scholarly journals Hepatitis Delta Virus RNA Encoding the Large Delta Antigen Cannot Sustain Replication due to Rapid Accumulation of Mutations Associated with RNA Editing

2003 ◽  
Vol 77 (22) ◽  
pp. 12048-12056 ◽  
Author(s):  
Thomas B. Macnaughton ◽  
Yi-Ija Li ◽  
Alison L. Doughty ◽  
Michael M. C. Lai
Keyword(s):  
Rna Editing ◽  
Hepatitis Delta Virus ◽  
Rna Replication ◽  
Site Directed Mutagenesis ◽  
Editing Event ◽  
Putative Promoter Region ◽  
Hepatitis Delta ◽  
Time Points ◽  
Rna Transfection ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains two RNA species (HDV-S and HDV-L), which encode the small and large forms of hepatitis delta antigens (S- and L-HDAg), respectively. HDV-L RNA is a result of an RNA editing event occurring at an amber/W site of HDV-S RNA. RNA editing must be regulated to prevent premature and excessive accumulation of HDV-L RNA in the viral life cycle. In this study, we used an RNA transfection procedure to study the replication abilities of HDV-L and HDV-S RNA. While HDV-S led to robust RNA replication, HDV-L could not replicate even after 6 days following transfection. The failure of HDV-L to replicate was not due to insufficient amounts of S-HDAg, as identical results were obtained in a cell line that stably overexpresses S-HDAg. Also, it was not due to possible inhibition by L-HDAg, as HDV-S RNA replication was not affected when both HDV-L and HDV-S RNA were cotransfected. Further, when L-HDAg expression from HDV-L RNA was abolished by site-directed mutagenesis, the mutant HDV-L RNA also failed to replicate. Unexpectedly, when the kinetics of RNA replication was examined daily, HDV-L was found to replicate at a low level at the early time points (1 to 2 days posttransfection) but then lose this capability at later time points. Sequence analysis of the replicated HDV-L RNA at day 1 posttransfection showed that it had undergone multiple nucleotide changes, particularly in the region near the putative promoter region of HDV RNA replication. In contrast, very few mutations were found in HDV-S RNA. These results suggest that the editing at the amber/W site triggers a series of additional mutations which rapidly reduce the replication efficiency of the resultant HDV genome and thus help regulate the amount of HDV-L RNA in infected cells. They also explain why L-HDAg is not produced early in HDV infection, despite the fact that HDV-L RNA is present in the virion.

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

2002 ◽  
Vol 76 (8) ◽  
pp. 3819-3827 ◽  
Author(s):  
Geetha C. Jayan ◽  
John L. Casey
Keyword(s):  
Rna Editing ◽  
Hepatitis Delta Virus ◽  
Rna Replication ◽  
Rna Packaging ◽  
High Level Expression ◽  
Hepatitis Delta ◽  
Huh7 Cells ◽  
Hepatitis Delta Antigen ◽  
High Level ◽  
Delta Virus

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 non-amber/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.

scholarly journals Effects of Conserved RNA Secondary Structures on Hepatitis Delta Virus Genotype I RNA Editing, Replication, and Virus Production

2005 ◽  
Vol 79 (17) ◽  
pp. 11187-11193 ◽  
Author(s):  
Geetha C. Jayan ◽  
John L. Casey
Keyword(s):  
Secondary Structure ◽  
Rna Editing ◽  
Hepatitis Delta Virus ◽  
Virus Production ◽  
Rna Replication ◽  
Base Pairing ◽  
Genotype I ◽  
Hepatitis Delta ◽  
Hdv Genotype ◽  
Delta Virus

ABSTRACT 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.

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

2003 ◽  
Vol 77 (14) ◽  
pp. 7786-7795 ◽  
Author(s):  
Qiufang Cheng ◽  
Geetha C. Jayan ◽  
John L. Casey
Keyword(s):  
Rna Editing ◽  
Hepatitis Delta Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Structural Elements ◽  
Virus Genotype ◽  
Hepatitis Delta ◽  
Genotype Iii ◽  
Hdv Genotype ◽  
Delta Virus

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.

scholarly journals Multiple genomic sequences of hepatitis delta virus are associated with cDNA promoter activity and RNA double rolling-circle replication

2012 ◽  
Vol 93 (3) ◽  
pp. 577-587 ◽  
Author(s):  
Fu-Tien Liao ◽  
Li-Sung Hsu ◽  
Jiunn-Liang Ko ◽  
Chun-Che Lin ◽  
Gwo-Tarng Sheu
Keyword(s):  
Hepatitis Delta Virus ◽  
Promoter Activity ◽  
Rna Synthesis ◽  
Stop Codon ◽  
Rna Replication ◽  
Site Directed Mutagenesis ◽  
Hepatitis Delta ◽  
Pol Ii ◽  
Rolling Circle ◽  
Delta Virus

To understand how DNA-dependent RNA polymerase II (pol II) recognizes hepatitis delta virus (HDV) RNA as a template, it is first necessary to identify the HDV sequence that acts as a promoter of pol II-initiated RNA synthesis. Therefore, we isolated the pol II-response element from HDV cDNA and examined the regulation by hepatitis delta antigens (HDAgs). Two HDV cDNA fragments containing bidirectional promoter activity were identified. One was located at nt 1582–1683 (transcription-promoter region 1, TR-P1) and the other at nt 1223–1363 (transcription-internal region 5, TR-I5). The promoter activities of these two regions were enhanced by HDAgs to differing degrees. Next, the role of these sequences in an HDV cDNA-free RNA replication system was characterized by site-directed mutagenesis. Our data showed that: (i) the AUG codon at the HDAg ORF of HDV RNA (nt 1599–1601) that mutates to UAG (amber stop codon) results in loss of dimeric but not monomeric HDV RNA synthesis. (ii) A 5 nt mutation of TR-P1 (P1-m5, nt 1670–1674) abolishes RNA replication completely. Two-nucleotide-mutated RNA (P1-m2, nt 1662–1663) is able to synthesize short RNAs but not monomeric HDV RNA. (iii) A mutation in 5 nt at the TR-I5 region (I5-m5, nt 1351–1355) also abolishes HDV replication. Mutants with 2 nt mutations (I5-m2, nt 1351–1352) or 3 nt mutations (I5-m3, nt 1353–1355) inhibit HDV dimeric but not monomeric RNA synthesis. Furthermore, large HDAg is expressed in cells transfected with I5-m3 and I5-m2 RNAs and that demonstrate the RNA-editing event in the monomeric HDV RNA. These results provide further understanding of the double rolling-circle mechanism in HDV RNA replication.

Hepatitis Delta Virus cDNA Monomer Can Be Used in Transfection Experiments to Initiate Viral RNA Replication

Virology ◽  
1993 ◽  
Vol 197 (1) ◽  
pp. 137-142 ◽  
Author(s):  
Fei-Ping Tai ◽  
Pei-Jer Chen ◽  
Fu-Lin Chang ◽  
Ding-Shinn Chen
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Hepatitis Delta ◽  
Virus Cdna ◽  
Delta Virus

scholarly journals The Large Delta Antigen of Hepatitis Delta Virus Potently Inhibits Genomic but Not Antigenomic RNA Synthesis: a Mechanism Enabling Initiation of Viral Replication

2000 ◽  
Vol 74 (16) ◽  
pp. 7375-7380 ◽  
Author(s):  
Lucy E. Modahl ◽  
Michael M. C. Lai
Keyword(s):  
Life Cycle ◽  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Dominant Negative ◽  
Genomic Rna ◽  
Inhibitory Effects ◽  
Hepatitis Delta ◽  
Artificial Dna ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains two types of hepatitis delta antigens (HDAg) in the virion. The small form (S-HDAg) is required for HDV RNA replication, whereas the large form (L-HDAg) potently inhibits it by a dominant-negative inhibitory mechanism. The sequential appearance of these two forms in the infected cells regulates HDV RNA synthesis during the viral life cycle. However, the presence of almost equal amounts of S-HDAg and L-HDAg in the virion raised a puzzling question concerning how HDV can escape the inhibitory effects of L-HDAg and initiate RNA replication after infection. In this study, we examined the inhibitory effects of L-HDAg on the synthesis of various HDV RNA species. Using an HDV RNA-based transfection approach devoid of any artificial DNA intermediates, we showed that a small amount of L-HDAg is sufficient to inhibit HDV genomic RNA synthesis from the antigenomic RNA template. However, the synthesis of antigenomic RNA, including both the 1.7-kb HDV RNA and the 0.8-kb HDAg mRNA, from the genomic-sense RNA was surprisingly resistant to inhibition by L-HDAg. The synthesis of these RNAs was inhibited only when L-HDAg was in vast excess over S-HDAg. These results explain why HDV genomic RNA can initiate replication after infection even though the incoming viral genome is complexed with equal amounts of L-HDAg and S-HDAg. These results also suggest that the mechanisms of synthesis of genomic versus antigenomic RNA are different. This study thus resolves a puzzling question about the early events of the HDV life cycle.

scholarly journals Hepatitis Delta Virus Minimal Substrates Competent for Editing by ADAR1 and ADAR2

2001 ◽  
Vol 75 (18) ◽  
pp. 8547-8555 ◽  
Author(s):  
Shuji Sato ◽  
Swee Kee Wong ◽  
David W. Lazinski
Keyword(s):  
Hepatitis Delta Virus ◽  
Cultured Cells ◽  
Reporter System ◽  
Editing Event ◽  
Hepatitis Delta ◽  
Reading Frame ◽  
Adenosine Deaminases ◽  
Delta Antigen ◽  
Amber Codon ◽  
Delta Virus

ABSTRACT A host-mediated RNA-editing event allows hepatitis delta virus (HDV) to express two essential proteins, the small delta antigen (HDAg-S) and the large delta antigen (HDAg-L), from a single open reading frame. One or several members of the ADAR (adenosine deaminases that act on RNA) family are thought to convert the adenosine to an inosine (I) within the HDAg-S amber codon in antigenomic RNA. As a consequence of replication, the UIG codon is converted to a UGG (tryptophan [W]) codon in the resulting HDAg-L message. Here, we used a novel reporter system to monitor the editing of the HDV amber/W site in the absence of replication. In cultured cells, we observed that both human ADAR1 (hADAR1) and hADAR2 were capable of editing the amber/W site with comparable efficiencies. We also defined the minimal HDV substrate required for hADAR1- and hADAR2-mediated editing. Only 24 nucleotides from the amber/W site were sufficient to enable efficient editing by hADAR1. Hence, the HDV amber/W site represents the smallest ADAR substrate yet identified. In contrast, the minimal substrate competent for hADAR2-mediated editing contained 66 nucleotides.

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

2004 ◽  
Vol 78 (23) ◽  
pp. 13325-13334 ◽  
Author(s):  
Yi-Jia Li ◽  
Michael R. Stallcup ◽  
Michael M. C. Lai
Keyword(s):  
Subcellular Localization ◽  
Posttranslational Modification ◽  
Hepatitis Delta Virus ◽  
Rna Binding ◽  
Rna Replication ◽  
Genomic Rna ◽  
Hepatitis Delta ◽  
Arginine Residues ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains a circular RNA which encodes a single protein, hepatitis delta antigen (HDAg). HDAg exists in two forms, a small form (S-HDAg) and a large form (L-HDAg). S-HDAg can transactivate HDV RNA replication. Recent studies have shown that posttranslational modifications, such as phosphorylation and acetylation, of S-HDAg can modulate HDV RNA replication. Here we show that S-HDAg can be methylated by protein arginine methyltransferase (PRMT1) in vitro and in vivo. The major methylation site is at arginine-13 (R13), which is in the RGGR motif of an RNA-binding domain. The methylation of S-HDAg is essential for HDV RNA replication, especially for replication of the antigenomic RNA strand to form the genomic RNA strand. An R13A mutation in S-HDAg inhibited HDV RNA replication. The presence of a methylation inhibitor, S-adenosyl-homocysteine, also inhibited HDV RNA replication. We further found that the methylation of S-HDAg affected its subcellular localization. Methylation-defective HDAg lost the ability to form a speckled structure in the nucleus and also permeated into the cytoplasm. These results thus revealed a novel posttranslational modification of HDAg and indicated its importance for HDV RNA replication. This and other results further showed that, unlike replication of the HDV genomic RNA strand, replication of the antigenomic RNA strand requires multiple types of posttranslational modification, including the phosphorylation and methylation of HDAg.

scholarly journals Genomic but Not Antigenomic Hepatitis Delta Virus RNA Is Preferentially Exported from the Nucleus Immediately after Synthesis and Processing

2002 ◽  
Vol 76 (8) ◽  
pp. 3928-3935 ◽  
Author(s):  
Thomas B. Macnaughton ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis Delta Virus ◽  
Large Fraction ◽  
Rna Replication ◽  
Metabolic Labeling ◽  
Rolling Circle Replication ◽  
Cell Region ◽  
Hepatitis Delta ◽  
Rolling Circle ◽  
Rna Export ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains a viroid-like circular RNA that replicates via a double rolling circle replication mechanism. It is generally assumed that HDV RNA is synthesized and remains exclusively in the nucleus until being exported to the cytoplasm for virion assembly. Using a [32P]orthophosphate metabolic labeling procedure to study HDV RNA replication (T. B. Macnaughton, S. T. Shi, L. E. Modahl, and M. M. C. Lai. J. Virol. 76:3920-3927, 2002), we unexpectedly found that a significant amount of newly synthesized HDV RNA was detected in the cytoplasm. Surprisingly, Northern blot analysis revealed that the genomic-sense HDV RNA is present almost equally in both the nucleus and cytoplasm, whereas antigenomic HDV RNA was mostly retained in the nucleus, suggesting the specific and highly selective export of genomic HDV RNA. Kinetic studies showed that genomic HDV RNA was exported soon after synthesis. However, only the monomer and, to a lesser extent, the dimer HDV RNAs were exported to the cytoplasm; very little higher-molecular-weight HDV RNA species were detected in the cytoplasm. These results suggest that the cleavage and processing of HDV RNA may facilitate RNA export. The export of genomic HDV RNA was resistant to leptomycin B, indicating that a cell region maintenance 1 (Crm1)-independent pathway was involved. The large form of hepatitis delta antigen (L-HDAg), which is responsible for virus packaging, was not required for RNA export, as a mutant HDV RNA genome unable to synthesize L-HDAg was still exported. The proportions of genomic HDV RNA in the nucleus and cytoplasm remained relatively constant throughout replication, indicating that export of genomic HDV RNA occurred continuously. In contrast, while antigenomic HDV RNA was predominately in the nucleus, there was a proportionally large fraction of antigenomic HDV RNA in the cytoplasm at early time points of RNA replication. These findings uncover a previously unrecognized presence of HDV RNA in the cytoplasm, which may have implications for viral RNA synthesis and packaging.

scholarly journals RNA Editing and its Control in Hepatitis Delta Virus Replication

Viruses ◽  
2010 ◽  
Vol 2 (1) ◽  
pp. 131-146 ◽  
Author(s):  
Renxiang Chen ◽  
Sarah Linnstaedt ◽  
John Casey
Keyword(s):  
Rna Editing ◽  
Virus Replication ◽  
Hepatitis Delta Virus ◽  
Hepatitis Delta ◽  
Delta Virus
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