Fig. 3.

Junction of the HRV-14 cre stem and loop. (a) Structure of the first base mismatch at the stem-loop junction, showing G11 and A24. Although these two bases are in close proximity, the formation of hydrogen bonds is not possible because of the orientation of the donor-acceptor groups. (b) Influence of base substitutions at G11 and A24, including reversal of these two bases, within the cre of HRV-14 on RNA replication in the context of the subgenomic replicon, ΔP1LucCRE (see Fig. 1a). Replication was assessed by measuring the increase in luciferase activity (± SEM in triplicate assays) between 3 and 24 h after transfection of RNAs into HeLa cells, normalized to that observed with WT ΔP1LucCRE replicon (cre = 100%) (10). The replicon Δcre does not contain a cre and thus serves as a negative control. (Inset) Shown are the WT loop sequence and the position of the G11 and A24 residues at the bottom of the loop.