## 2 RNA Structural Requirements for Editing
### Structural Requirements for Editing the HDV Genotype 1 amber/W Site  
The unbranched rod structure is a characteristic feature of HDV RNAs. This structure, in which about 70% of nucleotides are paired, is required for HDV RNA replication [12, 28]. Site-directed mutagenesis has indicated that, for HDV genotype 1, this structure is the substrate for editing at the amber/W site [9, 29]. The type 1 amber/W site occurs as an A-C mismatch pair in the midst of eight canonical Watson-Crick base pairs (Figure 3). Both the A-C mismatch and the base pairs immediately surrounding the site have been shown to be critical for editing [9, 30]. A-C mismatches, which are found in some (but not all) other editing sites, have been found to maximize editing efficiency [9, 30, 31, 32, 33]. The role of base-paired regions outside the 8 bp immediately surrounding the type 1 amber/W site is not settled. [PERSON] _et al._ concluded that no additional secondary structures were required beyond the 8 bp and the A-C mismatch [34]. Inspection of the RNA secondary structure downstream of the type 1 HDV amber/W site indicates that it contains base-paired segments but is more frequently disrupted by  
Figure 2: Model for interaction between ADAR1 and substrates for site-specific editing. The RNA is indicated by the blue lines; closely spaced parallel segments indicate base pairing. The target adenosine is indicated by the large blue A. Binding of ADAR1 to the RNA is mediated by the DRBMs (shaded rectangles), which recognize the dsRNA elements both 3’ of and around the targeted adenosine and position the deaminase domain. bulges and mismatches than the region 3' of other editing substrates (see Figure 3). These disruptions raised the question of whether the quality of base pairing in this region was sufficient to play an important role in ADAR1 binding and activity. The role of base pairing in the region 3' of the editing site was examined by site-directed mutations that either further disrupted or increased base pairing in HDV RNA segments expressed in cells [10]. Mutations that substantially disrupted base pairing had little detectable effect on editing. Moreover, mutations that improved base-pairing, particularly in the region 15-25 nt 3' of the editing site, increased editing significantly [10,12]. These results suggest that base pairing in the region up to 25 nt 3' of the amber/W site is not sufficient to recruit ADAR1 to the editing site _via_ interactions with the DRBMs and appear to be consistent with the conclusion of [PERSON] _et al._ that structures outside the immediate vicinity of the amber/W site are not required for editing. In contrast to the above conclusion, more recent work in our lab suggests that sequences even further removed from the type 1 amber/W site play an important role in editing. While the previous mutational analysis indicated that base-pairing within ~10-15 nt 3' of the editing site might not be required for editing [10,34], analysis of RNA deletion mutants indicates that removal of sequences between 42 and 108 nt 3' of the amber/W site reduces activity (RC, unpublished). Truncation to within 77 nt on the 3' side of the amber/W site diminished editing to less than one-third the level of full-length RNA; further truncation to 42 nt 3' of the site led to nearly complete loss of activity (Figure 4). Inspection of the predicted secondary structure of the region 3' of the amber/W site indicates that the highest degree of base pairing is located from 64 to 100 nt downstream. This region includes 34 bp that are minimally disrupted by one symmetric single nucleotide internal loop and four asymmetric single nucleotide bulges. By comparison, the region 25 nt 3' of the GluR-B R/G site, which is known to be required for editing of this site, consists of 23 bp that are disrupted by two symmetric single nucleotide internal loops [27].