CRV substitutions inhibited the elongation step of replication.

<p>(A) Schematic diagram of the 3' end of the replication-specific minigenome. The two initiation sites in the <i>tr promoter</i> (+1, +3) are indicated with green arrows, and the small tr (sm tr) and genome RNA products are shown in blue. The regions contained within the 13–35 and 24–48 primers are indicated in red. (B, C) Primer extension analysis of RNA generated by wt and variant L proteins using the 13–35 and 24–48 primers, respectively. The position of the +1 and +3 initiation sites were determined by co-migration of end-labeled DNA oligonucleotides, corresponding in length and sequence to cDNAs representing initiation from those sites (Lanes 1 and 2). Lane 3 shows RNA from a control transfection lacking L expression plasmid. The white lines between lanes 2 and 3 indicate where empty lanes were excised. (D, E) Quantification of replicates of the experiments shown in panels B and C. Panel D shows levels of RNA initiated at +1 and +3, detected by primer extension with primer 13–35. Panel E shows levels of RNA initiated at +1 detected by primer 24–48. In D and E the data are normalized to a mean of the two wt samples included in each experiment at 100%. In panel D, the bars show the mean and range of two independent experiments. In panel E, the bars show the mean and standard deviation of three independent experiments.