## 2 RNA Structural Requirements for Editing
### Editing the HDV Type 3 amber/W Site Requires a Branched Structure  
HDV type 3 also forms an unbranched rod structure that is required for replication. However, inspection of this structure indicates that the base pairing in the immediate vicinity of the amber/W adenosine is much more disrupted than in type 1 and, in fact, the type 3 amber/W adenosine is not edited when the RNA is in this conformation [28, 36]. Nevertheless, this site is edited by ADAR1 during replication, just like the type 1 amber/W site [7]. What we have found is that the type 3 RNA is considerably rearranged into an alternative branched structure in order for editing to occur (Figure 5). Figure 4: Editing at the HDV type 1 amber/W site requires sequences more than 77 nt 3’ of the site. Schematics indicate unbranched rod RNAs analyzed for editing _in vitro_ by ADAR1. Numbers below indicate distance in nucleotides to the ends of the unbranched rod RNA; in the 5’ direction (left) the end is formed by a 6 nt loop, in the 3’ direction (right) the end is the 3’ end of the RNA. A 5-point star indicates the location of the amber/W site. Relative level of editing of each RNA is shown in the bar graph on the right. In this structure, ca. 220 nt of the unbranched rod structure that are involved in 86 predicted base pairs are rearranged to form a branched structure consisting of two \(\sim\)25 bp stem-loops (SL1 and SL2) flanking a central base paired region that includes the amber/W site, which is itself base paired (Figure 5). Whereas the amber/W site is opposite position 580 in the unbranched rod structure, in the branched structure the paired position is 509 (Figure 5). **Figure 5.** Editing at the HDV type 3 amber/W site requires a branched structure. _Left._ Schematic showing the unbranched rod structure and the RNA secondary structure in the vicinity of the target adenosine (indicated by an asterisk). This structure is not a substrate for editing by ADAR1 either in cells or _in vitro_[28, 36]. _Right._ Schematic of the branched structure required for editing. The base pairing immediately surrounding the amber/W site in this branched structure is required for editing [28]. Because editing occurs on the antigenome, which is a replication intermediate, and replication requires the unbranched rod structure a consequence of this model is that the RNA must undergo a conformational change after editing has occurred. This conformational change is energetically favored because the branched structure required for editing is less stable than the unbranched rod structure, which is the lowest energy structure formed by the RNA [36]. Nevertheless, the branched conformation is moderately stable _in vitro_, even at 37 \({}^{\circ}\)C. Perhaps HDAg, or even cellular factors, play a role in facilitating the conformational change. Analysis of the elements of the branched structure that contribute to editing activity _in vitro_ has indicated that SL2, which is 3' of the site, is essential for editing, but SL1 is required for neither editing nor ADAR1 binding (as long as the remainder of the structure is formed). Thus, the role of SL1 appears to be simply to help stabilize the structure required for editing; once the structure is formed, SL1 does not participate in the editing reaction. The requirement for SL2 is consistent with the model for ADAR1 binding to regions with double-stranded RNA character that are 3' of the editing site. Indeed, analysis of ADAR1 binding indicates that SL2 is required. However, deletion analysis has indicated that nearly all of SL2 is required for binding (and editing); thus the linear distance along the RNA of sequence required for editing is about 40 bp--not as long as we have observed for the type 1 amber/W site (Figure 4), but considerably longer than required on dsRNA substrates [21, 22] or for the gluR-B R/G site [26].