Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.245
Filtrar
1.
Am J Chin Med ; 47(6): 1307-1324, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31505936

RESUMO

Aloe vera ethanol extract (AVE) reportedly has significant anti-influenza virus activity, but its underlying mechanisms of action and constituents have not yet been completely elucidated. Previously, we have confirmed that AVE treatment significantly reduces the viral replication of green fluorescent protein-labeled influenza A virus in Madin-Darby canine kidney (MDCK) cells. In addition, post-treatment with AVE inhibited viral matrix protein 1 (M1), matrix protein 2 (M2), and hemagglutinin (HA) mRNA synthesis and viral protein (M1, M2, and HA) expressions. In this study, we demonstrated that AVE inhibited autophagy induced by influenza A virus in MDCK cells and also identified quercetin, catechin hydrate, and kaempferol as the active antiviral components of AVE. We also found that post-treatment with quercetin, catechin hydrate, and kaempferol markedly inhibited M2 viral mRNA synthesis and M2 protein expression. A docking simulation suggested that the binding affinity of quercetin, catechin hydrate, and kaempferol for the M2 protein may be higher than that of known M2 protein inhibitors. Thus, the inhibition of autophagy induced by influenza virus may explain the antiviral activity of AVE against H1N1 or H3N2. Aloe vera extract and its constituents may, therefore, be potentially useful for the development of anti-influenza agents.


Assuntos
Aloe/química , Antivirais , Autofagia/efeitos dos fármacos , Vírus da Influenza A/fisiologia , Vírus da Influenza A/patogenicidade , Extratos Vegetais/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Células Cultivadas , Cães , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A/metabolismo , Rim/citologia , Ligação Proteica/efeitos dos fármacos , Quercetina/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas da Matriz Viral/metabolismo
2.
Chem Pharm Bull (Tokyo) ; 67(11): 1201-1207, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31434835

RESUMO

Oleanolic acid (OA) was discovered as a mild influenza hemagglutinin (HA) inhibitor in our earlier studies. In the present work, 20 compounds were prepared by structural modifications of OA, and their antiviral activities against influenza A/WSN/33 (H1N1) virus in Madin-Darby canine kidney (MDCK) cells were evaluated. Based on the biological result, structure-activity relationship (SAR) was discussed. Compound 10 with six-carbon chain and a terminal hydroxyl group showed the strongest anti-influenza activity with an IC50 of 2.98 µM, which is an order of magnitude more potent than OA. Hemagglutination inhibition and Surface plasmon resonance (SPR) assay indicated that compound 10 might interfere with influenza invasion by interacting with HA protein.


Assuntos
Antivirais/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Ácido Oleanólico/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Cães , Relação Dose-Resposta a Droga , Hemaglutininas/efeitos dos fármacos , Hemaglutininas/metabolismo , Vírus da Influenza A/metabolismo , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/virologia , Estrutura Molecular , Ácido Oleanólico/síntese química , Ácido Oleanólico/química , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
3.
Nat Microbiol ; 4(10): 1671-1679, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31263181

RESUMO

Influenza viruses antagonize key immune defence mechanisms via the virulence factor non-structural protein 1 (NS1). A key mechanism of virulence by NS1 is blocking nuclear export of host messenger RNAs, including those encoding immune factors1-3; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor complex, nuclear RNA export factor 1-nuclear transport factor 2-related export protein 1 (NXF1-NXT1), which is the principal receptor mediating docking and translocation of mRNAs through the nuclear pore complex via interactions with nucleoporins4,5. We determined the crystal structure of NS1 in complex with NXF1-NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1-NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. We demonstrate that a mutant influenza virus deficient in binding NXF1-NXT1 does not block host mRNA export and is attenuated. This attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. In sum, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.


Assuntos
Núcleo Celular/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , RNA Mensageiro/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células A549 , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Células Cultivadas , Cristalografia por Raios X , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/genética
4.
Biochim Biophys Acta Biomembr ; 1861(8): 1421-1427, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31153909

RESUMO

The influenza A M2 protein is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle. The monomeric subunits of the M2 homotetramer consist of an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain. The transmembrane domain forms a four-helix proton channel that promotes uncoating of virions upon host cell entry. The membrane-proximal region of the C-terminal domain forms a surface-associated amphipathic helix necessary for viral budding. The structure of the remaining ~34 residues of the distal cytoplasmic tail has yet to be fully characterized despite the functional significance of this region for influenza infectivity. Here, we extend structural and dynamic studies of the poorly characterized M2 cytoplasmic tail. We used SDSL-EPR to collect site-specific information on the mobility, solvent accessibility, and conformational properties of residues 61-70 of the full-length, cell-expressed M2 protein reconstituted into liposomes. Our analysis is consistent with the predominant population of the C-terminal tail dynamically extending away from the membranes surface into the aqueous medium. These findings provide insight into the hypothesis that the C-terminal domain serves as a sensor that regulates how M2 protein participates in critical events in the viral infection cycle.


Assuntos
Citoplasma/metabolismo , Vírus da Influenza A/metabolismo , Canais Iônicos/metabolismo , Proteínas da Matriz Viral/metabolismo , Membrana Celular/metabolismo , Vírus da Influenza A/fisiologia , Proteínas da Matriz Viral/química , Montagem de Vírus , Liberação de Vírus
5.
Mol Pharmacol ; 96(2): 148-157, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31175183

RESUMO

The Food and Drug Administration-approved influenza A antiviral amantadine inhibits the wild-type (WT) AM2 channel but not the S31N mutant predominantly found in circulating strains. In this study, serial viral passages were applied to select resistance against a newly developed isoxazole-conjugated adamantane inhibitor that targets the AM2 S31N channel. This led to the identification of the novel drug-resistant mutation L46P located outside the drug-binding site, which suggests an allosteric resistance mechanism. Intriguingly, when the L46P mutant was introduced to AM2 WT, the channel remained sensitive toward amantadine inhibition. To elucidate the molecular mechanism, molecular dynamics simulations and binding free energy molecular mechanics-generalized born surface area (MM-GBSA) calculations were performed on WT and mutant channels. It was found that the L46P mutation caused a conformational change in the N terminus of transmembrane residues 22-31 that ultimately broadened the drug-binding site of AM2 S31N inhibitor 4, which spans residues 26-34, but not of AM2 WT inhibitor amantadine, which spans residues 31-34. The MM-GBSA calculations showed stronger binding stability for 4 in complex with AM2 S31N compared with 4 in complex with AM2 S31N/L46P, and equal binding free energies of amantadine in complex with AM2 WT and AM2 L46P. Overall, these results demonstrate a unique allosteric resistance mechanism toward AM2 S31N channel blockers, and the L46P mutant represents the first experimentally confirmed drug-resistant AM2 mutant that is located outside of the pore where drug binds. SIGNIFICANCE STATEMENT: AM2 S31N is a high-profile antiviral drug target, as more than 95% of currently circulating influenza A viruses carry this mutation. Understanding the mechanism of drug resistance is critical in designing the next generation of AM2 S31N channel blockers. Using a previously developed AM2 S31N channel blocker as a chemical probe, this study was the first to identify a novel resistant mutant, L46P. The L46P mutant is located outside of the drug-binding site. Molecular dynamics simulations showed that L46P causes a dilation of drug-binding site between residues 22 and 31, which affects the binding of AM2 S31N channel blockers, but not the AM2 WT inhibitor amantadine.


Assuntos
Amantadina/farmacologia , Antivirais/farmacologia , Vírus da Influenza A/metabolismo , Mutação , Proteínas da Matriz Viral/genética , Regulação Alostérica/efeitos dos fármacos , Motivos de Aminoácidos , Animais , Antivirais/química , Sítios de Ligação , Cães , Farmacorresistência Viral , Feminino , Humanos , Vírus da Influenza A/efeitos dos fármacos , Células Madin Darby de Rim Canino , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Inoculações Seriadas , Relação Estrutura-Atividade , Proteínas da Matriz Viral/química , Xenopus laevis
6.
BMC Bioinformatics ; 20(1): 297, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159726

RESUMO

BACKGROUND: Host factors of influenza virus replication are often found in key topological positions within protein-protein interaction networks. This work explores how protein states can be manipulated through controllability analysis: the determination of the minimum manipulation needed to drive the cell system to any desired state. Here, we complete a two-part controllability analysis of two protein networks: a host network representing the healthy cell state and an influenza A virus-host network representing the infected cell state. In this context, controllability analyses aim to identify key regulating host factors of the infected cell's progression. This knowledge can be utilized in further biological analysis to understand disease dynamics and isolate proteins for study as drug target candidates. RESULTS: Both topological and controllability analyses provide evidence of wide-reaching network effects stemming from the addition of viral-host protein interactions. Virus interacting and driver host proteins are significant both topologically and in controllability, therefore playing important roles in cell behavior during infection. Functional analysis finds overlap of results with previous siRNA studies of host factors involved in influenza replication, NF-kB pathway and infection relevance, and roles as interferon regulating genes. 24 proteins are identified as holding regulatory roles specific to the infected cell by measures of topology, controllability, and functional role. These proteins are recommended for further study as potential antiviral drug targets. CONCLUSIONS: Seasonal outbreaks of influenza A virus are a major cause of illness and death around the world each year with a constant threat of pandemic infection. This research aims to increase the efficiency of antiviral drug target discovery using existing protein-protein interaction data and network analysis methods. These results are beneficial to future studies of influenza virus, both experimental and computational, and provide evidence that the combination of topology and controllability analyses may be valuable for future efforts in drug target discovery.


Assuntos
Antivirais/farmacologia , Sistemas de Liberação de Medicamentos , Descoberta de Drogas , Interações Hospedeiro-Patógeno , Mapas de Interação de Proteínas , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/metabolismo , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Replicação Viral/efeitos dos fármacos
7.
Anal Biochem ; 572: 52-57, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844367

RESUMO

Since 2013, the H7 subtype avian influenza virus (AIV-H7) has seriously endangered human life and health, and has had a serious impact on the poultry industry in China. A competitive enzyme-linked immunosorbent assay (C-ELISA) which detects the antibody for AIV-H7 was developed, basing on a monoclonal antibody (mAb) against the neutralizing epitopes on hemagglutinin (HA)gene. Twelve hybridoma cell lines were screened by cell fusion. Hemagglutination inhibition (HI) assay and indirect ELISA were used to identify the competitive effect of the mAbs. High-affinity mAb 1H11 was selected as a competitive antibody. The reaction conditions for the C-ELISA were optimized for AIV-H7 antibody detection. The cross-reactivity of the C-ELISA was determined by AIV-(H1H15), NDV, IBV and IBDV positive serum. A total of 1294 field samples (chicken (462), duck (318), goose (219), quail (203) and pigeon (92) were simultaneously detected by C-ELISA and HI assay. The C-ELISA was found to have a high specificity of 93.23% and a sensitivity of 96.24%. These results reveal a positive coincidence between C-ELISA and HI assay at a coincidence rate of 97.52%. In addition, It confirmed that this method can be used for the diagnosis of AIV-H7 antibodies from chicken, ducks, goose, quail and pigeons.


Assuntos
Anticorpos Antivirais/análise , Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Influenza A/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Galinhas , Patos , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/imunologia , Influenza Aviária/diagnóstico , Sensibilidade e Especificidade
8.
Emerg Microbes Infect ; 8(1): 327-338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866786

RESUMO

Influenza virus haemagglutinin (HA) and neuraminidase (NA) are involved in the recognition and modulation of sialic acids on the cell surface as the virus receptor. Although the balance between two proteins functions has been found to be crucial for viral fitness, the interplay between the proteins has not been well established. Herein we present evidence for interplay between influenza HA and NA, which may affect the balance between two glycoprotein functions. NA enzymatic activities against sialoglycans were promoted by the presence of HA, which is in accordance with the level of co-existing HA. Such activity enhancement was lost when the HA-receptor binding properties were abolished by low-pH treatment or by mutations at the HA receptor binding domain. Sialidase activities of NA-containing virus-like particles and native influenza viruses were detected using different NA-assays and sialic acid substrates. Most pronounced HA-mediated NA enhancement was found when intact virions were confronted with multivalent surface-anchored substrates, which mimics the physiological conditions on cell membranes. Using recombinant viruses with altered HA bindings preference between α2,3- and α2,6-linked sialic acids, we also found that NA function against different substrates is correlated with the HA-receptor specificity. The effect of HA-receptor specificities on NA functions, together with the HA-mediated NA enhancement, may play a role in virus evasion of the mucus barrier, as well as in cross-species adaptation. Our data also indicate the importance of using multivalent substrates in future studies of NA functions.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/metabolismo , Neuraminidase/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Virais/metabolismo , Animais , Cães , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Ligação Proteica , Especificidade por Substrato
9.
J Mol Model ; 25(4): 99, 2019 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-30904969

RESUMO

Though oseltamivir and zanamivir are the active anti-influenza drugs, the emergence of different strains of influenza A virus with mutations creates drug-resistance to these drugs. Therefore, it is essential to find a suitable approach to stop the viral infection. The present study focuses on understanding the conformational changes of the HA2 protein at different pH levels (pH 7, pH 6, pH 5) and on blocking the low pH-induced conformational changes of the HA2 protein with a suitable ligand using molecular docking and molecular dynamics (MD) simulation methods. As the pH value decreases to pH 5, the protein undergoes large conformational changes with less stability in the order of pH 7 > pH 6 > pH 5. The fusion peptide (residues 1-20) and the extended loop (residues 58-75) deviate more at pH 5. The ligand stachyflin bound between the N- and C-terminal helix regions retains the stability of the HA2 protein at pH 5 and blocks the low pH-induced conformational transition. The performance of stachyflin is increased when it directly interacts with residues at the intramonomer binding site rather than the intermonomer binding site. The susceptibility of the HA2 protein of different subtypes to stachyflin is in the order of H1 > H7 > H5 > H2 > H3. Stachflin has a higher binding affinity for H1 (at pH 7, pH 6, pH 5) and H7 subtypes than others. Lys47, Lys58, and Glu103 are the key residues that favor the binding and highly stabilize the HA2 protein at low pH. Graphical abstract Low pH-induced conformational change of influenza HA2 protein.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Conformação Proteica , Algoritmos , Antivirais/química , Antivirais/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Ligações de Hidrogênio , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
10.
Bioconjug Chem ; 30(4): 1192-1198, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30860815

RESUMO

The precise design of synthetic polymer ligands using controlled polymerization techniques provides an advantage for the field of nanoscience. We report the topological design of glyco-ligands based on synthetic polymers for targeting hemagglutinin (HA, lectin on the influenza virus). To achieve precise arrangement of the glycounits toward the sugar-binding pockets of HA, triarm star glycopolymers were synthesized. The interaction of the star glycopolymers with HA was found to depend on the length of the polymer arms and was maximized when the hydrodynamic diameter of the star glycopolymer was comparable to the distance between the sugar-binding pockets of HA. Following the formula of multivalent interaction, the number of binding sites in the interaction of the glycopolymers with HA was estimated as 1.8-2.7. Considering one HA molecule has three sugar-binding pockets, these values were reasonable. The binding mode of synthetic glycopolymer-ligands toward lectins could be tuned using controlled radical polymerization techniques.


Assuntos
Vírus da Influenza A/metabolismo , Polímeros/química , Química Click , Hemaglutininas Virais/metabolismo , Ligantes , Ligação Proteica
11.
Bioorg Med Chem Lett ; 29(9): 1113-1119, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30852083

RESUMO

Nonstructural protein 1 (NS1) plays a crucial function in the replication, spread, and pathogenesis of influenza virus by inhibiting the host innate immune response. Here we report the discovery and optimization of novel pyrazolopyridine NS1 antagonists that can potently inhibit influenza A/PR/8/34 replication in MDCK cells, rescue MDCK cells from cytopathic effects of seasonal influenza A strains, reverse NS1-dependent inhibition of IFN-ß gene expression, and suppress the slow growth phenotype in NS1-expressing yeast. These pyrazolopyridines will enable researchers to investigate NS1 function during infection and how antagonists can be utilized in the next generation of treatments for influenza infection.


Assuntos
Antivirais/síntese química , Desenho de Drogas , Vírus da Influenza A/metabolismo , Pirazóis/química , Piridinas/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Cães , Células HEK293 , Meia-Vida , Humanos , Interferon beta/metabolismo , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirazóis/metabolismo , Pirazóis/farmacologia , Piridinas/metabolismo , Piridinas/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
12.
J Virol ; 93(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30787149

RESUMO

Alveolar macrophages (AM) play pivotal roles in modulating host defense, pulmonary inflammation, and tissue injury following respiratory viral infections. However, the transcriptional regulation of AM function during respiratory viral infections is still largely undefined. Here we have screened the expression of 84 transcription factors in AM in response to influenza A virus (IAV) infection. We found that the transcription factor PPAR-γ was downregulated following IAV infection in AM through type I interferon (IFN)-dependent signaling. PPAR-γ expression in AM was critical for the suppression of exaggerated antiviral and inflammatory responses of AM following IAV and respiratory syncytial virus (RSV) infections. Myeloid PPAR-γ deficiency resulted in enhanced host morbidity and increased pulmonary inflammation following both IAV and RSV infections, suggesting that macrophage PPAR-γ is vital for restricting severe host disease development. Using approaches to selectively deplete recruiting monocytes, we demonstrate that PPAR-γ expression in resident AM is likely important in regulating host disease development. Furthermore, we show that PPAR-γ was critical for the expression of wound healing genes in AM. As such, myeloid PPAR-γ deficiency resulted in impaired inflammation resolution and defective tissue repair following IAV infection. Our data suggest a critical role of PPAR-γ expression in lung macrophages in the modulation of pulmonary inflammation, the development of acute host diseases, and the proper restoration of tissue homeostasis following respiratory viral infections.IMPORTANCE Respiratory viral infections, like IAV and respiratory syncytial virus (RSV) infections, impose great challenges to public health. Alveolar macrophages (AM) are lung-resident immune cells that play important roles in protecting the host against IAV and RSV infections. However, the underlying molecular mechanisms by which AM modulate host inflammation, disease development, and tissue recovery are not very well understood. Here we identify that PPAR-γ expression in AM is crucial to suppress pulmonary inflammation and diseases and to promote fast host recovery from IAV and RSV infections. Our data suggest that targeting macrophage PPAR-γ may be a promising therapeutic option in the future to suppress acute inflammation and simultaneously promote recovery from severe diseases associated with respiratory viral infections.


Assuntos
Vírus da Influenza A/metabolismo , Macrófagos Alveolares/metabolismo , Infecções por Orthomyxoviridae/metabolismo , PPAR gama/biossíntese , Pneumonia Viral/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Animais , Regulação da Expressão Gênica , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/virologia , Macrófagos Alveolares/patologia , Macrófagos Alveolares/virologia , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , PPAR gama/genética , Pneumonia Viral/genética , Pneumonia Viral/patologia , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/patologia
13.
Proc Natl Acad Sci U S A ; 116(7): 2577-2582, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30626642

RESUMO

Uncoating is an obligatory step in the virus life cycle that serves as an antiviral target. Unfortunately, it is challenging to study viral uncoating due to methodology limitations for detecting this transient and dynamic event. The uncoating of influenza A virus (IAV), which contains an unusual genome of eight segmented RNAs, is particularly poorly understood. Here, by encapsulating quantum dot (QD)-conjugated viral ribonucleoprotein complexes (vRNPs) within infectious IAV virions and applying single-particle imaging, we tracked the uncoating process of individual IAV virions. Approximately 30% of IAV particles were found to undergo uncoating through fusion with late endosomes in the "around-nucleus" region at 30 to 90 minutes postinfection. Inhibition of viral M2 proton channels and cellular endosome acidification prevented IAV uncoating. IAV vRNPs are released separately into the cytosol after virus uncoating. Then, individual vRNPs undergo a three-stage movement to the cell nucleus and display two diffusion patterns when inside the nucleus. These findings reveal IAV uncoating and vRNP trafficking mechanisms, filling a critical gap in knowledge about influenza viral infection.


Assuntos
Vírus da Influenza A/metabolismo , Desenvelopamento do Vírus , Animais , Núcleo Celular/metabolismo , Cães , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino , Transporte Proteico , Pontos Quânticos , RNA Viral/genética , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Vírion/metabolismo , Vírion/patogenicidade , Replicação Viral
14.
Bioorg Med Chem Lett ; 29(5): 744-748, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30655214

RESUMO

Sialyllactose (SL)-modified trimer DNAs with a similar SL presentation as their binding sites on influenza virus hemagglutinin (HA) trimer were designed and synthesized. These trimer DNAs showed high affinity for various influenza viruses, including A/Puerto Rico/08/34 (H1N1), A/Beijing/262/95 (H1N1), A/Yokohama/77/2008 (H1N1), and A/Panama/2007/99 (H3N2). Thus, presentation of SL residues on three vertexes of the scaffold as well as sialic acid binding sites on the HA trimer regardless of a tri-branched or triangular scaffold are important for high affinity for influenza viruses. These compounds have the potential for use in detection and as inhibitors of a broad spectrum of influenza viruses.


Assuntos
Antivirais/farmacologia , DNA/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Lactose/análogos & derivados , Ácidos Siálicos/química , Ligação Viral/efeitos dos fármacos , DNA/química , Humanos , Vírus da Influenza A/metabolismo , Lactose/química
15.
Comput Biol Chem ; 78: 273-281, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30597438

RESUMO

Developing antivirals for influenza A virus (FluA) has become more challenging due to high range of antigenic mutation and increasing numbers of drug-resistant viruses. Finding a selective inhibitor to target highly conserved region of protein-protein interactions interface, thereby increasing its efficiency against drug resistant virus could be highly beneficial. In this study, we used in silico approach to derive FluAPep1 from highly conserved region, PAN-PB1C interface and generated 121 FluAPep1 analogues. Interestingly, we found that the FluAPep1 interaction region in the PAN domain are highly conserved in many FluA subtypes. Especially, FluAPep1 targets two pandemic FluA strains, H1N1/avian/2009 and H3N2/Victoria/1975. All of these FluA subtypes PAN domain (H1N1/H3N2CAN/H3N2VIC/H7N1/H7N2) were superimposed with PAN domain from H17N10 and the calculated root mean standards deviations were less than 3 Å. FlexPepDock analysis revealed that FluAPep1 exhibited higher binding affinity (score -246.155) with the PAN domain. In addition, around 86% of non-hot spot mutated peptides (FluAPep28-122) showed enhanced binding affinity with PAN domain. ToxinPred analysis confirmed that designed peptides were non-toxic. Thus, FluAPep1 and its analogues has potential to be further developed into an antiviral treatment against FluA infection.


Assuntos
Antivirais/farmacologia , Simulação por Computador , RNA Polimerases Dirigidas por DNA/metabolismo , Desenho de Drogas , Influenza Humana/tratamento farmacológico , Peptídeos/metabolismo , Antivirais/síntese química , Antivirais/química , RNA Polimerases Dirigidas por DNA/química , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/metabolismo , Influenza Humana/virologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Peptídeos/química , Ligação Proteica/efeitos dos fármacos
16.
Biomed Pharmacother ; 111: 740-750, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30611999

RESUMO

Albeit microRNAs (miRNAs) have become increasingly appreciated for their essential roles in innate immune responses to viral infections; however, it is unknown how host miRNAs regulate influenza A virus (IAV)-induced inflammation. The aim of our study was to investigate the role of miR-146a in IAV replication in vitro and in vivo. In vitro, we found miR-146a was significantly upregulated in A549 cells with IAV infection. Overexpression of miR-146a promoted IAV replication, while downregulation of miR-146a repressed replication. We found that miR-146a diminished type I interferon (IFN) responses by decreasing IFN-ß production and IFN-stimulated gene (ISG) expression. Furthermore, we found the IFNs level and IAV replication regulated by miR-146a inhibitor was partially reversed by depletion of interferon receptor (IFNAR) 1 or 2. In addition, we found that miR-146a directly targets tumor necrosis factor receptor association factor 6 (TRAF6), which is involved in the production of type I IFN, and TRAF6 overexpression reversed the replication-promoting effect of miR-146a on IAV. In vivo, inhibition of miR-146a alleviated IAV-induced mice lung injury and promoted survival rates by promoting type I antiviral activities. It is, therefore, concluded that downregulation of miR-146a inhibits IAV replication by enhancing type I IFN response through its target gene TRAF6 in vitro and in vivo, suggesting miR-146a antagomir might be a potential therapeutic target during IAV infection.


Assuntos
Regulação para Baixo/fisiologia , Vírus da Influenza A/metabolismo , Interferon Tipo I/farmacologia , MicroRNAs/metabolismo , Replicação Viral/fisiologia , Células A549 , Animais , Antagomirs/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células HEK293 , Humanos , Vírus da Influenza A/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , Distribuição Aleatória , Células Vero , Replicação Viral/efeitos dos fármacos
17.
Biochem J ; 476(3): 467-481, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30617221

RESUMO

MOV10 has emerged as an important host antiviral factor. MOV10 not only inhibits various viruses, including human immunodeficiency virus type 1, hepatitis C virus and vesicular stomatitis virus, but also restricts the activity of retroelements long interspersed nucleotide element-1, Alu, SVA and intracisternal A particles. Here, we report that MOV10 suppresses influenza A virus infection through interacting with viral nucleoprotein (NP), sequestering viral RNP in the cytoplasm and causing the degradation of viral vRNA. The antiviral activity of MOV10 depends on the integrity of P-bodies. We also found that the antiviral activity of MOV10 is partially countered by viral NS1 protein that interferes with the interaction of MOV10 with viral NP and causes MOV10 degradation through the lysosomal pathway. Moreover, NS1-defective influenza A virus is more susceptible to MOV10 restriction. Our data not only expand the antiviral spectrum of MOV10 but also reveal the NS1 protein as the first viral antagonist of MOV10.


Assuntos
Citoplasma/metabolismo , Vírus da Influenza A/metabolismo , Proteólise , RNA Helicases/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células A549 , Citoplasma/genética , Células HEK293 , Humanos , Vírus da Influenza A/genética , Lisossomos/genética , Lisossomos/metabolismo , RNA Helicases/genética , Ribonucleoproteínas/genética , Proteínas não Estruturais Virais/genética
19.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30463972

RESUMO

Defective interfering particles (DIPs) replicate at the expense of coinfecting, fully infectious homologous virus. Typically, they contain a highly deleted form of the viral genome. Utilizing single-cell analysis, here we report the discovery of a yet-unknown DIP type, derived from influenza A viruses (IAVs), termed OP7 virus. Instead of deletions, the genomic viral RNA (vRNA) of segment 7 (S7) carried 37 point mutations compared to the reference sequence, affecting promoter regions, encoded proteins, and genome packaging signals. Coinfection experiments demonstrated strong interference of OP7 virus with IAV replication, manifested by a dramatic decrease in the infectivity of released virions. Moreover, an overproportional quantity of S7 in relation to other genome segments was observed, both intracellularly and in the released virus population. Concurrently, OP7 virions lacked a large fraction of other vRNA segments, which appears to constitute its defect in virus replication. OP7 virus might serve as a promising candidate for antiviral therapy. Furthermore, this novel form of DIP may also be present in other IAV preparations.IMPORTANCE Defective interfering particles (DIPs) typically contain a highly deleted form of the viral genome, rendering them defective in virus replication. Yet upon complementation through coinfection with fully infectious standard virus (STV), interference with the viral life cycle can be observed, leading to suppressed STV replication and the release of mainly noninfectious DIPs. Interestingly, recent research indicates that DIPs may serve as an antiviral agent. Here we report the discovery of a yet-unknown type of influenza A virus-derived DIP (termed "OP7" virus) that contains numerous point mutations instead of large deletions in its genome. Furthermore, the underlying principles that render OP7 virions interfering and apparently defective seem to differ from those of conventional DIPs. In conclusion, we believe that OP7 virus might be a promising candidate for antiviral therapy. Moreover, it exerts strong effects, both on virus replication and on the host cell response, and may have been overlooked in other IAV preparations.


Assuntos
Vírus Defeituosos/genética , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Células A549 , Animais , Antivirais , Cães , Genoma Viral/genética , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Nucleotídeos/genética , RNA Viral/genética , Vírion/genética , Replicação Viral/genética
20.
Cell ; 176(1-2): 281-294.e19, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30503209

RESUMO

Influenza viruses inhabit a wide range of host environments using a limited repertoire of protein components. Unlike viruses with stereotyped shapes, influenza produces virions with significant morphological variability even within clonal populations. Whether this tendency to form pleiomorphic virions is coupled to compositional heterogeneity and whether it affects replicative fitness remains unclear. Here, we address these questions by developing a strain of influenza A virus amenable to rapid compositional characterization through quantitative, site-specific labeling of viral proteins. Using this strain, we find that influenza A produces virions with broad variations in size and composition from even single infected cells. This phenotypic variability contributes to virus survival during environmental challenges, including exposure to antivirals. Complementing genetic adaptations that act over larger populations and longer times, this "low-fidelity" assembly of influenza A virus allows small populations to survive environments that fluctuate over individual replication cycles.


Assuntos
Vírus da Influenza A/metabolismo , Montagem de Vírus/fisiologia , Linhagem Celular , Células Cultivadas , Humanos , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Proteínas Virais , Vírion , Replicação Viral/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA