Initiation of hepatitis delta virus (HDV) replication: HDV RNA encoding the large delta antigen cannot replicate

The hepatitis delta virus (HDV) nucleocapsid consists of a genomic-length RNA of 1·7 kb and approximately equimolar amounts of the small and large forms of the hepatitis delta antigen (S-HDAg and L-HDAg, respectively). Since HDV RNA particles contain not only a genomic RNA species encoding S-HDAg but also an RNA species encoding L-HDAg, which is produced by an RNA-editing process, the question arises as to whether RNAs encoding either L-HDAg or S-HDAg can initiate replication. To study this, two cDNA-free transfection methods were employed: HDV RNA cotransfected with either the S-HDAg-encoding mRNA species or the ribonucleocapsid protein complex, comprising HDV RNA and recombinant S-HDAg. Results showed that the genomic-sense RNA encoding S-HDAg could promote HDV replication, whereas the L-HDAg-encoding RNA species was unable to replicate under the same conditions. The antigenomic RNA species encoding either S-HDAg or L-HDAg could not replicate by either of these procedures. In addition, L-HDAg alone could not promote replication of the genomic RNA but, by supplementing an equal amount of S-HDAg, replication occurred. These data indicate that L-HDAg-encoding RNA species are probably not involved in the initiation of HDV RNA synthesis; instead, their main function may be to serve as template for producing L-HDAg, which regulates HDV RNA synthesis and virion assembly. These results suggest that the genomic RNA species encoding S-HDAg is the only functional genome for HDV infection and explain why the presence of the edited HDV RNA encoding L-HDAg does not interfere with HDV infection.

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Transcription of Subgenomic mRNA of Hepatitis Delta Virus Requires a Modified Hepatitis Delta Antigen That Is Distinct from Antigenomic RNA Synthesis

Journal of Virology ◽

10.1128/jvi.00428-08 ◽

2008 ◽

Vol 82 (19) ◽

pp. 9409-9416 ◽

Cited By ~ 20

Author(s):

Chung-Hsin Tseng ◽

King-Song Jeng ◽

Michael M. C. Lai

Keyword(s):

Hepatitis Delta Virus ◽

Rna Synthesis ◽

Nuclear Bodies ◽

Genomic Rna ◽

Mrna Transcription ◽

Hepatitis Delta ◽

Qrt Pcr ◽

Delta Antigen ◽

Hepatitis Delta Antigen ◽

Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains a viroid-like, 1.7-kb circular RNA genome, which replicates via a double-rolling-circle model. However, the exact mechanism involved in HDV genome RNA replication and subgenomic mRNA transcription is still unclear. Our previous studies have shown that the replications of genomic and antigenomic HDV RNA strands have different sensitivities to α-amanitin and are associated with different nuclear bodies, suggesting that these two strands are synthesized in different transcription machineries in the cells. In this study, we developed a unique quantitative reverse transcription-PCR (qRT-PCR) procedure for detection of various HDV RNA species from an RNA transfection system. Using this qRT-PCR procedure and a series of HDV mutants, we demonstrated that Arg-13 methylation, Lys-72 acetylation, and Ser-177 phosphorylation of small hepatitis delta antigen (S-HDAg) are important for HDV mRNA transcription. In addition, these three S-HDAg modifications are dispensable for antigenomic RNA synthesis but are required for genomic RNA synthesis. Furthermore, the three RNA species had different sensitivities to acetylation and deacetylation inhibitors, showing that the metabolic requirements for the synthesis of HDV antigenomic RNA are different from those for the synthesis of genomic RNA and mRNA. In sum, our data support the hypothesis that the cellular machinery involved in the synthesis of HDV antigenomic RNA is different from that of genomic RNA synthesis and mRNA transcription, even though the antigenomic RNA and the mRNA are made from the same RNA template. We propose that acetylation and deacetylation of HDAg may provide a molecular switch for the synthesis of the different HDV RNA species.

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

Journal of Virology ◽

10.1128/jvi.76.19.9910-9919.2002 ◽

2002 ◽

Vol 76 (19) ◽

pp. 9910-9919 ◽

Cited By ~ 19

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.

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New Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Hepatitis Delta Virus Using a Hepatitis Delta Antigen Derived from a Taiwanese Clone and Comparison to the Abbott Radioimmunoassay

Clinical and Vaccine Immunology ◽

10.1128/cvi.05687-11 ◽

2012 ◽

Vol 19 (5) ◽

pp. 817-819 ◽

Cited By ~ 5

Author(s):

Yung-Bin Kuo ◽

Mei Chao ◽

Yi-Hsuan Lee ◽

Chau-Ting Yeh ◽

Err-Cheng Chan

Keyword(s):

Immunosorbent Assay ◽

Hepatitis Delta Virus ◽

Detection Efficiency ◽

Enzyme Linked Immunosorbent Assay ◽

Hepatitis Delta ◽

Hepatitis D ◽

Delta Antigen ◽

Hepatitis Delta Antigen ◽

Hdv Infection ◽

Delta Virus

ABSTRACTAn anti-hepatitis delta (HD) enzyme-linked immunosorbent assay (ELISA) using a specific recombinant hepatitis delta antigen derived from a local dominant hepatitis delta virus (hepatitis D virus; HDV) strain in Taiwan has been established. The detection efficiency of this assay was comparable to that of the commercially available Abbott anti-HD radioimmunoassay (RIA) and could be useful in routine laboratory diagnoses of HDV infection.

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By Inhibiting Replication, the Large Hepatitis Delta Antigen Can Indirectly Regulate Amber/W Editing and Its Own Expression

Journal of Virology ◽

10.1128/jvi.78.15.8120-8134.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 8120-8134 ◽

Cited By ~ 30

Author(s):

Shuji Sato ◽

Cromwell Cornillez-Ty ◽

David W. Lazinski

Keyword(s):

Hepatitis Delta Virus ◽

Wild Type ◽

Hepatitis Delta ◽

Farnesyl Transferase ◽

Virion Assembly ◽

Large Form ◽

Delta Antigen ◽

Farnesyl Transferase Inhibitor ◽

Hepatitis Delta Antigen ◽

Delta Virus

ABSTRACT Hepatitis delta virus (HDV) expresses two essential proteins with distinct functions. The small hepatitis delta antigen (HDAg-S) is expressed throughout replication and is needed to promote that process. The large form (HDAg-L) is farnesylated, is expressed only at later times via RNA editing of the amber/W site, and is required for virion assembly. When HDAg-L is artificially expressed at the onset of replication, it strongly inhibits replication. However, there is controversy concerning whether HDAg-L expressed naturally at later times as a consequence of editing and replication can similarly inhibit replication. Here, by stabilizing the predicted secondary structure downstream from the amber/W site, a replication-competent HDV mutant that exhibited levels of editing higher than those of the wild type was created. This mutant expressed elevated levels of HDAg-L early during replication, and at later times, its replication aborted prematurely. No further increase in amber/W editing was observed following the cessation of replication, indicating that editing was coupled to replication. A mutation in HDAg-L and a farnesyl transferase inhibitor were both used to abolish the ability of HDAg-L to inhibit replication. Such treatments rescued the replication defect of the overediting mutant, and even higher levels of amber/W editing resulted. It was concluded that when expressed naturally during replication, HDAg-L is able to inhibit replication and thereby inhibit amber/W editing and its own synthesis. In addition, the structure adjacent to the amber/W site is suboptimal for editing, and this creates a window of time in which replication can occur in the absence of HDAg-L.

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Modification of Small Hepatitis Delta Virus Antigen by SUMO Protein

Journal of Virology ◽

10.1128/jvi.01034-09 ◽

2009 ◽

Vol 84 (2) ◽

pp. 918-927 ◽

Cited By ~ 33

Author(s):

Chung-Hsin Tseng ◽

Tai-Shan Cheng ◽

Chiung-Yueh Shu ◽

King-Song Jeng ◽

Michael M. C. Lai

Keyword(s):

Posttranslational Modifications ◽

Hepatitis Delta Virus ◽

Rna Synthesis ◽

Rna Replication ◽

Virus Antigen ◽

Genomic Rna ◽

Hepatitis Delta ◽

Lysine Residues ◽

Hepatitis Delta Antigen ◽

Delta Virus

ABSTRACT 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.

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

Journal of Virology ◽

10.1128/jvi.72.7.5449-5456.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 5449-5456 ◽

Cited By ~ 52

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.

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

Journal of Virology ◽

10.1128/jvi.74.16.7375-7380.2000 ◽

2000 ◽

Vol 74 (16) ◽

pp. 7375-7380 ◽

Cited By ~ 37

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.

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

Journal of Virology ◽

10.1128/jvi.00245-06 ◽

2006 ◽

Vol 80 (13) ◽

pp. 6469-6477 ◽

Cited By ~ 12

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.

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