## 3 Control of HDV RNA Editing
### The Type 1 and Type 3 amber/W Sites are Sub-optimal for Editing by ADAR1
This binding likely interferes sterically with the ability of ADAR1 to bind the RNA; consistent with  
Figure 6: The HDV type 1 and type 3 amber/W sites are edited _in vitro_ by ADAR1 less efficiently than the HHV8 K12 transcript. HDV-3P and HDV-3E are two different HDV type 3 isolates [13]. this interpretation, recent _in vitro_ results have indicated that HDAg binding blocks degradation of the RNA by microococcal nuclease [43]. Both HDAg-S and HDAg-L inhibit editing equally well, indicating that this mechanism for controlling editing is not directly responsive to editing levels. The HDV type 1 and type 3 amber/W sites are controlled by different mechanisms. Editing of this site in type 1 RNA (blue) requires the unbranched rod structure and is inhibited by HDAg (green shape), which binds this structure. Editing of the HDV type 3 RNA (red) requires a branched structure that is less stable than the unbranched rod and can therefore form only following transcription. Editing is limited by the fraction of RNA that folds into the branched conformation during transcription. The mechanism employed by HDV type 3 to control editing is quite different. For HDV type 3, editing is not inhibited by HDAg-S, either in transfected cells or _in vitro_[46]. The mechanistic explanation for this lack of inhibition is not yet clear. Possibly, HDAg does not bind the branched structure required for editing. Our recent results indicate that, for HDV type 3, editing is controlled by RNA structural dynamics - specifically, the ability of the RNA to fold into the branched structure [13]. The possibility that such a mechanism could be important was raised by the observation that the branched structure is less energetically stable than the unbranched rod structure and, therefore, can only be formed following transcription of the RNA [36]. We explored this idea using two independent HDV type 3 isolates [13]. As shown in Figure 6, _in vitro_, ADAR1 edits the amber/W site of HDV-3P RNA more effectively than that of HDV-3E RNA. However, the converse is true when editing levels attained during replication are compared. Computational analysis of the secondary structures formed by HDV-3E and HDV-3P [36] suggested that this apparent paradox might be explained by differences in the RNA structural dynamics of these two RNAs - HDV-3E RNA could form the branched structure required for editing more readily than HDV-3P RNA. Consistent with the predictions, following transcription _in vitro_ HDV-3E and HDV-3P RNAs formed both the branched structure required for editing and the unbranched rod (which is not edited), but HDV-3E RNA formed the branched editing structure 3-4 fold more efficiently than did HDV-3P [13]. Based on these results, our model for  
Figure 7: The HDV type 1 and type 3 amber/W sites are controlled by different mechanisms. Editing of this site in type 1 RNA (blue) requires the unbranched rod structure and is inhibited by HDAg (green shape), which binds this structure. Editing of the HDV type 3 RNA (red) requires a branched structure that is less stable than the unbranched rod and can therefore form only following transcription. Editing is limited by the fraction of RNA that folds into the branched conformation during transcription. down-modulation of editing of the HDV type 3 editing site is that substrate availability is limited by the fraction of the RNA that folds into the branched structure following transcription (Figure 7).