## 3 Control of HDV RNA Editing
### The Type 1 and Type 3 amber/W Sites are Sub-optimal for Editing by ADAR1  
As mentioned above, previous mutational analyses had shown that increased base pairing 3' of the type 1 editing site increased editing and substantially decreased HDV RNA replication [10,12]. These observations led to the suggestion that the secondary structure formed by HDV type 1 RNA is sub-optimal for amber/W site editing by ADAR1 [10,12]. Direct comparison of the efficiencies of ADAR1 editing of the type 1 and type 3 amber/W sites with that of another viral substrate, the HHV-8 K12 transcript [42], supports this conclusion and extends it to include the HDV type 3 amber/W site (Figure 6). The features of the sites responsible for the sub-optimal activity appear to be different for type 1 and type 3. For type 1, the lack of sufficient base pairing within \(\sim\)25 nt 3' of the site limits activity, as described above. Increasing base pairing in this region increases both binding by ADAR1 and editing [10,43]. In contrast, base pairing is more substantial in the branched structure involved in type 3 amber/W site editing and this structure binds ADAR1 more tightly than does the type 1 unbranched rod structure (RC, SDL, unpublished). On the other hand, in the branched structure used by HDV type 3, the amber/W site exists as an A-U base pair, which is edited less efficiently than the much more common A-C mismatch [9,30,31,32,33]. This difference likely contributes to the sub-optimal nature of the site. Furthermore, we found that additional sequence and structural variations within the region \(\sim\)10 nt 3' of the amber/W site contributed to differences in editing between the structures formed by two type 3 sequences [13] (also see Figure 6). It remains to be seen how these variations contribute to differences in editing activity. It also remains to be seen whether such variations occur among HDV type 1 amber/W sites. Finally, given the critical role of editing in the HDV replication cycle, these variations raise the obvious question of whether such differences affect pathogenesis. Control of Editing by Limiting Substrate Availability--Different Mechanisms for Different Structures  
In addition to limiting editing by forming sub-optimal editing sites, we have found that HDV controls editing levels by limiting the availability of this sub-optimal substrate to ADAR1. Perhaps not surprisingly, the mechanisms employed by types 1 and 3 to limit substrate availability are as different as the structures used for editing. Just as the use of different structures by these genotypes underscores the important functional role of editing in the HDV replication cycle, the use of different control mechanisms emphasizes the essential need to manage the process. Comparison of editing occurring on non-replicating HDV type 1 RNAs with that occurring on replicating RNAs in cells indicated that editing levels were much higher for the former than the latter at early times (2-3 days) post-transfection [38]. The low level of editing occurring on the replicating RNA is consistent with the slow accumulation of replicating HDV RNA. If editing levels were too high early, replication would be inhibited by HDAg-L before sufficient HDV RNA accumulated for packaging. We and others have shown that, in transfected cells, HDAg can dramatically inhibit editing of the HDV type 1 amber/W site [12,38]. HDAg is known to bind the HDV RNA unbranched rod structure [43, 44, 45]. Recent work in our lab has shown that HDAg inhibits editing of HDV RNA by ADAR1 _in vitro_. Editing of the K12 RNA is not affected (RC, SDL, unpublished), indicating that the inhibition occurs by a direct interaction between HDAg and the RNA, rather than binding to ADAR1. Thus, our model for the down-modulation of editing during HDV type 1 replication is that the availability of the RNA for editing by ADAR1 is limited by binding of HDAg to the RNA (Figure 7).