RESUMEN
Stem-loop SL2 is a self-interacting palindromic sequence that has been identified within the hepatitis C virus genome (HCV). While, RNA dimerization of the HCV genome has been observed in vitro with short RNA sequences, the role of a putative RNA dimerization during viral replication has not been elucidated. To determine the effect of genomic dimerization on viral replication, we introduced mutations into SL2 predicted to disrupt genomic dimerization. Using surface plasmon resonance, we show that mutations within the SL2 bulge impact dimerization in vitro. Transfection of Huh7 cells with luciferase-encoding full-length genomes containing SL2 mutations abolishes viral replication. Luciferase expression indicates that viral translation is not or slightly affected and that the viral RNA is properly encapsidated. However, RT-qPCR analysis demonstrates that viral RNA synthesis is drastically decreased. In vitro synthesis experiments using the viral recombinant polymerase show that modifications of intra-molecular interactions have no effect on RNA synthesis, while impairing inter-molecular interactions decreases polymerase activity. This confirms that dimeric templates are preferentially replicated by the viral polymerase. Altogether, these results indicate that the dimerization of the HCV genomic RNA is a crucial step for the viral life cycle especially for RNA replication. RNA dimerization could explain the existence of HCV recombinants in cell culture and patients reported recently in other studies.
Asunto(s)
Genoma Viral/genética , Hepacivirus/genética , Secuencias Invertidas Repetidas/genética , Mutación/genética , Replicación Viral/genética , Emparejamiento Base , Línea Celular , Cartilla de ADN/genética , Dimerización , Vectores Genéticos/genética , Humanos , Luciferasas , Oligonucleótidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resonancia por Plasmón de Superficie , Replicación Viral/fisiologíaAsunto(s)
Discapacidades del Desarrollo/genética , Ataxias Espinocerebelosas/genética , Degeneraciones Espinocerebelosas/genética , Femenino , Factores de Crecimiento de Fibroblastos/genética , Eliminación de Gen , Humanos , Discapacidad Intelectual/etiología , Discapacidad Intelectual/genética , Imagen por Resonancia Magnética , Microcefalia/etiología , Microcefalia/genética , Mutación Puntual/genética , Degeneraciones Espinocerebelosas/complicaciones , Degeneraciones Espinocerebelosas/fisiopatología , Adulto JovenRESUMEN
The phase inversion of water-toluene emulsions stabilized with a single thermo- and pH-sensitive copolymer occurs through the formation of multiple emulsions. At low pH and ambient temperature, oil in water emulsions are formed which transform into highly stable multiple emulsions at pHs immediately lower than the inversion border. At higher pHs, the emulsion turns into a water in oil one.
