RESUMEN
Mitochondria are highly dynamic organelles that undergo continuous cycles of fission and fusion to maintain essential cellular functions. An imbalance between these two processes can result in many pathophysiological outcomes. Dengue virus (DENV) interacts with cellular organelles, including mitochondria, to successfully replicate in cells. This study used live-cell imaging and found an increase in mitochondrial length and respiration during DENV infection. The level of mitochondrial fission protein, Dynamin-related protein 1 (Drp1), was decreased on mitochondria during DENV infection, as well as Drp1 phosphorylated on serine 616, which is important for mitochondrial fission. DENV proteins NS4b and NS3 were also associated with subcellular fractions of mitochondria. Induction of fission through uncoupling of mitochondria or overexpression of Drp1 wild-type and Drp1 with a phosphomimetic mutation (S616D) significantly reduced viral replication. These results demonstrate that DENV infection causes an imbalance in mitochondrial dynamics by inhibiting Drp1-triggered mitochondrial fission, which promotes viral replication.
Asunto(s)
Virus del Dengue/fisiología , Dengue/metabolismo , Dengue/fisiopatología , GTP Fosfohidrolasas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismo , Secuencias de Aminoácidos , Línea Celular , Dengue/virología , Virus del Dengue/química , Virus del Dengue/genética , Dinaminas , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/virología , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Cell culture models are used widely to study the effects of dengue virus (DENV) on host cell function. Current methods of identification of cells infected with an unmodified DENV requires fixation and permeablization of cells to allow DENV-specific antibody staining. This method does not permit imaging of viable cells over time. In this report, a plasmid-based reporter was developed to allow non-destructive identification of DENV-infected cells. The plasmid-based reporter was demonstrated to be broadly applicable to the four DENV serotypes, including low-passaged strains, and was specifically cleaved by the viral protease with minimal interference on viral production. This study reveals the potential for this novel reporter system to advance the studies of virus-host interactions during DENV infection.