FIGURE 4.

HCV NS5B initiation in the presence of NNI2. A, time course of primer formation (10-mer) was monitored on denaturing PAGE gel. 10 μm NS5B was preincubated with 14 μm concentrations of each NNI2 for 30 min, and primer formation was initiated by adding 1 mm GTP and 50 μm ATP, UTP, and ddCTP and quenched at the indicated time intervals. Abortive intermediates were observed due to de novo initiation catalyzed by NS5B. The arrows denote the abortive intermediates (four and five nucleotides) formed in reactions where NNI2s were present. The initiation assay was also performed with either [α-³²P]ATP or [α-³²P]UTP as the radioactive probe. By comparing radiolabeled products between [α-³²P]ATP (upper panel) and [α-³²P]UTP (lower panel), we established the identities of different abortive intermediates. B, fraction of the abortive intermediates species (4- and 5-mer) relative to total product was plotted against reaction time. C, the amount of extended primer as well as abortive intermediates with various lengths was plotted versus time. Data were fit based on simulated model as shown in Scheme 2 using KinTek Explorer.