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. 2008 Aug 29;283(35):24089–24102. doi: 10.1074/jbc.M803422200

FIGURE 5.

FIGURE 5.

mAb 8B2 blocks primer-independent (de novo) RdRp activity of HCV NS5B in vitro. A, standard de novo RdRp assay was performed using an in vitro transcribed Con1/luc RNA template and increasing concentrations of mAbs 7G8 (lanes 1-6) and 8B2 (lanes 7-12). After a 2-h incubation at 25 °C, RNA was precipitated and analyzed on denaturing formaldehyde-agarose gel. Shown are ethidium bromide staining and the autoradiogram of the same gel. The critical additives and ssRNA yield normalized to the RdRp reaction without mAbs (lane 13) are indicated below the panels. The position of the Con1/luc RNA template is indicated on the right. M is the marker lane with transcribed and purified Con1/luc ssRNA (9510 nucleotides) used for the RdRp assay. D318N (lane 14) is an inactive form of the NS5B. The mAb:RdRp molar ratio value of 1 corresponds to 0.36 μm mAb concentration. B, quantitative PhosphorImager analysis of the reactions shown in A. The analysis of a single gel is presented. The curves show the efficiency of the ssRNA synthesis on Con1/luc RNA template by NS5B in the presence of mAbs 7G8 and 8B2.