FIG. 4.
Stability of NS5B-RNA complexes. (A) RNA synthesis was monitored with the T20-C16 template, which allows the incorporation and excision of chain-terminating 3′-dCMP at a single position (+16), as outlined in the experimental design under the image. Heparin was used as a protein trap to analyze enzyme dissociation under different conditions, including preincubation of heparin and NS5B (Apo-E) prior to the addition of RNA and the start of the reaction, preincubation of heparin with NS5B bound to the template and the GG primer, and the addition of heparin after elongation and chain termination. In the last case, the activity of NS5B was measured in the presence of 125 μM PPi and a mixture of 10 μM CTP and 3′-dUTP that is required to rescue RNA synthesis for a single-nucleotide incorporation event. The loss of activity is a measure of enzyme dissociation, which was monitored in the presence of increasing concentrations of heparin. Lanes 0 to 7, 0, 0.037, 0.073, 0.146, 0.293, 0.586, 1.17, and 2.34 μM heparin. (B) The results were quantified and expressed as concentrations of heparin required to inhibit 50% of the enzymatic activity (IC50s).