Increased RNA Editing and Inhibition of Hepatitis Delta Virus Replication by High-Level Expression of ADAR1 and ADAR2

Abstract

Hepatitis delta virus (HDV) is a subviral human pathogen that uses specific RNA editing activity of the host to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the longer form (HDAg-L), which is required for RNA packaging but which is a potent trans-dominant inhibitor of HDV RNA replication. Editing in infected cells is thought to be catalyzed by one or more of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). We examined the effects of increased ADAR1 and ADAR2 expression on HDV RNA editing and replication in transfected Huh7 cells. We found that both ADARs dramatically increased RNA editing, which was correlated with strong inhibition of HDV RNA replication. While increased HDAg-L production was the primary mechanism of inhibition, we observed at least two additional means by which ADARs can suppress HDV replication. High-level expression of both ADAR1 and ADAR2 led to extensive hyperediting at nonamber/W sites and subsequent production of HDAg variants that acted as trans-dominant inhibitors of HDV RNA replication. Moreover, we also observed weak inhibition of HDV RNA replication by mutated forms of ADARs defective for deaminase activity. Our results indicate that HDV requires highly regulated and selective editing and that the level of ADAR expression can play an important role: overexpression of ADARs inhibits HDV RNA replication and compromises virus viability.Hepatitis delta virus (HDV) is a subviral satellite of hepatitis B virus (HBV) that increases the severity of HBV-related disease (33). The HDV particle has three components: the HDV RNA genome, hepatitis delta antigen (HDAg), which is the sole HDV protein, and the hepatitis B surface antigen (HBsAg), which is the sole helper function provided by the helper virus, HBV (4,20,33). The RNA genome of HDV is a single-stranded circular molecule in which about 70% of the nucleotides can form Watson-Crick base pairs in an unbranched rod structure (18,40). The RNA may resemble an imperfect double-stranded RNA (dsRNA) with short (Ͻ15 bp) double-stranded regions interspersed with numerous mismatches, bulges, and internal loops.

Adenosine Deaminases Acting on RNA, RNA Editing, and Interferon Action

“…3) near the N-terminal portion (Rueter and others 1999). Viral RNA species that are targeted for A-to-I modifications by both ADAR1 and ADAR2 include the site-selective editing at the amber/W site of HDV RNA (Sato and others 2001;Jayan and Casey 2002a). However, the p110 form of ADAR1 appears to be the predominant enzyme responsible for the HDV RNA editing (Wong and Lazinski 2002), and the exact role of ADAR2 in editing viral RNAs is currently unclear.…”

“…Without editing, genome packaging does not occur; ADAR1, therefore, is necessary for virus replication. However, high levels of ADAR1 protein inhibit HDV replication ( Jayan and Casey 2002a;Hartwig and others 2004;Casey 2006). Overexpression of ADAR1 or ADAR2 in Huh-7 cells, or treatment with IFN that induces the p150 form of ADAR1, results in increased HDV RNA editing and decreased HDV replication ( Jayan and Casey 2002a;Hartwig and others 2004).…”

“…However, high levels of ADAR1 protein inhibit HDV replication ( Jayan and Casey 2002a;Hartwig and others 2004;Casey 2006). Overexpression of ADAR1 or ADAR2 in Huh-7 cells, or treatment with IFN that induces the p150 form of ADAR1, results in increased HDV RNA editing and decreased HDV replication ( Jayan and Casey 2002a;Hartwig and others 2004). Likewise, a replication-competent HDV virus mutant that shows increased editing at the amber/W site produces increased amounts of large HDAg early in infection, and replication of the mutant virus terminates prematurely (Sato and others 2004;Casey 2006).…”

“…Site-selective A-to-I editing is exemplified by the editing of viral RNAs, including the hepatitis delta virus (HDV) antigenome RNA ( Jayan and Casey 2002a) and the human herpes virus 8 (HHV8) kaposin K12 transcript RNA (Gandy and others 2007), and editing of cellular mRNA transcripts in the nervous system that encode neurotransmitter receptors for l-glutamate (GluR) and serotonin (5-HT2cR) (Higuchi and others 1993;Burns and others 1997;Liu and others 1999;Seeburg and Hartner 2003). In these cases, the high positional selectivity of the A-to-I RNA editing events leads to the synthesis of specific protein products with altered function.…”

Host Response to Polyomavirus Infection Is Modulated by RNA Adenosine Deaminase ADAR1 but Not by ADAR2

“…To achieve the full editing of 5HT-2cR pre-mRNA transcripts that results in three amino acid substitutions in exon 3, both ADAR1 and ADAR2 are necessary (2,12,16,24,34,49). The constitutively expressed form of ADAR1 is responsible primarily for the editing of the highly structured HDV antigenomic site that leads to the conversion of an amber stop codon to a tryptophan codon, allowing the synthesis of large delta antigen (18,53).…”