The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis

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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Hepatitis delta virus genome RNA synthesis initiates at position 1646 with a non-templated guanosine

Journal of Virology ◽

10.1128/jvi.02017-21 ◽

2021 ◽

Author(s):

Susannah Stephenson-Tsoris ◽

John L. Casey

Keyword(s):

Liver Disease ◽

Rna Polymerase ◽

Hepatitis Delta Virus ◽

Rna Synthesis ◽

Viral Rna ◽

Start Site ◽

Hepatitis Delta ◽

Pol Ii ◽

Infected Cells ◽

Delta Virus

Hepatitis delta virus (HDV) is a significant human pathogen that causes acute and chronic liver disease; there is no licensed therapy. HDV is a circular negative-sense ssRNA virus that produces three RNAs in infected cells: genome, antigenome and mRNA; the latter encodes hepatitis delta antigen, the viral protein. These RNAs are synthesized by host DNA-dependent RNA polymerase acting as an RNA-dependent RNA polymerase. Although HDV genome RNA accumulates to high levels in infected cells, the mechanism by which this process occurs remains poorly understood. For example, the nature of the 5’ end of the genome, including the synthesis start site and its chemical composition, are not known. Analysis of this process has been challenging because the initiation site is part of an unstable precursor in the rolling circle mechanism by which HDV genome RNA is synthesized. In this study, circular HDV antigenome RNAs synthesized in vitro were used to directly initiate HDV genome RNA synthesis in transfected cells, thus enabling detection of the 5’ end of the genome RNA. The 5’ end of this RNA is capped, as expected for a Pol II product. Initiation begins at position 1646 on the genome, which is located near the loop end proximal to the start site for HDAg mRNA synthesis. Unexpectedly, synthesis begins with a guanosine that is not conventionally templated by the HDV RNA. IMPORTANCE Hepatitis delta virus (HDV) is a unique virus that causes severe liver disease. It uses host RNA Polymerase II to copy its circular RNA genome in a unique and poorly understood process. Although the virus RNA accumulates to high levels within infected cells, it is not known how synthesis of the viral RNA begins, nor even where on the genome synthesis starts. Here, we identify the start site for the initiation of HDV genome RNA synthesis as position 1646, which is at one end of the closed hairpin-like structure of the viral RNA. The 5’ end of the RNA is capped, as expected for Pol II products. However, RNA synthesis begins with a guanosine that is not present in the genome. Thus, although HDV uses Pol II to synthesize the viral genome, some details of the initiation process are different. These differences could be important for successfully targeting virus replication.

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

Journal of General Virology ◽

10.1099/0022-1317-83-10-2507 ◽

2002 ◽

Vol 83 (10) ◽

pp. 2507-2513 ◽

Cited By ~ 6

Author(s):

Gwo-Tarng Sheu

Keyword(s):

Hepatitis Delta Virus ◽

Rna Synthesis ◽

Main Function ◽

Genomic Rna ◽

Hepatitis Delta ◽

Virion Assembly ◽

Delta Antigen ◽

Hepatitis Delta Antigen ◽

Hdv Infection ◽

Delta Virus

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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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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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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Transcription of Hepatitis Delta Virus RNA by RNA Polymerase II

Journal of Virology ◽

10.1128/jvi.01758-07 ◽

2007 ◽

Vol 82 (3) ◽

pp. 1118-1127 ◽

Cited By ~ 56

Author(s):

Jinhong Chang ◽

Xingcao Nie ◽

Ho Eun Chang ◽

Ziying Han ◽

John Taylor

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Hepatitis Delta Virus ◽

Cell System ◽

Hepatitis Delta ◽

Pol Ii ◽

Delta Antigen ◽

Nuclear Extracts ◽

Virus Rna ◽

Delta Virus

ABSTRACT Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

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

Virology ◽

10.1006/viro.1993.1574 ◽

1993 ◽

Vol 197 (1) ◽

pp. 137-142 ◽

Cited By ~ 13

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

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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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Origin of Hepatitis Delta Virus mRNA

Journal of Virology ◽

10.1128/jvi.74.16.7204-7210.2000 ◽

2000 ◽

Vol 74 (16) ◽

pp. 7204-7210 ◽

Cited By ~ 45

Author(s):

Severin Gudima ◽

Shwu-Yuan Wu ◽

Cheng-Ming Chiang ◽

Gloria Moraleda ◽

John Taylor

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Hepatitis Delta Virus ◽

Rna Synthesis ◽

Genome Replication ◽

Rna Transcription ◽

Hepatitis Delta ◽

New Findings ◽

Delta Virus

ABSTRACT Hepatitis delta virus (HDV) is unique relative to all known animal viruses, especially in terms of its ability to redirect host RNA polymerase(s) to transcribe its 1,679-nucleotide (nt) circular RNA genome. During replication there accumulates not only more molecules of the genome but also its exact complement, the antigenome. In addition, there are relatively smaller amounts of an 800-nt RNA of antigenomic polarity that is polyadenylated and considered to act as mRNA for translation of the single and essential HDV protein, the delta antigen. Characterization of this mRNA could provide insights into the in vivo mechanism of HDV RNA-directed RNA transcription and processing. Previously, we showed that the 5′ end of this RNA was located in the majority of species, at nt 1630. The present studies show that (i) at least some of this RNA, as extracted from the liver of an HDV-infected woodchuck, behaved as if it contained a 5′-cap structure; (ii) in the infected liver there were additional polyadenylated antigenomic HDV RNA species with 5′ ends located at least 202 nt and even 335 nt beyond the nt 1630 site, (iii) the 5′ end at nt 1630 was not detected in transfected cells, following DNA-directed HDV RNA transcription, in the absence of genome replication, and (iv) nevertheless, using in vitro transcription with purified human RNA polymerase II holoenzyme and genomic RNA template, we did not detect initiation of template-dependent RNA synthesis; we observed only low levels of 3′-end addition to the template. These new findings support the interpretation that the 5′ end detected at nt 1630 during HDV replication represents a specific site for the initiation of an RNA-directed RNA synthesis, which is then modified by capping.

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