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1.
Immunohorizons ; 3(1): 1-12, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31356172

RESUMO

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection and hospitalization in infants. It is well established that both CD4+ and CD8+ T cells are critical for mediating viral clearance but also contribute to the induction of immunopathology following RSV infection. C57BL/6 mice are often used to study T cell responses following RSV infection given the wide variety of genetically modified animals available. To date, few RSV-derived CD4+ and CD8+ T cell epitopes have been identified in C57BL/6 mice. Using an overlapping peptide library spanning the entire RSV proteome, intracellular cytokine staining for IFN-γ was performed to identify novel CD4+ and CD8+ T cell epitopes in C57BL/6 mice. We identified two novel CD4+ T cell epitopes and three novel CD8+ T cell epitopes located within multiple RSV proteins. Additionally, we characterized the newly described T cell epitopes by determining their TCR Vß expression profiles and MHC restriction. Overall, the novel RSV-derived CD4+ and CD8+ T cell epitopes identified in C57BL/6 mice will aid in future studies of RSV-specific T cell responses.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/metabolismo , Infecções por Vírus Respiratório Sincicial/patologia , Vírus Sincicial Respiratório Humano/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Interferon gama/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Biblioteca de Peptídeos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas Virais/metabolismo
3.
J Proteome Res ; 18(3): 1145-1161, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30706717

RESUMO

Respiratory syncytial virus (RSV) is a significant cause of mortality and morbidity in infants, the elderly, immunocompromised individuals, and patients with congenital heart diseases. Despite extensive efforts, a vaccine against RSV is still not available. We have previously reported the development of a subunit vaccine (ΔF/TriAdj) composed of a truncated version of the fusion protein (ΔF) and a polymer-based combination adjuvant (TriAdj). We compared inflammatory responses of ΔF/TriAdj-vaccinated and unvaccinated mice following intranasal challenge with RSV. Rapid and early inflammatory responses were observed in lung samples from both groups but modulated in the vaccinated group 7 days after the viral challenge. The underlying mechanism of action of ΔF/TriAdj was further studied through LC-MS-based metabolomic profiling by using 12C- or 13C-dansyl labeling for the amine/phenol submetabolome. RSV infection predominantly affected the amino acid biosynthesis pathways and urea cycle, whereas ΔF/TriAdj modulated the concentrations of almost all of the altered metabolites. Tryptophan metabolites were significantly affected, including indole, l-kynurenine, xanthurenic acid, serotonin, 5-hydroxyindoleacetic acid, and 6-hydroxymelatonin. The results from the present study provide further mechanistic insights into the mode of action of this RSV vaccine candidate and have important implications in the design of metabolic therapeutic interventions.


Assuntos
Imunização/métodos , Metabolômica/métodos , Infecções por Vírus Respiratório Sincicial/metabolismo , Vacinas de Subunidades/metabolismo , Adjuvantes Imunológicos/metabolismo , Administração Intranasal , Animais , Anticorpos Antivirais/imunologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/virologia , Camundongos , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/metabolismo , Vírus Sincicial Respiratório Humano/patogenicidade , Vacinas de Subunidades/genética
4.
J Med Chem ; 62(7): 3206-3227, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30411898

RESUMO

Respiratory syncytial virus (RSV) is a globally prevalent viral infection with limited treatment options which hospitalizes millions each year. Treatment options have been limited to palivizumab, a monoclonal antibody, approved for prophylaxis in high-risk infants and ribavirin with very limited efficacy and significant safety concerns. This Perspective surveys the range of direct acting antiviral agents (DAAs) that target key steps in the viral life cycle. A number of approaches to DAAs have produced landmark clinical studies over the past few years, notably in fusion and nucleoside inhibitors, and an update of the clinical status of these compounds is provided. Non-nucleoside inhibitors of replication are reviewed in addition to inhibitors of other mechanisms, notably the RSV N and G proteins. This article will provide an informative perspective of the current status of drug discovery targeted at providing an effective therapy for RSV infection.


Assuntos
Antivirais/química , Descoberta de Drogas , Bibliotecas de Moléculas Pequenas/química , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Humanos , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Palivizumab/imunologia , Palivizumab/uso terapêutico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo
5.
Viruses ; 10(10)2018 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-30274351

RESUMO

The virus⁻host protein interactions that underlie respiratory syncytial virus (RSV) assembly are still not completely defined, despite almost 60 years of research. RSV buds from the apical surface of infected cells, once virion components have been transported to the budding sites. Association of RSV matrix (M) protein with the actin cytoskeleton may play a role in facilitating this transport. We have investigated the interaction of M with actin in vitro and cell culture. Purified wildtype RSV M protein was found to bind directly to polymerized actin in vitro. Vero cells were transfected to express full-length M (1⁻256) as a green fluorescent protein-(GFP) tagged protein, followed by treatment with the microfilament destabilizer, cytochalasin D. Destabilization of the microfilament network resulted in mislocalization of full-length M, from mostly cytoplasmic to diffused across both cytoplasm and nucleus, suggesting that M interacts with microfilaments in this system. Importantly, treatment of RSV-infected cells with cytochalasin D results in lower infectious virus titers, as well as mislocalization of M to the nucleus. Finally, using deletion mutants of M in a transfected cell system, we show that both the N- and C-terminus of the protein are required for the interaction. Together, our data suggest a possible role for M⁻actin interaction in transporting virion components in the infected cell.


Assuntos
Actinas/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas da Matriz Viral/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citocalasina D/farmacologia , Citoplasma/metabolismo , Deleção de Genes , Humanos , Ligação Proteica/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Vírion/metabolismo , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
6.
Virology ; 523: 129-139, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30144786

RESUMO

Experimental results in vitro and in animal models are used to guide researchers in testing vaccines or treatment in humans. However, viral kinetics are different in vitro, in animals, and in humans, so it is sometimes difficult to translate results from one system to another. In this study, we use a mathematical model to fit experimental data from multiple cycle respiratory syncytial virus (RSV) infections in vitro, in african green monkey (AGM), and in humans in order to quantitatively compare viral kinetics in the different systems. We find that there are differences in viral clearance rate, productively infectious cell lifespan, and eclipse phase duration between in vitro and in vivo systems and among different in vivo systems. We show that these differences in viral kinetics lead to different estimates of drug effectiveness of fusion inhibitors in vitro and in AGM than in humans.


Assuntos
Modelos Estatísticos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Inibidores de Proteínas Virais de Fusão/uso terapêutico , Proteínas Virais de Fusão/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Adulto , Idoso , Animais , Criança , Chlorocebus aethiops , Expressão Gênica , Especificidade de Hospedeiro , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/virologia , Fusão de Membrana/efeitos dos fármacos , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
7.
J Mol Biol ; 430(6): 777-792, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-29414675

RESUMO

RNA transcription of mononegavirales decreases gradually from the 3' leader promoter toward the 5' end of the genome, due to a decay in polymerase processivity. In the respiratory syncytial virus and metapneumovirus, the M2-1 protein ensures transcription anti-termination. Despite being a homotetramer, respiratory syncytial virus M2-1 binds two molecules of RNA of 13mer or longer per tetramer, and temperature-sensitive secondary structure in the RNA ligand is unfolded by stoichiometric interaction with M2-1. Fine quantitative analysis shows positive cooperativity, indicative of conformational asymmetry in the tetramer. RNA binds to M2-1 through a fast bimolecular association followed by slow rearrangements corresponding to an induced-fit mechanism, providing a sequential description of the time events of cooperativity. The first binding event of half of the RNA molecule to one of the sites increases the affinity of the second binding event on the adjacent contacting protomer by 15-fold, product of increased effective concentration caused by the entropic link. This mechanism allows for high-affinity binding with an otherwise relaxed sequence specificity, and instead suggests a yet undefined structural recognition signature in the RNA for modulating gene transcription. This work provides a basis for an essential event for understanding transcription antitermination in pneumoviruses and its counterpart Ebola virus VP30.


Assuntos
Proteínas de Transporte/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Ebolavirus/metabolismo , Regulação Viral da Expressão Gênica , Genes Virais , Cinética , Metapneumovirus/genética , Metapneumovirus/metabolismo , Modelos Moleculares , Conformação Proteica , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/metabolismo , Transcrição Genética , Proteínas Virais/genética
8.
PLoS Pathog ; 14(2): e1006837, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29470533

RESUMO

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Epitopos/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Ligação Competitiva , Reações Cruzadas , Mapeamento de Epitopos , Humanos , Cinética , Metapneumovirus/imunologia , Metapneumovirus/metabolismo , Microscopia Eletrônica , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/antagonistas & inibidores , Proteínas Virais de Fusão/genética
9.
PLoS Pathog ; 13(12): e1006803, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29281742

RESUMO

The large polymerase subunit (L) of non-segmented negative strand RNA viruses transcribes viral mRNAs and replicates the viral genome. Studies with VSV have shown that conserved region V (CRV) of the L protein is part of the capping domain. However, CRV folds over and protrudes into the polymerization domain, suggesting that it might also have a role in RNA synthesis. In this study, the role of respiratory syncytial virus (RSV) CRV was evaluated using single amino acid substitutions and a small molecule inhibitor called BI-D. Effects were analyzed using cell-based minigenome and in vitro biochemical assays. Several amino acid substitutions inhibited production of capped, full-length mRNA and instead resulted in accumulation of short transcripts of approximately 40 nucleotides in length, confirming that RSV CRV has a role in capping. In addition, all six variants tested were either partially or completely defective in RNA replication. This was due to an inability of the polymerase to efficiently elongate the RNA within the promoter region. BI-D also inhibited transcription and replication. In this case, polymerase elongation activity within the promoter region was enhanced, such that the small RNA transcribed from the promoter was not released and instead was elongated past the first gene start signal. This was accompanied by a decrease in mRNA initiation at the first gene start signal and accumulation of aberrant RNAs of varying length. Thus, in addition to its function in mRNA capping, conserved region V modulates the elongation properties of the polymerase to enable productive transcription and replication to occur.


Assuntos
RNA Replicase/genética , RNA Replicase/metabolismo , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Antivirais/farmacologia , Linhagem Celular , Sequência Conservada , Descoberta de Drogas , Genes Virais , Humanos , Modelos Moleculares , Regiões Promotoras Genéticas , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Replicase/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/patogenicidade , Elongação da Transcrição Genética , Proteínas Virais/química
10.
PLoS One ; 12(10): e0184629, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29028839

RESUMO

Human Respiratory Syncytial Virus (HRSV) is a leading cause of bronchopneumonia in infants and the elderly. To date, knowledge of viral and host protein interactions within HRSV is limited and are critical areas of research. Here, we show that HRSV Matrix (M) protein interacts with the cellular adaptor protein complex 3 specifically via its medium subunit (AP-3Mu3A). This novel protein-protein interaction was first detected via yeast-two hybrid screen and was further confirmed in a mammalian system by immunofluorescence colocalization and co-immunoprecipitation. This novel interaction is further substantiated by the presence of a known tyrosine-based adaptor protein MU subunit sorting signal sequence, YXXФ: where Ф is a bulky hydrophobic residue, which is conserved across the related RSV M proteins. Analysis of point-mutated HRSV M derivatives indicated that AP-3Mu3A- mediated trafficking is contingent on the presence of the tyrosine residue within the YXXL sorting sequence at amino acids 197-200 of the M protein. AP-3Mu3A is up regulated at 24 hours post-infection in infected cells versus mock-infected HEp2 cells. Together, our data suggests that the AP-3 complex plays a critical role in the trafficking of HRSV proteins specifically matrix in epithelial cells. The results of this study add new insights and targets that may lead to the development of potential antivirals and attenuating mutations suitable for candidate vaccines in the future.


Assuntos
Complexo 3 de Proteínas Adaptadoras/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas da Matriz Viral/metabolismo , Complexo 3 de Proteínas Adaptadoras/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Células HeLa , Humanos , Ligação Proteica , Estabilidade Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transporte Proteico , Vírus Sincicial Respiratório Humano/fisiologia , Regulação para Cima , Proteínas da Matriz Viral/química , Montagem de Vírus
11.
Nat Commun ; 8(1): 563, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28916773

RESUMO

Infection of cells by respiratory syncytial virus induces the formation of cytoplasmic inclusion bodies (IBs) where all the components of the viral RNA polymerase complex are concentrated. However, the exact organization and function of these IBs remain unclear. In this study, we use conventional and super-resolution imaging to dissect the internal structure of IBs. We observe that newly synthetized viral mRNA and the viral transcription anti-terminator M2-1 concentrate in IB sub-compartments, which we term "IB-associated granules" (IBAGs). In contrast, viral genomic RNA, the nucleoprotein, the L polymerase and its cofactor P are excluded from IBAGs. Live imaging reveals that IBAGs are highly dynamic structures. Our data show that IBs are the main site of viral RNA synthesis. They further suggest that shortly after synthesis in IBs, viral mRNAs and M2-1 transiently concentrate in IBAGs before reaching the cytosol and suggest a novel post-transcriptional function for M2-1.Respiratory syncytial virus (RSV) induces formation of inclusion bodies (IBs) sheltering viral RNA synthesis. Here, Rincheval et al. identify highly dynamic IB-associated granules (IBAGs) that accumulate newly synthetized viral mRNA and the viral M2-1 protein but exclude viral genomic RNA and RNA polymerase complexes.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Corpos de Inclusão/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Nucleoproteínas/metabolismo
12.
Nat Commun ; 8(1): 167, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28761099

RESUMO

Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure-activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection.Respiratory syncytial virus causes lung infections in children, immunocompromised adults, and in the elderly. Here the authors show that a chemical inhibitor to a viral fusion protein is effective in reducing viral titre and ameliorating infection in rodents and neonatal lambs.


Assuntos
Imidazolidinas/metabolismo , Indóis/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Inibidores de Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular Tumoral , Chlorocebus aethiops , Células Epiteliais , Humanos , Imidazolidinas/farmacologia , Imidazolidinas/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Estrutura Molecular , Pneumonia Viral/tratamento farmacológico , Ratos , Mucosa Respiratória/citologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sinciciais Respiratórios/metabolismo , Ovinos , Relação Estrutura-Atividade , Células Vero , Inibidores de Proteínas Virais de Fusão/farmacologia , Inibidores de Proteínas Virais de Fusão/uso terapêutico
13.
Infect Genet Evol ; 52: 1-9, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28414106

RESUMO

In this study, we examined the molecular evolution of the fusion protein (F) gene in human respiratory syncytial virus subgroup B (HRSV-B). First, we performed time-scale evolution analyses using the Bayesian Markov chain Monte Carlo (MCMC) method. Next, we performed genetic distance, linear B-cell epitope prediction, N-glycosylation, positive/negative selection site, and Bayesian skyline plot analyses. We also constructed a structural model of the F protein and mapped the amino acid substitutions and the predicted B-cell epitopes. The MCMC-constructed phylogenetic tree indicated that the HRSV F gene diverged from the bovine respiratory syncytial virus gene approximately 580years ago and had a relatively low evolutionary rate (7.14×10-4substitutions/site/year). Furthermore, a common ancestor of HRSV-A and -B diverged approximately 290years ago, while HRSV-B diverged into three clusters for approximately 60years. The genetic similarity of the present strains was very high. Although a maximum of 11 amino acid substitutions were observed in the structural model of the F protein, only one strain possessed an amino acid substitution located within the palivizumab epitope. Four epitopes were predicted, although these did not correspond to the neutralization sites of the F protein including the palivizumab epitope. In addition, five N-glycosylation sites of the present HRSV-B strains were inferred. No positive selection sites were identified; however, many sites were found to be under negative selection. The effective population size of the gene has remained almost constant. On the basis of these results, it can be concluded that the HRSV-B F gene is highly conserved, as is the F protein of HRSV-A. Moreover, our prediction of B-cell epitopes does not show that the palivizumab reaction site may be recognized as an epitope during naturally occurring infections.


Assuntos
Vírus Sincicial Respiratório Humano/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Substituição de Aminoácidos , Teorema de Bayes , Epitopos de Linfócito B/metabolismo , Evolução Molecular , Glicosilação , Humanos , Cadeias de Markov , Modelos Moleculares , Filogenia , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/genética , Proteínas do Envelope Viral/metabolismo
14.
J Med Chem ; 60(6): 2305-2325, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28245119

RESUMO

Respiratory syncytial virus (RSV) represents a threat to infants, the elderly, and the immunocompromised. RSV entry blockers are in clinical trials, but escape mutations challenge their potential. In search of RSV inhibitors, we have integrated a signature resistance mutation into a recombinant RSV virus and applied the strain to high-throughput screening. Counterscreening of candidates returned 14 confirmed hits with activities in the nano- to low-micromolar range. All blocked RSV polymerase activity in minigenome assays. Compound 1a (GRP-74915) was selected for development based on activity (EC50 = 0.21 µM, selectivity index (SI) 40) and scaffold. Resynthesis confirmed the potency of the compound, which suppressed viral RNA synthesis in infected cells. However, metabolic testing revealed a short half-life in the presence of mouse hepatocyte fractions. Metabolite tracking and chemical elaboration combined with 3D-quantitative structure-activity relationship modeling yielded analogues (i.e., 8n: EC50 = 0.06 µM, SI 500) that establish a platform for the development of a therapeutic candidate.


Assuntos
Antivirais/química , Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Desenho de Fármacos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/enzimologia , Animais , Antivirais/metabolismo , Linhagem Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Relação Quantitativa Estrutura-Atividade , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo
15.
Virology ; 504: 63-72, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28157546

RESUMO

Whether respiratory syncytial virus (RSV) induces severe infantile pulmonary disease may depend on viral strain and expression of types I and III interferons (IFNs). These IFNs impact disease severity by inducing expression of many anti-viral IFN-stimulated genes (ISGs). To investigate the impact of RSV strain on IFN and ISG expression, we stimulated human monocyte-derived DCs (MDDCs) with either RSV A2 or Line 19 and measured expression of types I and III IFNs and ISGs. At 24h, A2 elicited higher ISG expression than Line 19. Both strains induced MDDCs to express genes for IFN-ß, IFN-α1, IFN-α8, and IFN-λ1-3, but only A2 induced IFN-α2, -α14 and -α21. We then show that IFN-α8 and IFN-α14 most potently induced MDDCs and bronchial epithelial cells (BECs) to express ISGs. Our findings demonstrate that RSV strain may impact patterns of types I and III IFN expression and the magnitude of the ISG response by DCs and BECs.


Assuntos
Células Dendríticas/imunologia , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Adulto , Brônquios/citologia , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/virologia , Células Epiteliais/citologia , Humanos , Inflamação/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/metabolismo
16.
Sci Rep ; 7: 42794, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28220811

RESUMO

The identification of the infectious agents is pivotal for appropriate care of patients with viral diseases. Current viral diagnostics rely on selective detection of viral nucleic acid or protein components. In general, detection of proteins rather than nucleic acids is technically more suitable for rapid tests. However, protein-based virus identification methods depend on antibodies limiting the practical applicability of these approaches. Aptamers rival antibodies in target selectivity and binding affinity, and excel in terms of robustness and cost of synthesis. Although aptamers have been generated for virus identification in laboratory settings, their introduction into routine virus diagnostics has not been realized, yet. Here, we demonstrate that the rationally designed SELEX protocol can be applied on whole virus to select aptamers, which can potentially be applied for viral diagnostics. This approach does not require purified virus protein or complicated virus purification. The presented data also illustrate that corroborating the functionality of aptamers with various approaches is essential to pinpoint the most appropriate aptamer amongst the panel of candidates obtained by the selection. Our protocol yielded aptamers capable of detecting respiratory syncytial virus (RSV), an important pathogen causing severe disease especially in young infants, at clinically relevant concentrations in complex matrices.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Vírus Sincicial Respiratório Humano/isolamento & purificação , Aptâmeros de Nucleotídeos/química , Polarização de Fluorescência , Humanos , Faringe/virologia , Ligação Proteica , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/metabolismo , Técnica de Seleção de Aptâmeros , Proteínas Virais/química , Proteínas Virais/metabolismo
17.
J Biol Chem ; 292(6): 2120-2131, 2017 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28031463

RESUMO

Phosphoprotein is the main cofactor of the viral RNA polymerase of Mononegavirales It is involved in multiple interactions that are essential for the polymerase function. Most prominently it positions the polymerase complex onto the nucleocapsid, but also acts as a chaperone for the nucleoprotein. Mononegavirales phosphoproteins lack sequence conservation, but contain all large disordered regions. We show here that N- and C-terminal intrinsically disordered regions account for 80% of the phosphoprotein of the respiratory syncytial virus. But these regions display marked dynamic heterogeneity. Whereas almost stable helices are formed C terminally to the oligomerization domain, extremely transient helices are present in the N-terminal region. They all mediate internal long-range contacts in this non-globular protein. Transient secondary elements together with fully disordered regions also provide protein binding sites recognized by the respiratory syncytial virus nucleoprotein and compatible with weak interactions required for the processivity of the polymerase.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Fosfoproteínas/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Sequência de Aminoácidos , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas Intrinsicamente Desordenadas/química , Ressonância Magnética Nuclear Biomolecular , Fosfoproteínas/química , Ligação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos
18.
Artigo em Inglês | MEDLINE | ID: mdl-28018859

RESUMO

Human respiratory syncytial virus (RSV) is a lung tropic virus causing severe airway diseases including bronchiolitis and pneumonia among infants, children, and immuno-compromised individuals. RSV triggers transforming growth factor-ß (TGF-ß) production from lung epithelial cells and TGF-ß facilitates RSV infection of these cells. However, it is still unknown whether RSV infected myeloid cells like macrophages produce TGF-ß and the role of TGF-ß if any during RSV infection of these cells. Our study revealed that RSV infected macrophages produce TGF-ß and as a consequence these cells activate TGF-ß dependent SMAD-2/3 signaling pathway. Further mechanistic studies illustrated a role of autophagy in triggering TGF-ß production from RSV infected macrophages. In an effort to elucidate the role of TGF-ß and SMAD-2/3 signaling during RSV infection, we surprisingly unfolded the requirement of TGF-ß-SMAD2/3 signaling in conferring optimal innate immune antiviral response during RSV infection of macrophages. Type-I interferon (e.g., interferon-ß or IFN-ß) is a critical host factor regulating innate immune antiviral response during RSV infection. Our study revealed that loss of TGF-ß-SMAD2/3 signaling pathway in RSV infected macrophages led to diminished expression and production of IFN-ß. Inhibiting autophagy in RSV infected macrophages also resulted in reduced production of IFN-ß. Thus, our studies have unfolded the requirement of autophagy-TGF-ß-SMAD2/3 signaling network for optimal innate immune antiviral response during RSV infection of macrophages.


Assuntos
Interferon beta/imunologia , Macrófagos/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Antivirais , Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Modelos Animais de Doenças , Imunidade Inata , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Interferon beta/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/metabolismo , Transdução de Sinais
19.
J Mol Model ; 22(12): 286, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27817112

RESUMO

The human respiratory syncytial virus (hRSV) is the major cause of lower respiratory tract infection in children and elderly people worldwide. Its genome encodes 11 proteins including SH protein, whose functions are not well known. Studies show that SH protein increases RSV virulence degree and permeability to small compounds, suggesting it is involved in the formation of ion channels. The knowledge of SH structure and function is fundamental for a better understanding of its infection mechanism. The aim of this study was to model, characterize, and analyze the structural behavior of SH protein in the phospholipids bilayer environment. Molecular modeling of SH pentameric structure was performed, followed by traditional molecular dynamics (MD) simulations of the protein immersed in the lipid bilayer. Molecular dynamics with excited normal modes (MDeNM) was applied in the resulting system in order to investigate long time scale pore dynamics. MD simulations support that SH protein is stable in its pentameric form. Simulations also showed the presence of water molecules within the bilayer by density distribution, thus confirming that SH protein is a viroporin. This water transport was also observed in MDeNM studies with histidine residues of five chains (His22 and His51), playing a key role in pore permeability. The combination of traditional MD and MDeNM was a very efficient protocol to investigate functional conformational changes of transmembrane proteins that act as molecular channels. This protocol can support future investigations of drug candidates by acting on SH protein to inhibit viral infection. Graphical Abstract The ion channel of the human respiratory syncytial virus (hRSV) small hydrophobic protein (SH) transmembrane domainᅟ.


Assuntos
Canais Iônicos/química , Simulação de Dinâmica Molecular , Domínios e Motivos de Interação entre Proteínas , Proteínas Virais/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Canais Iônicos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Conformação Proteica , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/metabolismo
20.
PLoS One ; 11(10): e0164789, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27764150

RESUMO

Infection with Respiratory Syncytial Virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the infection process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an engineered RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4°C or higher is a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site Ø, and a novel antibody 4D7, which was found to bind antigenic site I on the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4°C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly lose the site Ø epitope and gain the ability to bind 4D7.


Assuntos
Antígenos/imunologia , Vírus Sincicial Respiratório Humano/metabolismo , Vacinas de Subunidades/imunologia , Proteínas Virais de Fusão/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo/imunologia , Antígenos/metabolismo , Epitopos/imunologia , Células HEK293 , Humanos , Microscopia Eletrônica de Transmissão , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/imunologia , Ressonância de Plasmônio de Superfície , Vacinas de Subunidades/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo
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