FIG. 2.

Inhibition of genotype III RNA editing by HDAg is not affected by mutations that stabilize the branched double-hairpin RNA structure relative to the unbranched rod structure. (A) Schematic of the branched double-hairpin structure required for genotype III RNA editing (left) and the unbranched rod structure. In the “stem-flip” mutant M8/9 (3) the 8 or 9 bp nearest the loops of stem-loops SL1 and SL2 are reversed, destabilizing the unbranched rod structure but leaving the predicted stability of the double-hairpin structure unaffected. In this mutant the distribution of the RNA between the unbranched rod and branched double-hairpin structures is probably altered, as indicated by the arrows. The amber/W site adenosine is indicated by “A,” and the editing event is indicated by a vertical arrow extending from “A” to “I”; an “X” is placed on this arrow above the unbranched rod structure because editing does not occur in this conformation (3). (B) Inhibition of amber/W editing in M8/9 mutant RNA by genotype III HDAg-S and HDAg-L. Huh-7 cells were cotransfected with the nonreplicating genotype III RNA expression construct pHDV-III-NR-M8/9 (3), and various amounts of the genotype III HDAg-S or HDAg-L expression constructs pHDAg-S-III (lanes 2 to 4) and pHDAg-L-III (lanes 5 to 7). Amounts of HDAg expression construct cotransfected were: lane 1, none; lanes 2 and 5, 0.5 ng; lanes 3 and 6, 5 ng; lanes 4 and 7, 50 ng. RNA was harvested and editing analyzed as in Fig. 1. The numbers at the bottom of the panel are the average editing values obtained from duplicate transfections.