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

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.

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The Conserved Serine 177 in the Delta Antigen of Hepatitis Delta Virus Is One Putative Phosphorylation Site and Is Required for Efficient Viral RNA Replication

Journal of Virology ◽

10.1128/jvi.75.19.9087-9095.2001 ◽

2001 ◽

Vol 75 (19) ◽

pp. 9087-9095 ◽

Cited By ~ 27

Author(s):

Jung-Jung Mu ◽

Ding-Shinn Chen ◽

Pei-Jer Chen

Keyword(s):

Hepatitis Delta Virus ◽

Critical Role ◽

Phosphorylation Site ◽

Biological Significance ◽

Rna Replication ◽

Viral Rna ◽

Hepatitis Delta ◽

Delta Antigen ◽

Putative Phosphorylation Site ◽

Delta Virus

ABSTRACT Hepatitis delta virus (HDV) small delta antigen (S-HDAg) plays a critical role in virus replication. We previously demonstrated that the S-HDAg phosphorylation occurs on both serine and threonine residues. However, their biological significance and the exact phosphorylation sites of S-HDAg are still unknown. In this study, phosphorylated S-HDAg was detected only in the intracellular compartment, not in viral particles. In addition, the number of phosphorylated isoforms of S-HDAg significantly increased with the extent of viral replication in transfection system. Site-directed mutagenesis showed that alanine replacement of serine 177, which is conserved among all the known HDV strains, resulted in reduced phosphorylation of S-HDAg, while the mutation of the other two conserved serine residues (2 and 123) had little effect. The S177A mutant dramatically decreased its capability in assisting HDV RNA replication, with a preferential and profound impairment of the antigenomic RNA replication. Furthermore, the viral RNA editing, a step relying upon antigenomic RNA replication, was also abolished by this mutation. These results suggested that phosphorylation of S-HDAg, with serine 177 as a presumable site, plays a critical role in viral RNA replication, especially in augmenting the replication of antigenomic RNA.

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

Journal of Virology ◽

10.1128/jvi.78.23.13325-13334.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13325-13334 ◽

Cited By ~ 60

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.

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

Journal of Virology ◽

10.1128/jvi.76.8.3928-3935.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 3928-3935 ◽

Cited By ~ 38

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.

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Hepatitis delta virus cDNA sequence from an acutely HBV-infected chimpanzee: Sequence conservation in experimental animals

Journal of Medical Virology ◽

10.1002/jmv.1890340412 ◽

1991 ◽

Vol 34 (4) ◽

pp. 268-279 ◽

Cited By ~ 15

Author(s):

Ton Kos ◽

Anco Molijn ◽

Leen-Jan Van Doorn ◽

Alex Van Belkum ◽

Martin Dubbeld ◽

...

Keyword(s):

Hepatitis Delta Virus ◽

Cdna Sequence ◽

Sequence Conservation ◽

Hepatitis Delta ◽

Experimental Animals ◽

Infected Chimpanzee ◽

Chimpanzee Sequence ◽

Virus Cdna ◽

Delta Virus

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

Journal of Virology ◽

10.1128/jvi.76.8.3819-3827.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 3819-3827 ◽

Cited By ~ 50

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.

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RNA Replication without RNA-Dependent RNA Polymerase: Surprises from Hepatitis Delta Virus

Journal of Virology ◽

10.1128/jvi.79.13.7951-7958.2005 ◽

2005 ◽

Vol 79 (13) ◽

pp. 7951-7958 ◽

Cited By ~ 75

Author(s):

Michael M. C. Lai

Keyword(s):

Rna Polymerase ◽

Hepatitis Delta Virus ◽

Rna Replication ◽

Rna Dependent Rna Polymerase ◽

Hepatitis Delta ◽

Delta Virus

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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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