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. 2010 Aug 20;285(43):32906–32918. doi: 10.1074/jbc.M110.151316

FIGURE 3.

FIGURE 3.

Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin (Mr 4000–6000, 200 μg/ml) was then added followed by [α-32P]CTP and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A, reactions were performed with G1-C and H77_NS5b (upper panel) or J4_NS5b (lower panel: empty diamonds, preincubation without NTP; filled squares, preincubation with GTP; filled triangles, preincubation with CTP and GTP). B, reactions were performed with G3-U and H77_NS5b (upper panel) or J4_NS5b (lower panel: filled squares, preincubation with GTP; filled circles, preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.