The ORF encoding HDAg runs almost 75% of the length of the HDV rod RNA structure (Fig. 1) and is the reason why HDV RNA is so much larger than viroid RNA. This ORF exists in two sizes, a small and a large form, on different RNA molecules, with coding capacities for 195 and 214 amino acids, respectively. The difference between these two ORFs is base mutation at position 1015. This results from a specific editing event that is linked to HDV replication and catalyzed by the cellular enzyme ADAR-1 (adenosine deaminase that acts on RNA-1; Jayan and Casey 2002; Wong and Lazinski 2002). The nucleotide at position 1015 can be an A, which leads to a termination codon UAG, or G, giving UGG (Trp), which allows for the read-through of 19 additional amino acids. The resultant proteins are referred to as small-and large-HDAg, respectively (S-HDAg; L-HDAg), and are translated from an approximately 0.8 kb unspliced, antigenomic-sense, subgenomic transcript which is structurally identical to the conventional cellular mRNAs with both a 5' 7-methylguanosine -cap structure and a poly A+ 3-tail (Gudima et al. 2000; Fig. 1). Notably, only HDV genomes containing the smaller version of the ORF can initiate replication (Glen and White 1991; Macnaughton et al. 2003; see later) such that early in infection only S-HDAg is synthesized.

With the exception of the19 aa C-terminal extension in the L-HDAg, the two HDAg species are identical in amino acid sequence and share many biochemical properties. Despite this, these proteins play very different roles in the HDV life cycle. S-HDAg is a crucial activator for the initiation and maintenance of HDV RNA replication in vivo. L-HDAg, which is only synthesized late in infection, is essential for virus packaging. Another function ascribed to L-HDAg is the suppression of HDV RNA replication (Chao et al. 1990). However, more recent studies in our laboratory indicated that L-HDAg-mediated suppression occurred only when this protein was expressed abnormally early in the HDV replication cycle (Macnaughton and Lai 2002b). Moreover, when expressed early, only suppression of synthesis of genomic RNA from the antigenomic RNA template, but not vice versa, was observed (Modahl and Lai 2000). When L-HDAg is expressed in the context of the natural replication cycle, it does not influence steady-state, cellular concentrations of either genomic or antige-nomic HDV RNA (Macnaughton and Lai 2002b). These results were disputed recently (Sato et al. 2004), following observations using a mutant HDV genome designed to undergo rapid editing. This mutant expressed a higher level of L-HDAg early after initiation of replication, and HDV replication was rapidly terminated. While these results are consistent with the effects of early, unregulated L-HDAg expression, they do not establish that L-HDAg performs an inhibitory role in natural replication, in which L-HDAg is expressed only late in the infection. This point was noted by the authors, who concluded that at least 'L-HDAg does not regulate the expression of wild type HDV' (Sato et al. 2004). Despite the differences between S- and L-HDAg, these proteins also share some common functions, such as stabilization of HDV RNA (Lazinski and Taylor 1994), enhancement of ribozyme activity (Jengetal. 1996) andRNA chaperon activity (Huang and Wu 1998). A number of functional domains have been identified within HDAg (Fig. 2), most of which are present on both S- and L-HDAg. Within the amino-terminal one-third there is a coiled-coil domain that promotes protein-protein interactions and is essential for the activator functions of S-HDAg (Xia and Lai 1992; Lazinski and Taylor 1993). Within the middle third are two domains, a nuclear localization sequence (NLS, Fig 2; Xia et al. 1992; Lazinski and Taylor 1993) and an RNA-binding

60 69 75 88 97 107

195 214

Coiled Coil Domain

Core ARM 1 ARM 2 Prolin c/Glycine-Rich Domain

Whole RNA Binding Domain



Fig. 2 Functional domain map of hepatitis delta antigen. NLS, nuclear localization signal; ARM, arginine rich motif;HLH,helix-loop-helixmotif; S-HDAg, small hepatitis delta antigen; L-HDAg, large hepatitis delta antigen motif that is semi-specific for HDV RNA (Lin et al. 1990; Chao et al. 1991). The latter domain is comprised of two arginine-rich motifs (ARM, Fig. 2), which are separated by a spacer region that includes a helix-loop-helix (HLH) motif (Lee et al. 1993; Chang et al. 1993). Both ARMs and the spacer region are required for RNA binding and the activation function of S-HDAg (Lee et al. 1993). The C-terminal third is characterized by a stretch of amino acids that is rich in proline and glycine residues, the function of which is not clear. Finally at the very C terminus of L-HDAg there is a four-amino-acid motif (CXXQ) that serves as a substrate for prenylation, with the modification occurring at the cystine residue of this quartet (Glenn et al. 1992; Lee et al. 1994; Otto and Casey 1996; also see chapter by J.S. Glenn, this volume). Prenylation alters the conformation of L-HDAg, resulting in the masking of a C-terminal epitope (Hwang and Lai 1994) present on the native S-HDAg. This modification also promotes membrane binding and is essential for the interaction between the L-HDAg and HBsAg (de Bruin et al. 1994; Hwang and Lai 1993), which is a critical step in HDV particle assembly. While the C-terminal 19 amino acids of L-HDAg are necessary and sufficient for virus assembly (Lee et al. 1995), this process is enhanced by the presence of S-HDAg (Wang et al. 1994). This enhancement is likely due to a combination of direct effects such as protein-protein binding of S-HDAg with L-HDAg (Wang et al. 1994) and indirect effects such as the recently demonstrated enhancement of prenylation of L-HDAg by the presence of S-HDAg (O'Malley and Lazinski 2005).

In addition to prenylation, HDAg undergoes a number of other post-translational modifications, which likely play key regulatory roles during the HDV life cycle (see chapter by W.-H. Huang et al., this volume). These modifications are by phosphorylation (Chang et al. 1988; Mu et al., 1999,et al. 2001), acetylation (Mu et al. 2004) and methylation (Li et al. 2004). Specifically, Methylation of Arg13, acetylation of Lys72 and phosphorylation of Ser177 and Ser123 have been reported to affect the subcellular localization of HDAg and most of these modifications are important for antigenomic but not genomic HDV RNA replication (see later).

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