Differential Inhibition of RNA Editing in Hepatitis Delta Virus Genotype III by the Short and Long Forms of Hepatitis Delta Antigen

Abstract

Hepatitis delta virus (HDV) produces two essential forms of the sole viral protein from the same open reading frame by using host RNA editing activity at the amber/W site in the antigenomic RNA. The roles of these two forms, HDAg-S and HDAg-L, are opposed. HDAg-S is required for viral RNA replication, whereas HDAg-L, which is produced as a result of editing, inhibits viral RNA replication and is required for virion packaging. Both the rate and amount of editing are important because excessive editing will inhibit viral RNA replication, whereas insufficient editing will reduce virus secretion. Here we show that for HDV genotype III, which is associated with severe HDV disease, HDAg-L strongly inhibits editing of a nonreplicating genotype III reporter RNA, while HDAg-S inhibits only when expressed at much higher levels. The different inhibitory efficiencies are due to RNA structural elements located ca. 25 bp 3 of the editing site in the double-hairpin RNA structure required for editing at the amber/W site in HDV genotype III RNA. These results are consistent with regulation of amber/W editing in HDV genotype III by a negative-feedback mechanism due to differential interactions between structural elements in the HDV genotype III RNA and the two forms of HDAg.RNA editing at the viral amber/W site plays a central role in the replication cycle of hepatitis delta virus (HDV), a subviral human pathogen that increases the risk of severe liver disease in those infected with its helper, hepatitis B virus (HBV) (37). The virus uses adenosine-to-inosine RNA editing activity of the host cell, catalyzed by the cellular enzyme ADAR1 (17,35,36,40), to produce two forms of the sole viral protein, hepatitis delta antigen (HDAg), from the same open reading frame (4,26,35). These forms, HDAg-S and HDAg-L, differ by the presence of an additional 19 to 20 amino acids at the C terminus of HDAg-L. During the course of virus replication, the UAG (amber) stop codon of HDAg-S is changed to a UGG (trp) codon as a result of editing at adenosine 1012 (the amber/W site) in the antigenome (7,35). This change directs the translation of the C-terminal 19 to 20 amino acids unique to HDAg-L and alters the form and function of the viral protein (42, 44). HDAg-S is required for RNA replication, whereas HDAg-L is required for packaging the viral RNA with the envelope of hepatitis B surface antigen but inhibits RNA replication (2, 9, 18, 39, 44).Varying the extent of editing at the amber/W site, either by altering ADAR expression or by site-directed mutagenesis near the site, can have important consequences for HDV replication and virus production (3, 16). Excessive editing at the amber/W site results in reduced levels of RNA replication and reduced production of viable virions because edited antigenomes encode HDAg-L, which is a trans-dominant inhibitor of HDV RNA replication (13,14). Insufficient editing can lead to increased intracellular HDV RNA replication but inhibits virion production (3). Thus, the kinetics and extent of editing are likely reg...

The fraction of RNA that folds into the correct branched secondary structure determines hepatitis delta virus type 3 RNA editing levels

“…For HDV genotype I (HDV-1), editing is down-modulated in part by HDAg-S (Polson et al 1998;Sato et al 2004), most likely by binding to the RNA. However, we previously observed that HDAg-S is not an effective inhibitor of amber/W site editing for HDV-3, which differs from HDV-1 by z40% in nucleic acid sequence (Cheng et al 2003), thus indicating that an alternative mechanism must exist for the regulation of editing for this genotype. Unlike HDV-1, amber/W site editing in HDV-3 requires a branched secondary structure comprised of a central base-paired region containing the amber/W site flanked by two z25-base-pair stem-loops, designated SL1 and SL2 (Casey 2002;Linnstaedt et al 2006).…”

“…RNAs were named based on the parent sequence plus the source of the exchanged nucleotides; thus, mE-SF / P EDIT contains miniaturized Ecuadorian sequences in which the four sequence variations from the Peruvian RNA near the editing site have been substituted for Ecuadorian sequences. Construct pHDV-III-NR M24/25 expresses a nonreplicating HDV-3 Peru RNA in which the secondary structure of the edited conformation around the amber/W site is energetically favored by having removed SL1 and SL2 sequences (Cheng et al 2003); we refer to that expression construct here as Pnr. In the construct Pnr / P EDIT , the 4 nt in Pnr corresponding to positions 272, 273, 275, and 276 in mP (Fig.…”

“…The former contains Peruvian HDV sequences, the latter contains the four sequences of the Ecuadorian variant between positions 272 and 276. RNAs were harvested 2 d post-transfection and analyzed for amber/W site editing, as described previously (Cheng et al 2003). level than that of the Ecuadorian isolate (Fig. 2C).…”

“…RNA editing at the amber/W site was detected by Sty I digestion of RT-PCR products as previously described (Casey and Gerin 1995;Polson et al 1996;Jayan and Casey 2002b;Linnstaedt et al 2006). Amber/W site editing in HDV RNA isolated from transfected Huh-7 cells was determined as before (Cheng et al 2003). For analysis of amber/W site editing in viral RNA in patient sera, RNA was isolated by proteinase K digestion, extraction, and ethanol precipitation, as described (Niro et al 1997); editing at the amber/ W site was determined by RT-PCR amplification using primers 59-GAAGGAAGGCCCTCGAGAACAAGA-39 and 59-GAGATGC CATGCCGACCCGAAGAG-39 followed by 32 P labeling and Sty I digestion, as described (Polson et al 1996).…”

“…For this analysis we used expression constructs for nonreplicating RNAs from which SL1 and SL2 have been removed (Cheng et al 2003). These deletions are predicted to favor the formation of the central base-paired region containing the amber/W site in the branched editing conformation over the formation of the structure around the amber/W site that occurs in the native unbranched rod structure and, consistent with this prediction, were shown to increase editing in transfected cells (Cheng et al 2003). Replacement of the 4 nt in the nonreplicating Peruvian RNA, Pnr, with Ecuadorian sequences, Pnr / E EDIT , resulted in a threefold reduction in editing (Fig.…”