Development of a cell culture system with consistently expression of whole hepatitis C virus gene and Renilla luciferase and its application / 中华传染病杂志

ABSTRACT

ObjectiveTo develop a cell culture system with consistent expression of whole hepatitis C virus (HCV) gene and Renilla luciferase gene and to facilitate the study on HCV pathogenesis and the screening of new antiviral drugs.MethodsRenilla luciferase (RLuc) reporter gene and a mutation that could yield higher virus gene expression were introduced into the C-terminus of non-structural protein 5A (NS5A) of the JFH1 viral genome by using recombinant PCR.The viral RNA was transfected into Huh7.5 cells.Naǐve Huh7.5 cells were infected by the supernatant from the viral RNA transfected cells.HCV replication and infection were determined by virus titration,Renilla luciferase assay,immunofluorescence assay and western blotting.IFN-α was used to evaluate the feasibility of this system for anti-HCV new drug screening.ResultsThe viral RNA replicated efficiently in transfected cells.These cells could produce high titer of HCV-Rluc reporter virus and the virus titer reached to 1.5 × 104 FFU/ml at day 15 of posttransfection.The activity of Renilla luciferase was inhibited by IFN-α in a dose dependent manner in Huh7.5 cells infected by HCV-Rluc reporter virus.ConclusionThe recombinant HCV-JFH1-Rluc reporter gene system is sensitive and efficient.It can be a useful tool for high throughput screening of anti-HCV drugs.