Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Sci Rep ; 10(1): 18101, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33093566

RESUMO

HIV encodes an aspartyl protease that is activated during, or shortly after, budding of viral particles from the surface of infected cells. Protease-mediated cleavage of viral polyproteins is essential to generating infectious viruses, a process known as 'maturation' that is the target of FDA-approved antiretroviral drugs. Most assays to monitor protease activity rely on bulk analysis of millions of viruses and obscure potential heterogeneity of protease activation within individual particles. In this study we used nanoscale flow cytometry in conjunction with an engineered FRET reporter called VIral ProteasE Reporter (VIPER) to investigate heterogeneity of protease activation in individual, patient-derived viruses. We demonstrate previously unappreciated interpatient variation in HIV protease processing efficiency that impacts viral infectivity. Additionally, monitoring of protease activity in individual virions distinguishes between drug sensitivity or resistance to protease inhibitors in patient-derived samples. These findings demonstrate the feasibility of monitoring enzymatic processes using nanoscale flow cytometry and highlight the potential of this technology for translational clinical discovery, not only for viruses but also other submicron particles including exosomes, microvesicles, and bacteria.


Assuntos
Farmacorresistência Viral , Citometria de Fluxo/métodos , Infecções por HIV/virologia , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , HIV-1/enzimologia , Vírion/enzimologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , HIV-1/efeitos dos fármacos , HIV-1/isolamento & purificação , Humanos , Células Jurkat , Vírion/efeitos dos fármacos , Vírion/isolamento & purificação
2.
Commun Biol ; 3(1): 452, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814813

RESUMO

Bedaquiline (BDQ, Sirturo) has been approved to treat multidrug resistant forms of Mycobacterium tuberculosis. Prior studies suggested that BDQ was a selective inhibitor of the ATP synthase from M. tuberculosis. However, Sirturo treatment leads to an increased risk of cardiac arrhythmias and death, raising the concern that this adverse effect results from inhibition at a secondary site. Here we show that BDQ is a potent inhibitor of the yeast and human mitochondrial ATP synthases. Single-particle cryo-EM reveals that the site of BDQ inhibition partially overlaps with that of the inhibitor oligomycin. Molecular dynamics simulations indicate that the binding mode of BDQ to this site is similar to that previously seen for a mycobacterial enzyme, explaining the observed lack of selectivity. We propose that derivatives of BDQ ought to be made to increase its specificity toward the mycobacterial enzyme and thereby reduce the side effects for patients that are treated with Sirturo.


Assuntos
Diarilquinolinas/farmacologia , Inibidores Enzimáticos/farmacologia , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Sítios de Ligação , Microscopia Crioeletrônica , Diarilquinolinas/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Proteínas Fúngicas , Humanos , ATPases Mitocondriais Próton-Translocadoras/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Reprodutibilidade dos Testes , Relação Estrutura-Atividade
3.
Vaccine ; 36(16): 2147-2154, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29550194

RESUMO

Plant-made virus-like particle (VLP) vaccines that display wild-type influenza hemagglutinin (HA) are rapidly advancing through clinical trials. Produced by transient transfection of Nicotiana benthamiana, these novel vaccines are unusually immunogenic, eliciting both humoral and cellular responses. Here, we directly visualized VLPs bearing either HA trimers derived from strains A/California/7/2009 or A/Indonesia/5/05 using cryo-electron microscopy and determined the 3D organization of the VLPs using cryo-electron tomography. More than 99.9% of the HA trimers in the vaccine preparations were found on discoid and ovoid-shaped particles. The discoid-shaped VLPs presented HA trimers on their outer diameter. The ovoid-shaped VLPs contained HA trimers evenly distributed at their surface. The VLPs were stable for 12 months at 4 °C. Early interactions of the VLPs with mouse dendritic and human monocytoid (U-937) cells were visualized by electron microscopy after resin-embedding and sectioning. The VLP particles were observed bound to plasma membranes as well as inside vesicles. Mouse dendritic cells exposed to VLPs displayed classic morphological changes associated with activation including the extensive formation of dendrites. Our findings demonstrate that plant-made VLPs bearing influenza HA trimers are morphologically stable over time and raise the possibility that these VLPs may interact with and activate antigen-presenting cells in a manner similar to the intact virus.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/ultraestrutura , Antígenos Virais/imunologia , Linhagem Celular , Microscopia Crioeletrônica , Células Dendríticas/imunologia , Células Dendríticas/ultraestrutura , Glicoproteínas de Hemaglutininação de Vírus da Influenza/administração & dosagem , Humanos , Imunização , Vacinas contra Influenza/administração & dosagem , Influenza Humana/prevenção & controle , Camundongos , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/ultraestrutura
4.
Virology ; 505: 80-90, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28235684

RESUMO

Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release.


Assuntos
Citometria de Fluxo/métodos , HIV-1/isolamento & purificação , Vírion/isolamento & purificação , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Vesículas Extracelulares/fisiologia , Fluorescência , Humanos , Limite de Detecção
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...