FIG. 6.

Site-directed mutagenesis has no effect on HCV replication. (a) Real-time qRT-PCR analysis of the HCV RNA titer using total cellular RNA collected at the indicated time points from cells transfected with JFH1^E2FL (WT), JFH1^C128A (C128A), JFH1^C184A (C184A), JFH1^C128/184A (C128/184A), or JFH1^PP/AA (PP/AA) RNA. (b) Immunoblot analysis of NS5A protein and GAPDH in whole-cell lysate collected from cells transfected with WT, C128A, C184A, or C128/184A RNA at day 3 posttransfection. (c) Confocal microscopy of the subcellular localization of the LD (green), core (blue), NS5A protein (red), and nucleus (4′,6-diamidino-2-phenylindole [DAPI]) (gray) in WT and C128A replicating cells on day 3 posttransfection. Bars, 10 μm. (d) An RNA-protein binding precipitation assay was performed with in vitro-translated core^WT and core^C128A using poly(U) agarose as the resin. +RNase and −RNase, samples with and without RNase treatment, respectively, as described in Materials and Methods. Input indicates that 1/40 of the amount of translated product was used in this assay. Data represent the means ± standard deviations from three independent experiments (a) or are representative of those from three independent experiments (b to d).