Figure 1. LAP blocks replication of JFH1 HCV in tissue culture.

(A) Schematic representation of wt JFH1 and wt and pol-null luciferase reporter (NRLFC) constructs. (B–K) Huh-7.5.1 cells were pretreated with buffer (panels B and C), various concentrations of wt LAP (panels D–G), or Y23Q mutant LAP (panels H–K) as described in materials and methods. Excess LAP was then removed, and cells were washed with MEM, followed by infection of cells with JFH1 virus (moi=1). Infection was stopped at 72h post-infection, and one half of the cells were stained with DAPI, and the other half was scored for NS5A immunofluorescence using anti-NS5A antibody. (L)10 μg of in vitro transcribed genomic RNA of wild-type NRLFC reporter virus was introduced into Huh-7.5.1 cells (by electroporation) that were pretreated with indicated concentrations of wt LAP, or Y23Q mutant or the non-specific peptide (NSP). The reporter virus lacking polymerase activity (pol-null) is included as negative control. The transfected cells were lysed at indicated time points using Promega passive lysis buffer and the levels of Renilla luciferase were quantified. The experiment was done in triplicate and the mean values with standard deviation of Renilla luciferase (corrected for transfection efficiency and extract protein concentration) values are presented as a bar graph in log₁₀ scale. The lacZ gene (encodes beta-galactosidase protein) under CMV immediate early promoter was used to determine the percentage of transfected cells by comparing the number of cells expressing the reporter protein to the total number of cells in the population.