scholarly journals Genotype-Specific Complementation of Hepatitis Delta Virus RNA Replication by Hepatitis Delta Antigen

1998 ◽  
Vol 72 (4) ◽  
pp. 2806-2814 ◽  
Author(s):  
John L. Casey ◽  
John L. Gerin
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Replication ◽  
Cdna Clones ◽  
Genotype I ◽  
Hepatitis Delta ◽  
Genotype Iii ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen ◽  
Hdv Genotype ◽  
Delta Virus

ABSTRACT Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.

scholarly journals Large Hepatitis Delta Antigen Is Not a Suppressor of Hepatitis Delta Virus RNA Synthesis once RNA Replication Is Established

2002 ◽  
Vol 76 (19) ◽  
pp. 9910-9919 ◽  
Author(s):  
Thomas B. Macnaughton ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
State Level ◽  
Rna Replication ◽  
Replication Cycle ◽  
Steady State Level ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen ◽  
Delta Virus

ABSTRACT Moderation of hepatitis delta virus (HDV) replication is a likely prerequisite in the establishment of chronic infections and is thought to be mediated by the intracellular accumulation of large hepatitis delta antigen (L-HDAg). The regulatory role of this protein was suggested from several studies showing that cotransfection of plasmid cDNAs expressing both L-HDAg and HDV RNA results in a potent inhibition of HDV RNA replication. However, since this approach differs significantly from natural HDV infections, where HDV RNA replication is initiated from an RNA template, and L-HDAg appears only late in the replication cycle, it remains unclear whether L-HDAg can modulate HDV RNA replication in the natural HDV replication cycle. In this study, we investigated the effect of L-HDAg, produced as a result of the natural HDV RNA editing event, on HDV RNA replication. The results showed that following cDNA-free HDV RNA transfection, a steady-state level of RNA was established at 3 to 4 days posttransfection. The same level of HDV RNA was reached when a mutant HDV genome unable to make L-HDAg was used, suggesting that L-HDAg did not play a role. The rates of HDV RNA synthesis, as measured by metabolic labeling experiments, were identical at 4 and 8 days posttransfection and in the wild type and the L-HDAg-deficient mutant. We further examined the effect of overexpression of L-HDAg at various stages of the HDV replication cycle, showing that HDV RNA synthesis was resistant to L-HDAg when it was overexpressed 3 days after HDV RNA replication had initiated. Finally, we showed that, contrary to conventional thinking, L-HDAg alone, at a certain molar ratio with HDV RNA, can initiate HDV RNA replication. Thus, L-HDAg does not inherently inhibit HDV RNA synthesis. Taken together, these results indicated that L-HDAg affects neither the rate of HDV RNA synthesis nor the final steady-state level of HDV RNA and that L-HDAg is unlikely to act as an inhibitor of HDV RNA replication in the natural HDV replication cycle.

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 Transcription of Hepatitis Delta Antigen mRNA Continues throughout Hepatitis Delta Virus (HDV) Replication: a New Model of HDV RNA Transcription and Replication

1998 ◽  
Vol 72 (7) ◽  
pp. 5449-5456 ◽  
Author(s):  
Lucy E. Modahl ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Hepatitis Delta ◽  
New Model ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen ◽  
Genomic Length ◽  
Polyadenylated Mrna ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) replicates by RNA-dependent RNA synthesis according to a double rolling circle model. Also synthesized during replication is a 0.8-kb, polyadenylated mRNA encoding the hepatitis delta antigen (HDAg). It has been proposed that this mRNA species represents the initial product of HDV RNA replication; subsequent production of genomic-length HDV RNA relies on suppression of the HDV RNA polyadenylation signal by HDAg. However, this model was based on studies which required the use of an HDV cDNA copy to initiate HDV RNA replication in cell culture, thus introducing an artificial requirement for DNA-dependent RNA synthesis. We have now used an HDV cDNA-free RNA transfection system and a method that we developed to detect specifically the mRNA species transcribed from the HDV RNA template. We established that this polyadenylated mRNA is 0.8 kb in length and its 5′ end begins at nucleotide 1631. Surprisingly, kinetic studies showed that this mRNA continued to be synthesized even late in the viral replication cycle and that the mRNA and the genomic-length RNA increased in parallel, even in the presence of HDAg. Thus, a switch from production of the HDAg mRNA to the full-length HDV RNA does not occur in this system, and suppression of the polyadenylation site by HDAg may not significantly regulate the synthesis of the HDAg mRNA, as previously proposed. These findings reveal novel insights into the mechanism of HDV RNA replication. A new model of HDV RNA replication and transcription is proposed.

scholarly journals Genetic Changes in Hepatitis Delta Virus from Acutely and Chronically Infected Woodchucks

2006 ◽  
Vol 80 (13) ◽  
pp. 6469-6477 ◽  
Author(s):  
John L. Casey ◽  
Bud C. Tennant ◽  
John L. Gerin
Keyword(s):  
Hepatitis Delta Virus ◽  
Hepatitis Virus ◽  
Consensus Sequence ◽  
Genotype I ◽  
Hepatitis Delta ◽  
Genetic Changes ◽  
Host Immune Responses ◽  
Hepatitis Delta Antigen ◽  
Hdv Infection ◽  
Delta Virus

ABSTRACT A woodchuck-derived hepatitis delta virus (HDV) inoculum was created by transfection of a genotype I HDV cDNA clone directly into the liver of a woodchuck that was chronically infected with woodchuck hepatitis virus. All woodchucks receiving this inoculum became positive for HDV RNA in serum, and 67% became chronically infected, similar to the rate of chronic HDV infection in humans. Analysis of HDV sequences obtained at 73 weeks postinfection indicated that changes had occurred at a rate of 0.5% per year; many of these modifications were consistent with editing by host RNA adenosine deaminase. The appearance of sequence changes, which were not evenly distributed on the genome, was correlated with the course of HDV infection. A limited number of modifications occurred in the consensus sequence of the viral genome that altered the sequence of the hepatitis delta antigen (HDAg). All chronically infected animals examined exhibited these changes 73 weeks following infection, but at earlier times, only one of the HDV carriers exhibited consensus sequence substitutions. On the other hand, sequence modifications in animals that eventually recovered from HDV infection were apparent after 27 weeks. The data are consistent with a model in which HDV sequence changes are selected by host immune responses. Chronic HDV infection in woodchucks may result from a delayed and weak immune response that is limited to a small number of epitopes on HDAg.

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 Interaction and Replication Activation of Genotype I and II Hepatitis Delta Antigens

2004 ◽  
Vol 78 (6) ◽  
pp. 2693-2700 ◽  
Author(s):  
Sheng-Chieh Hsu ◽  
Jaw-Ching Wu ◽  
I-Jane Sheen ◽  
Wan-Jr Syu
Keyword(s):  
Genotype I ◽  
Hepatitis Delta ◽  
Hepatitis D ◽  
Virus Surface ◽  
Delta Antigen ◽  
B Virus ◽  
Hepatitis Delta Antigen ◽  
Different Origins ◽  
Genotype Ii ◽  
Hdv Genotype

ABSTRACT The nucleotide sequences of hepatitis D viruses (HDV) vary 5 to 14% among isolates of the same genotype and 23 to 34% among different genotypes. The only viral-genome-encoded antigen, hepatitis delta antigen (HDAg), has two forms that differ in size. The small HDAg (HDAg-S) trans-activates viral replication, while the large form (HDAg-L) is essential for viral assembly. Previously, it has been shown that the packaging efficiency of HDAg-L is higher for genotype I than for genotype II. In this study, the question of whether other functional properties of the HDAgs are affected by genotype differences is addressed. By coexpression of the two antigens in HuH-7 cells followed by specific antibody precipitation, it was found that HDAgs of different origins interacted without genotypic discrimination. Moreover, in the presence of hepatitis B virus surface antigen, HDAg-S was incorporated into virion-like particles through interaction with HDAg-L without genotype restriction. As to the differences in replication activation of genotype I HDV RNA, all HDAg-S clones tested had some trans-activation activity, and this activity varied greatly among isolates. As to the support of HDV genotype II replication, only clones of HDAg-S from genotype II showed trans-activation activity, and this activity also varied among isolates. In conclusion, genotype has no effect on HDAg interaction and genotype per se only partly predicts how much the HDAg-S of an HDV isolate affects the replication of a second HDV isolate.

scholarly journals Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

2013 ◽  
Vol 87 (15) ◽  
pp. 8665-8674 ◽  
Author(s):  
L. H. Daigh ◽  
B. L. Griffin ◽  
A. Soroush ◽  
M. R. Mamedov ◽  
J. L. Casey
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Binding ◽  
Binding Activity ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
Virus Rna ◽  
Hepatitis Delta Antigen ◽  
Delta Virus
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