Figure 4.

Real-time fluorescence-based polymerization assay using HCV RdRp and FMDV RdRp. (A) SDS-PAGE analysis of different purification stages of HCV NS5bΔ21 and HCV NS5bΔ21-GNN polymerases after expression in E. coli. Lane 1, a lysate of E. coli BL21(DE3)-pRIL containing plasmid pET28-HCV NS5bΔ21 and grown in the absence of IPTG is shown. Lane 2, same as lane 1 but grown in the presence of 500 μM IPTG. Purified proteins HCV NS5bΔ21 (lane 3) and HCV NS5bΔ21-GNN (lane 4) obtained after purification through Ni-NTA resin. M, molecular marker. The molecular weight of each band is indicated in kDa. (B) De novo polymerase activity performed by recombinant HCV RdRp in the presence of [α-³²P]ATP using poly-U as template. Each lane represents a reaction stopped at a different time point (from 0 to 2 hours; lanes 2 to 6). Polymerization assays in the presence of MgCl₂ (lane 7), in the absence of any metal donor (lane 8), or using GNN inactive mutant (lane 9) showed no detectable activity. Lane 1 represents a negative control reaction in the absence of enzyme. The position of the labeled non-incorporated nucleotide is indicated (*). A vertical black line separates non-contiguous images from the same gel. (C) Real-time determination of fluorescence emitted in assays containing purified NS5bΔ21 wild-type (WT) and NS5bΔ21 GNN, and in the presence of different components indicated in the figure. Shown are average values obtained from five different replicas. (D) Same as in (C) but using FMDV 3D WT and GNN RdRps. Gel electrophoresis, visualization by autoradiography analysis and fluorometric measurements were performed as described in Methods.