Figure 2.

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.