Development of a simple system for screening anti-hepatitis C virus drugs utilizing mutants capable of vigorous replication.
J Virol Methods
; 169(2): 380-4, 2010 Nov.
Article
em En
| MEDLINE
| ID: mdl-20713089
ABSTRACT
Replication of infectious hepatitis C virus in Huh7 cells, a human hepatocyte cell line, has become possible due to the unique properties of the JFH1 isolate. Developing reporter virus systems for a simple titration has been attempted by integrating heterologous reporter genes into the JFH1 genome, resulting in a big infectivity reduction that limits the usefulness of such reporter systems. To overcome this problem, JFH1-infected Huh7 cells were cultured continuously for 2 years to obtain Huh7-adapted JFH1 variants capable of yielding up to 1000-fold higher titers. Sequence analysis of variant genome RNA suggested that this adapted population consisted mainly of two variants. By joining the 5'-half of the obtained representative viral complementary DNA (cDNA) fragments of the variants with the 3'-half of the wild-type's, two prototype clones, A/WT and B/WT, were constructed. Replication of A/WT and B/WT viruses in Huh7 cells showed up to 100-1000-fold higher titers than the wild-type. A Renilla luciferase cDNA was inserted into the Nonstructural Protein 5A region of the A/WT and B/WT cDNA to generate A/WT-Rluc and B/WT-Rluc, respectively. Transfection of Huh7 cells with in vitro-transcribed A/WT-Rluc and B/WT-Rluc RNA resulted in production of infectious viruses with approximately 15- and 25-fold higher titers, respectively, than the wild-type RNA. The replication of A/WT-Rluc and B/WT-Rluc viruses was more vigorous than the wild-type even with insertion of the luciferase cDNA showing a good correlation of luciferase activities with infectious titers. Furthermore, interferon-alpha inhibited the replication of A/WT-Rluc and B/WT-Rluc viruses in a dose-dependent manner as determined by a luciferase assay. These results imply that our system is potentially a tool useful for screening anti-hepatitis C virus drugs in a simple and time/cost-saving manner.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Antivirais
/
Replicação Viral
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Hepacivirus
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Avaliação Pré-Clínica de Medicamentos
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Revista:
J Virol Methods
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
China