RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antivirais/farmacologia , Técnicas Biossensoriais , Hepacivirus/efeitos dos fármacos , Interferon beta/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Linhagem Celular Tumoral , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Genes Reporter , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Interferon-alfa/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Replicon , Fatores de Tempo , Ativação Transcricional , Transfecção , Proteínas não Estruturais Virais/genéticaRESUMO
AIM: To express stably human CD81 gene on mouse hepatoma cell line Hepa 1-6 using liver specific promoter. METHODS: RNA were isolated from human HepG2 cells which could be infected with hepatitis C virus. RT-PCR was carried out using human CD81 gene specific primers. Amplified fragments were cloned into pGEM-T vector. Albumin promoter and enhancer which were liver tissue specific were ligated to the 5'end of human CD81 gene and SV40 polyA sequence was fused with 3'end of CD81. The fused CD81 gene was inserted into eukaryotic expression vector pcDNA3 to construct a recombinant vector pcDNA3-Alb p-CD81 which was then transfected into Hepa 1-6 cells through lipofectamine mediation. Human CD81 mRNA transcription and its protein expression were detected by RT-PCR and FACS, respectively. RESULTS: Sequence analysis showed that the cloned gene segment was human CD81 gene sequence. After transfection, transcripted human CD81 mRNA was obtained and human CD81 molecules were expressed stably on Hepa 1-6 cells. CONCLUSION: The obtained positive cell clones which stably express HCV receptor human CD81 lay the foundation for further study on interactions between HCV envelope proteins and human CD81, screening of HCV infection blocking drugs and development of HCV infection mouse model.