FIGURE 6.

Effect of S556 mutations on initiation and elongation of RNA synthesis. For quantification of initiation products, wild type or mutant NS5b and 341 nt (−) RNA were incubated in the RdRp reaction buffer with 0.5 mm GTP (G1-C) or ATP (G3-U) and 10 μm [α-³²P]CTP as the only added nucleotides. 10% (v/v) DMSO was also added in the experiments performed with G3-U as template (panel B). At different time points, four microliters were collected and analyzed on 22% polyacrylamide gels as described under “Experimental Procedures.” The gels were submitted to electronic autoradiography, and the radioactive bands were quantified with the Quantity one software. A, shown is J4 NS5b with G1-C. B, shown is J4 NS5b with G3-U. C, shown is H77_NS5b with G1-C. D, for quantification of elongation products, wild type or mutant J4 NS5b and G1-C were incubated in a RdRp reaction buffer with 500 μm ATP, GTP, 3′-dUTP, and 100 μm [α-³²P]CTP for 2 h at 25 °C. Four microliters of the reaction were analyzed on 20% polyacrylamide gels, and the products were quantified as above.