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La pérdida de función de la quinasa dependiente de ciclina 5 (CDK5) altera el citoesqueleto y reduce la infección in vitro por el virus del dengue 2
The loss of function of Cyclin-Dependent Kinase 5 (CDK5) alters the Cytoskeleton and decrease the in vitro Dengue Virus-2 infection
DOI:
https://doi.org/10.15446/abc.v24n3.79347Palabras clave:
Filamentos de actina, microtúbulos, quinasas dependiente de ciclina, roscovitina, silenciamiento génico, virus dengue (es)Cyclin-dependent kinase 5, cytoskeleton, dengue virus, gene silencing, roscovitine (en)
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La quinasa dependiente de ciclina 5 (CDK5) regula diversas funciones en neuronas, células endoteliales y epiteliales, entre ellas la dinámica del citoesqueleto. Así mismo, se ha reportado que componentes del citoesqueleto, tales como, filamentos de actina y microtúbulos juegan un rol importante durante la infección por el virus dengue (DENV). El objetivo del presente trabajo fue evaluar por dos métodos, inhibición química y silenciamiento génico, la participación de CDK5 durante la infección por DENV-2. La actividad antiviral de roscovitina fue evaluada usando ensayos de Unidades Formadoras de Placa (PFU). La eficiencia de transfección y el silenciamiento de CDK5, empleando miARNs artificiales, se determinó por citometría de flujo. El efecto sobre la proteína de envoltura viral y elementos del citoesqueleto se evidenció mediante microscopia avanzada de fluorescencia y análisis de imágenes. Roscovitina mostró actividad antiviral en etapas pre y post-infectivas en una forma dependiente de la dosis. El tratamiento con roscovitina y miRCDK5 mostró ser efectivo reduciendo la cantidad de CDK5 en células no infectadas. En células infectadas y transfectadas con miRCDK5, así como tratadas con el inhibidor, se observó una reducción significativa de la proteína de envoltura viral; sin embargo, no se encontró reducción significativa de CDK5. Además, el tratamiento con roscovitina indujo cambios celulares morfológicos evidentes en células infectadas. Los resultados indican la potencial participación de CDK5 durante la infección por DENV-2, posiblemente mediando la traducción proteica o la replicación del genoma viral a través de la regulación de la dinámica del citoesqueleto. Se requieren datos adicionales para esclarecer la mecanística del fenómeno usando métodos alternativos.
Cyclin-Dependent Kinase 5 (CDK5) regulates several functions in neurons, endothelial, and epithelial cells, including the cytoskeleton dynamics. Likewise, it has been reported that some cytoskeleton elements, such as actin filaments and microtubules, play an essential role during Dengue virus (DENV) infection. This work aimed to evaluate the role of CDK5 during DENV-2 infection by two methods, chemical inhibition, and gene silencing. The antiviral activity of roscovitine was evaluated using Plaque Forming Units (PFU) assay. The transfection efficiency and knockdown of CDK5, using artificial miRNAs, was carried out by flow cytometry. The effect on the viral envelope protein and cytoskeleton elements was evidenced by advanced fluorescence microscopy and image analysis. Roscovitine showed antiviral activity in pre and post-infection stages in a dose-dependent manner. Treatment with roscovitine and miRCDK5 decrease the amount of CDK5 in uninfected cells. In cells infected and transfected with miRCDK5, as well as treated with the inhibitor, a significant reduction of the viral envelope protein was observed; however, no significant reduction of CDK5 was found. Also, evident morphological cellular changes were observed during the treatment with roscovitine in infected cells. The results indicate the potential participation of CDK5 during DENV-2 infection, possibly mediating protein translation or replication of the viral genome through the cytoskeletal dynamics regulation. Additional data are required to clarify the mechanistic of these phenomena using alternative methods.
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1. Aimer Gutierrez-Diaz, Steve Hoffmann, Juan Carlos Gallego-Gómez, Clara Isabel Bermudez-Santana. (2024). Systematic computational hunting for small RNAs derived from ncRNAs during dengue virus infection in endothelial HMEC-1 cells. Frontiers in Bioinformatics, 4 https://doi.org/10.3389/fbinf.2024.1293412.
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