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.

Hepatitis delta virus (HDV) requires host RNA editing at the viral RNA amber/W site. Of the two host genes responsible for RNA editing via deamination of adenosines in double-stranded RNAs, short inhibitory RNAmediated knockdown of host ADAR1 expression but not that of ADAR2 led to decreased HDV amber/W editing and virus production. Despite substantial sequence and structural variation among the amber/W sites of the three HDV genotypes, ADAR1a was primarily responsible for editing all three. We conclude that ADAR1 is primarily responsible for editing HDV RNA at the amber/W site during HDV infection.Hepatitis delta virus (HDV) infection causes severe acute and chronic hepatitis in those infected with its helper, hepatitis B virus (32). A central event in the HDV replication cycle is an RNA editing event that allows the virus to produce two forms of the sole viral protein, hepatitis delta antigen (HDAg), that have different and opposed functions in the HDV replication cycle (reviewed in reference 14). Editing involves the specific deamination of the amber/W site adenosine to inosine and changes the stop codon of HDAg-S to a tryptophan codon for 7,26,30).In mammals, the ADAR1 and ADAR2 genes encode proteins that edit specific adenosines in double-stranded RNA segments (reviewed in references 15, 20, and 33), and ADAR1 and ADAR2 proteins can specifically edit the amber/W site in HDV RNA (18,33,36) as well as adenosines in several cellular pre-mRNA substrates (15,20,34). The product of a third related gene, ADAR3, has no apparent deaminase activity on other ADAR1 or ADAR2 substrates (9, 27) and is unlikely to edit HDV RNA. ADAR1 is expressed in many tissues, while the highest level of ADAR2 expression is found in the brain (21, 28). The relative levels of ADAR1 and ADAR2 RNA expression have been analyzed by Northern blotting for some tissues (9, 22) but not for the liver. Using Northern blot hybridization and reverse transcription-PCR (RT-PCR), we analyzed ADAR1 and ADAR2 expression both in cultured Huh-7 human hepatoma cells and in HDV-infected liver tissue and found that the expression level of ADAR1 is 10-to 20-fold higher than that of ADAR2. These data are consistent with the general pattern of ADAR1 and ADAR2 expression (9,21,27) and could suggest that ADAR1 is principally responsible for HDV amber/W editing in infected hepatocytes. However, these enzymes can exhibit differential activities on some substrates (28,33,36). Although previous studies (18,33,36) showed that both ADAR1 and ADAR2 can edit HDV RNA when overexpressed in Huh-7 cells, their relative activities on the HDV amber/W site were not investigated: amber/W editing activities were analyzed only at very high, possibly saturating, levels of ADAR expression.We sought to determine the extent to which ADAR1 and ADAR2 and their splice variants are responsible for HDV RNA editing in vivo by using short inhibitory RNAs (siRNAs) (2, 10) to specifically knock down expression of ADAR1 or ADAR2 in cultured Huh-7 cells. siRNAs (Table 1) were designed as double-s...

RNA editing of the hepatitis delta virus (HDV) antigenome at the amber/W site by the host RNA adenosine deaminase ADAR1 is a critical step in the HDV replication cycle. Editing is required for production of the viral protein hepatitis delta antigen long form (HDAg-L), which is necessary for viral particle production but can inhibit HDV RNA replication. The RNA secondary structural features in ADAR1 substrates are not completely defined, but base pairing in the 20-nucleotide (nt) region 3 of editing sites is thought to be important. The 25-nt region 3 of the HDV amber/W site in HDV genotype I RNA consists of a conserved secondary structure that is mostly base paired but also has asymmetric internal loops and single-base bulges. To understand the effect of this 3 region on the HDV replication cycle, mutations that either increase or decrease base pairing in this region were created and the effects of these changes on amber/W site editing, RNA replication, and virus production were studied. Increased base pairing, particularly in the region 15 to 25 nt 3 of the editing site, significantly increased editing; disruption of base pairing in this region had little effect. Increased editing resulted in a dramatic inhibition of HDV RNA synthesis, mostly due to excess HDAg-L production. Although virus production at early times was unaffected by this reduced RNA replication, at later times it was significantly reduced. Therefore, it appears that the conserved RNA secondary structure around the HDV genotype I amber/W site has been selected not for the highest editing efficiency but for optimal viral replication and secretion.Hepatitis delta virus (HDV) causes acute severe and chronic liver disease in humans. HDV encodes just one protein, hepatitis delta antigen (HDAg), and relies heavily on host functions and the structure of its RNA for replicative functions. One of these functions is RNA editing, which plays a central role in the HDV replication cycle. During HDV replication, the host RNA adenosine deaminase ADAR1 deaminates, or edits, the adenosine in the UAG (amber) stop codon for the short form of HDAg (HDAg-S) to inosine (11,27,31). As a result of editing this adenosine, referred to as the amber/W site (27), the HDAg reading frame is extended by an additional 19 to 20 amino acids to encode the long form of HDAg (HDAg-L). Editing is critical for HDV because HDAg-S is required for viral RNA replication and HDAg-L is required for packaging (6, 7). HDV must modulate the extent of editing for at least three reasons. First, both edited and unedited genomes are packaged, but only unedited genomes are likely to be infectious because HDAg-L does not support RNA replication; second, insufficient HDAg-L production will decrease virus secretion (11); and third, excessive and premature production of HDAg-L can inhibit RNA replication (10, 29), although the significance of this last point has been questioned (20).Like other substrates for editing by ADAR1, editing at the HDV amber/W site requires specific structures in the immediate...

The purposes of this study were to identify exogenous factors that would depress synthesis of saturated fats and enhance synthesis of unsaturated fats in the dairy cow’s mammary gland. Certain long‐chain exogenous fatty acids are known to modulate endogenous fat synthesis within tissues. We analyzed the effects of two different long‐chain monounsaturated fatty acids, namely oleic acid and trans‐vaccenic acid (TVA), on activities of acetyl‐CoA carboxylase (ACC), fatty acid synthetase (FAS) and stearoyl‐CoA desaturase (SCD) in bovine mammary epithelial cell cultures. The study was done using an established bovine mammary epithelial cell line, the MacT cells. ACC (EC 6.4.1.2) and FAS (EC 2.3.1.85) are two major enzymes involved in biosynthesis of saturated fatty acids in eucaryotic cells. SCD (EC 1.14.99.5) is the enzyme catalyzing the critical committed step in biosynthesis of unsaturated fatty acids from their saturated precursors. Data indicated depression of activity of enzymes responsible for mammary synthesis of saturated fatty acids (ACC and FAS), along with a simultaneous enhancement of mammary desaturase activity, by TVA.

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...