Immunoblot analysis demonstrates that the large and small forms of hepatitis delta virus antigen have different C-terminal amino acid sequences

Wang, Jia-Gang; Cullen, John M.; Lemon, Stanley M.

doi:10.1099/0022-1317-73-1-183

Cited by 34 publications

(34 citation statements)

References 17 publications

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“…Four days after RNA transfection, the cells were then transfected again, with or without the SUMO1-GG and Ubc9 expression plasmids. Two days later, the whole-cell extracts were prepared and then subjected to immunoblotting with a mouse monoclonal antibody against HDAg (lanes 1 and 2) or with a rabbit polyclonal antibody specific for L-HDAg (LP3) (49) (lanes 3 and 4).…”

Section: Fig 4 S-hdag Produced From Hdv Rna-replicating Cells Is a mentioning

confidence: 99%

Modification of Small Hepatitis Delta Virus Antigen by SUMO Protein

Tseng

Cheng

Shu

et al. 2010

J Virol

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Hepatitis delta antigen (HDAg) is a nuclear protein that is intimately involved in hepatitis delta virus (HDV) RNA replication. HDAg consists of two protein species, the small form (S-HDAg) and the large form (L-HDAg).Previous studies have shown that posttranslational modifications of S-HDAg, such as phosphorylation, acetylation, and methylation, can modulate HDV RNA replication. In this study, we show that S-HDAg is a small ubiquitin-like modifier 1 (SUMO1) target protein. Mapping data showed that multiple lysine residues are SUMO1 acceptors within S-HDAg. Using a genetic fusion strategy, we found that conjugation of SUMO1 to S-HDAg selectively enhanced HDV genomic RNA and mRNA synthesis but not antigenomic RNA synthesis. This result supports our previous proposition that the cellular machinery involved in the synthesis of HDV antigenomic RNA is different from that for genomic RNA synthesis and mRNA transcription, requiring different modified forms of S-HDAg. Sumoylation represents a new type of modification for HDAg.Hepatitis delta virus (HDV) causes chronic and, occasionally, fulminant hepatitis in humans (16). HDV is a satellite virus which requires hepatitis B virus (HBV) to supply envelope proteins for virus assembly and production (46). It contains a circular RNA genome of 1.7 kb which replicates through a double-rolling-circle mechanism in the nucleus (33). The viral genomic RNA (G-RNA) is first replicated into the full-length antigenomic RNA (AG-RNA) and is also transcribed into a 0.8-kb mRNA, which encodes the only HDV protein, hepatitis delta antigen (HDAg). The AG-RNA, in turn, is replicated into G-RNA by another round of rollingcircle replication. The production of HDAg, which is intimately involved in HDV RNA replication, is a unique feature distinguishing HDV from plant viroids, which do not encode any protein. HDAg consists of two species, the small delta antigen (S-HDAg) (195 amino acids [aa]; 24 kDa) and the large delta antigen (L-HDAg) (214 aa; 27 kDa), which play different roles in HDV replication. S-HDAg is an essential activator for HDV RNA replication (25). In contrast, L-HDAg inhibits certain stages of HDV RNA replication but is required for virion assembly (5,7,28,32). HDAg has been shown to be modified posttranslationally by phosphorylation (6, 9, 40, 42), acetylation (41), methylation (29), and in the case of L-HDAg, isoprenylation (14). Arg-13 methylation, Lys-72 acetylation, and Ser-177 phosphorylation are three major modifications of S-HDAg and are important for the functions of S-HDAg in HDV RNA replication (29,40,41,48). Isoprenylation on Cys-211 of L-HDAg is required for virus assembly (14). SUMO (small ubiquitin-related modifier) has been identified as a reversible posttranslational protein modifier (34, 35). The human genome encodes four SUMO proteins: SUMO1 to SUMO4 (13,17). Among them, SUMO1 to SUMO3 are ubiquitously expressed, whereas SUMO4 is expressed mainly in the kidneys, lymph nodes, and spleen (17). Sumoylation is carried out by an E1 activating enzyme (the heterodimer...

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Section: Fig 4 S-hdag Produced From Hdv Rna-replicating Cells Is a mentioning

confidence: 99%

Modification of Small Hepatitis Delta Virus Antigen by SUMO Protein

Tseng

Cheng

Shu

et al. 2010

J Virol

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“…Genomes synthesized from such edited antigenomes subsequently serve as templates for antigenomic sense mRNAs in which the amber stop codon has been replaced with a tryptophan codon. In mRNAs derived from edited antigenomes, an additional 19 or 20 codons are thereby translated to produce the long form of delta antigen, HDAg-L (Weiner et al 1988;Xia et al 1990;Casey et al 1992;Wang et al 1992). HDAg-L enables viral particle formation by interacting with the envelope protein of HBV, and inhibits replication (Kuo et al 1989;Chang et al 1991;Ryu et al 1992).…”

Section: Introductionmentioning

confidence: 99%

The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing

Linnstaedt

Kasprzak²,

Shapiro

et al. 2006

RNA

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RNA editing plays a critical role in the life cycle of hepatitis delta virus (HDV). The host editing enzyme ADAR1 recognizes specific RNA secondary structure features around the amber/W site in the HDV antigenome and deaminates the amber/W adenosine. A previous report suggested that a branched secondary structure is necessary for editing in HDV genotype III. This branched structure, which is distinct from the characteristic unbranched rod structure required for HDV replication, was only partially characterized, and knowledge concerning its formation and stability was limited. Here, we examine the secondary structures, conformational dynamics, and amber/W site editing of HDV genotype III RNA using a miniaturized HDV genotype III RNA in vitro. Computational analysis of this RNA using the MPGAfold algorithm indicated that the RNA has a tendency to form both metastable and stable unbranched secondary structures. Moreover, native polyacrylamide gel electrophoresis demonstrated that this RNA forms both branched and unbranched rod structures when transcribed in vitro. As predicted, the branched structure is a metastable structure that converts readily to the unbranched rod structure. Only branched RNA was edited at the amber/W site by ADAR1 in vitro. The structural heterogeneity of HDV genotype III RNA is significant because not only are both conformations of the RNA functionally important for viral replication, but the ratio of the two forms could modulate editing by determining the amount of substrate RNA available for modification.

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“…nation of this adenosine to inosine, which base pairs preferentially with C, leads to the production of an HDAg-encoding mRNA in which the UAG amber termination codon has been changed to UGG (tryptophan), thereby extending the open reading frame (ORF) to encode HDAg-L, which is 19 amino acids longer at the carboxyl terminus (38,41). Because of the codon change produced by this editing event, the editing site is called the amber/W site (31).…”

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confidence: 99%

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

Jayan

Casey

2002

J Virol

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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.

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Immunoblot analysis demonstrates that the large and small forms of hepatitis delta virus antigen have different C-terminal amino acid sequences

Cited by 34 publications

References 17 publications

Modification of Small Hepatitis Delta Virus Antigen by SUMO Protein

Modification of Small Hepatitis Delta Virus Antigen by SUMO Protein

The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing

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

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