Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Sci Rep ; 10(1): 2919, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076025

RESUMO

Oral rabies vaccination (ORV) is highly effective in foxes and raccoon dogs, whereas for unknown reasons the efficacy of ORV in other reservoir species is less pronounced. To investigate possible variations in species-specific cell tropism and local replication of vaccine virus, different reservoir species including foxes, raccoon dogs, raccoons, mongooses, dogs and skunks were orally immunised with a highly attenuated, high-titred GFP-expressing rabies virus (RABV). Immunofluorescence and RT-qPCR screenings revealed clear differences among species suggesting host specific limitations to ORV. While for responsive species the palatine tonsils (tonsilla palatina) were identified as a main site of virus replication, less virus dissemination was observed in the tonsils of rather refractory species. While our comparison of vaccine virus tropism emphasizes the important role that the tonsilla palatina plays in eliciting an immune response to ORV, our data also indicate that other lymphoid tissues may have a more important role than originally anticipated. Overall, these data support a model in which the susceptibility to oral live RABV vaccine infection of lymphatic tissue is a major determinant in vaccination efficacy. The present results may help to direct future research for improving vaccine uptake and efficacy of oral rabies vaccines under field conditions.


Assuntos
Reservatórios de Doenças/virologia , Tecido Linfoide/imunologia , Mucosa/imunologia , Vacina Antirrábica/imunologia , Raiva/imunologia , Vacinação , Administração Oral , Animais , Anticorpos Antivirais/imunologia , Raposas/imunologia , Raposas/virologia , Proteínas de Fluorescência Verde/metabolismo , Tecido Linfoide/virologia , Mucosa/virologia , Especificidade de Órgãos , Tonsila Palatina/imunologia , Tonsila Palatina/virologia , RNA Viral/genética , Raiva/sangue , Raiva/veterinária , Raiva/virologia , Vírus da Raiva/fisiologia , Especificidade da Espécie , Tropismo , Carga Viral , Replicação Viral/fisiologia
2.
Dev Cell ; 45(2): 262-275.e8, 2018 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-29689199

RESUMO

The complex architecture of neuronal networks in the brain requires tight control of the actin cytoskeleton. The actin nucleator Cobl is critical for neuronal morphogenesis. Here we reveal that Cobl is controlled by arginine methylation. Coprecipitations, coimmunoprecipitations, cellular reconstitutions, and in vitro reconstitutions demonstrated that Cobl associates with the protein arginine methyltransferase PRMT2 in a Src Homology 3 (SH3) domain-dependent manner and that this promotes methylation of Cobl's actin nucleating C-terminal domain. Consistently, PRMT2 phenocopied Cobl functions in both gain- and loss-of-function studies. Both PRMT2- and Cobl-promoted dendritogenesis relied on methylation. PRMT2 effects require both its catalytic domain and SH3 domain. Cobl-mediated dendritic arborization required PRMT2, complex formation with PRMT2, and PRMT2's catalytic activity. Mechanistic studies reveal that Cobl methylation is key for Cobl actin binding. Therefore, arginine methylation is a regulatory mechanism reaching beyond controlling nuclear processes. It also controls a major, cytosolic, cytoskeletal component shaping neuronal cells.


Assuntos
Citoesqueleto de Actina/metabolismo , Arginina/metabolismo , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurônios/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas/metabolismo , Animais , Células Cultivadas , Proteínas do Citoesqueleto , Feminino , Hipocampo/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos , Neurônios/citologia , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/genética , Proteínas/genética , Ratos , Ratos Wistar , Técnicas do Sistema de Duplo-Híbrido
3.
Vaccine ; 35(32): 3938-3944, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28641888

RESUMO

Oral vaccination using attenuated and recombinant rabies vaccines has been proven a powerful tool to combat rabies in wildlife. However, clear differences have been observed in vaccine titers needed to induce a protective immune response against rabies after oral vaccination in different reservoir species. The mechanisms contributing to the observed resistance against oral rabies vaccination in some species are not completely understood. Hence, the immunogenicity of the vaccine virus strain, SPBN GASGAS, was investigated in a species considered to be susceptible to oral rabies vaccination (red fox) and a species refractory to this route of administration (striped skunk). Additionally, the dissemination of the vaccine virus in the oral cavity was analyzed for these two species. It was shown that the palatine tonsils play a critical role in vaccine virus uptake. Main differences could be observed in palatine tonsil infection between both species, revealing a locally restricted dissemination of infected cells in foxes. The absence of virus infected cells in palatine tonsils of skunks suggests a less efficient uptake of or infection by vaccine virus which may lead to a reduced response to oral vaccination. Understanding the mechanisms of oral resistance to rabies virus vaccine absorption and primary replication may lead to the development of novel strategies to enhance vaccine efficacy in problematic species like the striped skunk.


Assuntos
Vacina Antirrábica/imunologia , Vacina Antirrábica/farmacocinética , Vírus da Raiva/imunologia , Raiva/veterinária , Administração Oral , Animais , Raposas , Mephitidae , Raiva/prevenção & controle , Vacina Antirrábica/administração & dosagem
4.
PLoS Biol ; 13(9): e1002233, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26334624

RESUMO

Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer-binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca2+ and multiple, direct associations of the Ca2+ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl's functions. Cobl-induced dendritic branch initiation was preceded by Ca2+ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca2+/CaM modulates Cobl's actin binding properties and furthermore promotes Cobl's previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca2+/CaM and reveal that the Ca2+/CaM-controlled molecular mechanisms we discovered are crucial for Cobl's cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca2+/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca2+ signals steer and power branch initiation during early arborization of nerve cells-a key process in neuronal network formation.


Assuntos
Citoesqueleto de Actina/metabolismo , Sinalização do Cálcio , Calmodulina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Plasticidade Neuronal , Actinas/metabolismo , Animais , Células COS , Proteínas de Transporte/metabolismo , Chlorocebus aethiops , Proteínas do Citoesqueleto , Células HEK293 , Humanos , Masculino , Camundongos , Ratos
5.
J Virol ; 89(18): 9591-600, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26157129

RESUMO

UNLABELLED: Rabies virus (RABV) polymerase L together with phosphoprotein P forms the PL polymerase complex that is essential for replication and transcription. However, its exact mechanism of action, interactions with cellular factors, and intracellular distribution are yet to be understood. Here by imaging a fluorescently tagged polymerase (mCherry-RABV-L), we show that L accumulates at acetylated and reorganized microtubules (MT). In silico analysis revealed a dynein light chain 1 (DLC1) binding motif in L that could mediate MT binding through dynein motors. As DLC1 binding by polymerase cofactor P is known, we compared the impact of the DLC1-binding motifs in P and L. Viruses with mutations in the respective motifs revealed that both motifs are required for efficient primary transcription, indicating that DLC1 acts as a transcription enhancer by binding to both P and L. Notably, also the levels of cellular DLC1 protein were regulated by both motifs, suggesting regulation of the DLC1 gene expression by both P and L. Finally, disruption of the motif in L resulted in a cell-type-specific loss of MT localization, demonstrating that DLC1 is involved in L-mediated cytoskeleton reorganization. Overall, we conclude that DLC1 acts as a transcription factor that stimulates primary RABV transcription by binding to both P and L. We further conclude that L influences MT organization and posttranslational modification, suggesting a model in which MT manipulation by L contributes to efficient intracellular transport of virus components and thus may serve as an important step in virus replication. IMPORTANCE: Regulation of rabies virus polymerase complex by viral and cellular factors thus far has not been fully understood. Although cellular dynein light chain 1 (DLC1) has been reported to increase primary transcription by binding to polymerase cofactor phosphoprotein P, the detailed mechanism is unknown, and it is also not known whether the large enzymatic polymerase subunit L is involved. By fluorescence microscopy analysis of fluorescence-tagged rabies virus L, in silico identification of a potential DLC1 binding site in L, and characterization of recombinant rabies virus mutants, we show that a DLC1 binding motif in L is involved in cytoskeleton localization and reorganization, primary transcription regulation by DLC1, and regulation of cellular DLC1 gene expression. By providing evidence for a direct contribution of a DLC1 binding motif in L, our data significantly increase the understanding of rabies virus polymerase regulation and host manipulation by the virus as well.


Assuntos
Dineínas do Citoplasma/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Raiva/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Motivos de Aminoácidos , Linhagem Celular Tumoral , Dineínas do Citoplasma/genética , RNA Polimerases Dirigidas por DNA/genética , Células HEK293 , Humanos , Fatores de Transcrição/genética , Proteínas Virais/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...