Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen

Abstract

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.Hepatitis delta virus (HDV) is a subviral human pathogen that increases the risk of severe liver disease in those infected with its helper, hepatitis B virus (34). The HDV genome is an ϳ1,680-nucleotide (nt) circular RNA that replicates through a circular RNA intermediate, the antigenome (21). Both the genome and antigenome possess extensive intramolecular complementarity and are predicted to form rod-shaped structures in which about 70% of the nucleotides are base paired (39).HDV produces two forms of the sole viral protein, hepatitis delta antigen (HDAg) (4), and both are translated from a single mRNA that is transcribed from the genomic RNA (16,18,40). The shorter form, HDAg-S, is required for RNA replication, while the longer form, HDAg-L, inhibits replication but is required for packaging the viral RNA with the envelope of hepatitis B surface antigen (19,35,42). The virus uses adenosine-to-inosine RNA editing activity of the host cell to produce HDAg-S and HDAg-L from the same open reading frame (6, 25). The editing does not occur on the mRNA directly but on adenosine 1012 of the antigenome (9, 32). The nucleotide change is subsequently passed to the genome during replication and to the mRNA during transcription. The ultimate effect is the conversion of the UAG amber termination codon of HDAg-S to a UGG tryptophan codon required to synthesize the slightly longer HDAg-L; because of the codon change produced by this editing event, the editing site is called the amber/W site (32).We previously sh...

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

“…As yet, the factors that determine these variations are not well understood. 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.…”

Hepatitis Delta Virus Antigen Is Methylated at Arginine Residues, and Methylation Regulates Subcellular Localization and RNA Replication

“…The 0.8-kb mRNA is translated into S-HDAg, which is required for continuous HDV RNA replication (21). In the later stages of viral replication, an RNA editing event occurs at the amber termination codon of the S-HDAg ORF to allow the synthesis of L-HDAg (40,41), which is required for virus assembly (2,24). L-HDAg was also thought to inhibit HDV RNA replication when expressed artificially early in viral replication (4,24), but a recent study showed that L-HDAg does not play a significant role in regulating viral RNA levels in cells (29).…”

Réécriture du matériel génétique : fonctions et mécanismes de l’édition de l’ARN

“…Les conséquences de ce processus sur le cycle viral pourraient être très importantes. En effet, l'Ag-S/p24 codé par l'ARNm non édité participe au déclenchement de la réplica-tion virale, tandis que l'Ag-L/p27 produit après l'édition par ADAR inhibe la réplication, ce qui suggère que l'efficacité du processus d'édition jouerait un rôle décisif au cours de l'infection virale [41]. que la modification C-U paraît procéder par un mécanis-me analogue à celui touchant l'ARNm ApoB.…”