G05 did not inhibit elongation step of RNA synthesis but inhibited RNA binding of the polymerase. (a) The G05 compound reduced the amount of the newly synthesized RNA strand in a dose-dependent manner. The compound was added to a [32P]-UMP incorporation reaction using recombinant NS5B and poly(A)-oligo(dT) template at a concentration of 1 5 10 or 15 μM (lanes 2-5). (b) Single processive cycle conditions were set up with heparin an RNA polymerase trapper. Lane 1; RNA product in the absence of NS5B lane 2; RNA product in the presence of NS5B lane 3; RNA product in the presence of NS5B with the addition of heparin prior to the template; lane 4; single processive reaction without G05 compound lanes 5-7; single processive reaction at a concentration of 1 5 or 10 μM G05 compound respectively. (c) Inhibition of binding between recombinant NS5B and template RNA was measured. Recombinant hexahistidine-tagged NS5B was preincubated with G05 at various concentrations before adding 3' YTP RNA. After incubation the mixture was pulled down with Ni-NTA resin and the RNA was analyzed in a gel electrophoresis. Lane 1; no inhibitor lane 2; 0.5 μM G05 added lane 3; 1 μM G05 added lane 4; 5 μM G05 added lane 5; 10 μM G05 added.