Fig 2.

Primer-dependent RNA synthesis. (a) Concentration dependence of primer-dependent RNA synthesis by wild-type BVDV NS5B. A 3′-recessed RNA primer-template duplex (duplex A) was used in this RdRp assay. The RdRp assay reaction mixtures contained between 0.5 and 5 μM wild-type BVDV NS5B, 0.1 μM 5′-end-labeled RNA, and 50 mM NaCl. (b) Primer extension by wild-type (WT) BVDV NS5B and its mutant derivatives in the presence of MgCl₂ (white bars) or MnCl₂ (gray bars) as the divalent metal ion. Reactions were carried out at 37°C for 2 h, using 0.250 μg of poly(C) template, 0.125 μg oligo(G)₁₂ primer, 0.4 μCi/μl [α-³²P]rGTP, and 50 nM NS5B in a total volume of 25 μl. [α-³²P]rGMP incorporation was quantified by a DEAE filter binding scintillation assay.