocratoxinas/química , Espectrometria de Fluorescência/métodos , Proteínas Virais/química , Vinho/análise
17.
Dokl Biochem Biophys ; 484(1): 52-54, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31012013

RESUMO

To enhance the synthesis of antigenic envelope proteins L1 of high-grade papillomavirus types HPV16, HPV18, HPV31, and HPV45, the sequence of the gene encoding the cucumber mosaic virus replicase (RdRP CMV) was inserted into the genetic construct. This made it possible to increase the production of these antigenic proteins to 25-27 µg/mg total soluble protein.


Assuntos
Cucumovirus , Epitopos , Lycopersicon esculentum , Papillomaviridae/genética , Vacinas contra Papillomavirus , Proteínas Virais , Cucumovirus/genética , Cucumovirus/metabolismo , Epitopos/biossíntese , Epitopos/química , Epitopos/genética , Lycopersicon esculentum/química , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/virologia , Vacinas contra Papillomavirus/biossíntese , Vacinas contra Papillomavirus/química , Vacinas contra Papillomavirus/genética , Proteínas Virais/biossíntese , Proteínas Virais/química , Proteínas Virais/genética
18.
BMC Plant Biol ; 19(1): 159, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023231

RESUMO

BACKGROUND: A disease of unknown etiology in water chestnut plants (Eleocharis dulcis) was reported in China between 2012 and 2014. High throughput sequencing of small RNA (sRNA) combined with bioinformatics, and molecular identification based on PCR detection with virus-specific primers and DNA sequencing is a desirable approach to identify an unknown infectious agent. In this study, we employed this approach to identify viral sequences in water chestnut plants and to explore the molecular interaction of the identified viral pathogen and its natural plant host. RESULTS: Based on high throughput sequencing of virus-derived small RNAs (vsRNA), we identified the sequence a new-to-science double-strand DNA virus isolated from water chestnut cv. 'Tuanfeng' samples, a widely grown cultivar in Hubei province, China, and analyzed its genomic organization. The complete genomic sequence is 7535 base-pairs in length, and shares 42-52% nucleotide sequence identity with viruses in the Caulimoviridae family. The virus contains nine predicated open reading frames (ORFs) encoding nine hypothetical proteins, with conserved domains characteristic of caulimoviruses. Phylogenetic analyses at the nucleotide and amino acid levels indicated that the virus belongs to the genus Soymovirus. The virus is tentatively named Water chestnut soymovirus-1 (WCSV-1). Phylogenetic analysis of the putative viral polymerase protein suggested that WCSV-1 is distinct to other well established species in the Soymovirus genus. This conclusion was supported by phylogenetic analyses of the amino acid sequences encoded by ORFs I, IV, VI, or VII. The sRNA bioinformatics showed that the majority of the vsRNAs are 22-nt in length with a preference for U at the 5'-terminal nucleotide. The vsRNAs are unevenly distributed over both strands of the entire WCSV-1 circular genome, and are clustered into small defined regions. In addition, we detected WCSV-1 in asymptomatic and symptomatic water chestnut samples collected from different regions of China by using PCR. RNA-seq assays further confirmed the presence of WCSV-1-derived viral RNA in infected plants. CONCLUSIONS: This is the first discovery of a dsDNA virus in the genus Soymovirus infecting water chestnuts. Data presented also add new information towards a better understanding of the co-evolutionary mechanisms between the virus and its natural plant host.


Assuntos
Caulimoviridae/fisiologia , Eleocharis/virologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Caulimoviridae/genética , China , Biologia Computacional , Sequência Conservada , Eleocharis/genética , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , RNA Viral/genética , Transcriptoma/genética , Proteínas Virais/química
19.
Anal Bioanal Chem ; 411(13): 2915-2924, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31016327

RESUMO

An upgraded nicking/polymerization strategy for ultrasensitive electrochemical detection of Watermelon mosaic virus (WMV) is proposed on the basis of the exonuclease and polymerase activity of T4 DNA polymerase and Mg2+-dependent DNAzyme-assisted and hemin/G-quadruplex DNAzyme-assisted cascade amplification strategies. Briefly, the hybridized DNA of the target WMV sequence, HP1, and P1 was recognized and nicked by nicking endonuclease Nb.BbvCI, and two DNA segments (P1-25 and P1-6) were produced. P1-25 was digested in the 3'→5' direction, and digestion was halted at the 3'-terminal G locus with the exonuclease activity of T4 DNA polymerase. When dNTP solution mix was added to the mixture, an intact enzymatic sequence of Mg2+-dependent DNAzyme was synthesized by T4 DNA polymerase, which hybridized with its substrate sequence in the loop segment of HP2 immobilized on a gold electrode and initiated the cleavage round. The caged G-quadruplex sequence was released and formed hemin/G-quadruplex-based DNAzyme, resulting in sharply increased electrochemical signals. A correlation between the differential pulse voltammetry signal and the concentration of target WMV sequence was obtained in the range from 50 fM to 1 nM, with 50 fM detection limit. Because the nicking and polymerization reactions are irreversible and share the same buffer, the cascade amplification strategy is an ultrasensitive and high-efficiency strategy, indicating potential for viral detection. Graphical abstract An upgrade nicking/polymerization strategy for ultrasensitive electrochemical detection of Watermelon mosaic virus (WMV) was proposed based on DNAzyme-assistant cascade amplification strategies.


Assuntos
Técnicas Biossensoriais/métodos , Citrullus/virologia , DNA Polimerase Dirigida por DNA/química , Quadruplex G , Hemina/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Potyvirus/isolamento & purificação , Proteínas Virais/química , DNA Catalítico/química , Técnicas Eletroquímicas/métodos , Hibridização de Ácido Nucleico , Doenças das Plantas/virologia , Polimerização , Potyvirus/genética
20.
J Microbiol Biotechnol ; 29(5): 696-703, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-30982317

RESUMO

Cronobacter sakazakii is an opportunistic pathogen causing serious infections in neonates. In this study, a bacteriophage ΦCS01, which infects C. sakazakii, was isolated from swine feces and its morphology, growth parameters, and genomic analysis were investigated. Transmission electron microscopy revealed that ΦCS01 has a spherical head and is 65.74 nm in diameter with a 98.75 nm contracted tail, suggesting that it belongs to the family Myoviridae. The major viral proteins are approximately 71 kDa and 64 kDa in size. The latent period of ΦCS01 was shown to be 60 min, and the burst size was 90.7 pfu (plaque-forming units)/ infected cell. Bacteriophage ΦCS01was stable at 4-60°C for 1 h and lost infectivity after 1 h of heating at 70°C. Infectivity remained unaffected at pH 4-9 for 2 h, while the bacteriophage was inactivated at pH <3 or >10. The double-stranded ΦCS01 DNA genome consists of 48,195 base pairs, with 75 predicted open reading frames. Phylogenetic analysis is closely related to that of the previously reported C. sakazakii phage ESP2949-1. The newly isolated ΦCS01 shows infectivity in the host bacterium C. sakazakii, indicating that it may be a promising alternative to antibacterial agents for the removal of C. sakazakii from powdered infant formulas.


Assuntos
Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/fisiologia , Cronobacter sakazakii/virologia , Genoma Viral , Filogenia , Animais , Bacteriófagos/isolamento & purificação , DNA , Fezes/microbiologia , Inocuidade dos Alimentos , Especificidade de Hospedeiro , Concentração de Íons de Hidrogênio , Myoviridae/classificação , Fases de Leitura Aberta , Suínos , Temperatura Ambiente , Proteínas Virais/química , Sequenciamento Completo do Genoma
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