RESUMEN
BACKGROUND: The development of small molecule inhibitors of hepatitis C virus (HCV) core protein as antiviral agents has been intensively pursued as a viable strategy to eradicate HCV infection. However, lack of a robust and convenient small animal model has hampered the assessment of in vivo efficacy of any antiviral compound. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this work was to develop a novel method to screen anti-core protein siRNA in the mouse liver by bioluminescence imaging. The inhibitory effect of two shRNAs targeting the highly conserved core region of the HCV genome, shRNA452 and shRNA523, was examined using this method. In the transient mouse model, the effect of shRNA-523 was detectable at as early as 24 h and became even more pronounced at later time points. The effect of shRNA-452 was not detectable until 48 h post-transduction. In a stable mouse model, shRNA523 reduced luciferase levels by up to 76.4±26.0% and 91.8±8.0% at 6 h and 12 h after injection respectively, and the inhibitory effect persisted for 1 day after a single injection while shRNA-Scramble did not seem to have an effect on the luciferase activity in vivo. CONCLUSIONS/SIGNIFICANCE: Thus, we developed a simple and quantitative assay for real-time monitoring of HCV core protein inhibitors in mice.
Asunto(s)
Mediciones Luminiscentes/métodos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas del Núcleo Viral/metabolismo , Alanina Transaminasa/sangre , Animales , Western Blotting , Línea Celular Tumoral , Vectores Genéticos/genética , Humanos , Interleucina-1beta/sangre , Interleucina-6/sangre , Hígado/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño/genética , Reproducibilidad de los Resultados , Factores de Tiempo , Transfección/métodos , Proteínas del Núcleo Viral/genéticaRESUMEN
AIM: To develop a sensitive assay for screening compounds against hepatitis C virus (HCV). METHODS: The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP), which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence. Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy. Cellular localization and protein levels were examined by Western blotting. RESULTS: HCV NS3/4A protease cleaved eYFP-MAVS from mitochondria to block the activation of interferon (IFN)-ß promoter, thus resulting in downregulation of SEAP activity. The decrease in SEAP activity was proportional to the dose of active NS3/4A protease. Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A. CONCLUSION: Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors. This system will constitute a new tool to allow the efficient screening of HCV inhibitors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antivirales/farmacología , Técnicas Biosensibles , Hepacivirus/efectos de los fármacos , Interferón beta/genética , Transducción de Señal/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Western Blotting , Línea Celular Tumoral , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Genes Reporteros , Hepacivirus/enzimología , Hepacivirus/genética , Humanos , Interferón-alfa/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Replicón , Factores de Tiempo , Activación Transcripcional , Transfección , Proteínas no Estructurales Virales/genéticaRESUMEN
By bioluminescence imaging and hydrodynamic gene transfer technology, the activities of hepatitis B virus (HBV) promoters and the effects of HBV enhancers on these promoters in mice under true physiological conditions have been assessed. Our studies reveal that either of the two HBV enhancers can stimulate HBV major promoter activity in hepa 1-6 cells (in vitro) and in mouse liver (in vivo), and the enhancer effects on the three promoters (S1, S2 and X promoter) are markedly greater in vivo than in vitro. The two HBV enhancers have no cooperative action on HBV promoters in vitro or in vivo.