Variations in AmberW Site Structures Among HDV Genotypes

It is interesting to note that both the RNA secondary structure around the amber/W site and the C-terminal sequences specific to L-HDAg are distinguishing features of some HDV genotypes (Casey 2002; Casey et al. 1993; Hsu et al. 2002; Ivaniushina et al. 2001; Niro et al. 1997; Shakil et al. 1997) (see the chapter by P. Deny, this volume). The genotypic differences in the RNA secondary structure required for editing fall into two categories: (1) the dif ferent portions of the HDV antigenome that form the structure; and (2) the specific RNA secondary structure formed around the amber/W site. Despite these variations, all three of the genotypes analyzed (I, II and III) are edited by the same enzyme—ADAR1 (Jayan and Casey 2002b).

In HDV genotype I the structure required for amber/W site editing is part of the unbranched rod structure characteristic of HDV RNA (Fig. 2; Casey et al. 1992). The eight Watson-Crick base-pairs flanking the amber/W site and the A-C mismatch pair involving the amber/W site are highly conserved among over 50 genotype I sequences (Niro et al. 1997; Yang et al. 1995). The role of this structure in editing has been confirmed by site-directed mutagenesis studies (Casey et al. 1992; Casey and Gerin 1995; Polson et al. 1996; Wong et al. 2001). HDV genotype III RNA also forms an unbranched rod structure, which is required for RNA replication; however, the base-pairing in the immediate vicinity of the amber/W site is disrupted such that this structure does not function as a substrate for amber/W editing (Casey 2002). Rather, editing in genotype III requires an alternative, 'double hairpin' structure that creates better base-pairing in the immediate vicinity of the amber/W site (Fig. 2; Casey 2002). This structure, which differs from the unbranched rod structure by about 80 base-pairs, contains two stem-loops that essentially shift the positions of the noncoding side of the HDV antigenome that are base-paired with the amber/W site region. In the unbranched rod structure the amber/W site is opposite position 580; in the double hairpin structure, the paired position is 509. The structure required for editing in the other HDV genotypes has not yet been determined. Comparative analysis of the predicted secondary structure in the vicinity of the amber/W site in the unbranched rod reveals structures similar to the genotype I structure but more disrupted (Hsu et al. 2002; Ivaniushina et al. 2001; Radjef et al. 2004).

Inspection of the predicted RNA secondary structures around the amber/W sites of genotypes I and III indicates that the genotype III amber/W site differs from the type I site. In some cases the differences occur at positions that have been shown to be essential for efficient editing in genotype I (Fig. 2). For example, the A-C mismatch pair that involves the amber/W adenosine and which is highly conserved among genotype I isolates, occurs as an AU pair in genotype III; when introduced by site-specific mutagenesis into a genotype I genome, this specific change substantially reduces both editing and virus production (Casey et al. 1992; Jayan and Casey 2005). The significance and effect of these variations on editing, RNA replication and virus production in genotype III remains to be determined. Perhaps the variations at the genotype III site can be explained by compensatory effects, such as changes elsewhere in the editing site region, including sequences/structures 3' of the editing site, or differences in the mechanisms by which HDV regulates editing during replication. Another (but not mutually exclusive) possibility is that the differences described are responsible for variations in the levels of editing among genotypes (Hsu et al. 2002).

Defining the structural determinants for editing remains an important goal. Despite recognitionofthe commonfeaturesamong editing sitesnoted in Sect. 3.1, the sequence and structural determinants for highly specific editing are still not well understood. Only a handful of substrates for highly specific editing have been identified to date in mammals, and it is anticipated that many more remain to be found (Paul and Bass 1998). Knowledge of sequence and structural requirements for editing will likely facilitate the prediction of potential adenosine deamination editing sites from analysis of genomic sequences. Moreover, it is reasonable to expect that differences in editing levels among different substrate adenosines are due in part to variations in the structure of the RNA in the vicinity of the editing sites. Understanding the effects of structural variations will contribute to our understanding of how this important post-transcriptional regulatory mechanism is modulated. Variations among the amber/W sites in the HDV genotypes may provide a valuable opportunity to evaluate the effects of different sequences and RNA secondary structures on editing at the HDV amber/W site.

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