Characterization of hepatitis delta antigen: specific binding to hepatitis delta virus RNA.

ABSTRACT RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.

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Recombinant Hepatitis Delta Antigen from E. coli Promotes Hepatitis Delta Virus RNA Replication Only from the Genomic Strand but Not the Antigenomic Strand

Virology ◽

10.1006/viro.2000.0680 ◽

2000 ◽

Vol 278 (2) ◽

pp. 578-586 ◽

Cited By ~ 13

Author(s):

Gwo-Tarng Sheu ◽

Michael M.C. Lai

Keyword(s):

Hepatitis Delta Virus ◽

Rna Replication ◽

Recombinant Hepatitis ◽

Hepatitis Delta ◽

E Coli ◽

Delta Antigen ◽

Virus Rna ◽

Hepatitis Delta Antigen ◽

Delta Virus

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Hepatitis Delta Antigen Requires a Flexible Quasi-Double-Stranded RNA Structure To Bind and Condense Hepatitis Delta Virus RNA in a Ribonucleoprotein Complex

Journal of Virology ◽

10.1128/jvi.00443-14 ◽

2014 ◽

Vol 88 (13) ◽

pp. 7402-7411 ◽

Cited By ~ 16

Author(s):

B. L. Griffin ◽

S. Chasovskikh ◽

A. Dritschilo ◽

J. L. Casey

Keyword(s):

Rna Structure ◽

Hepatitis Delta Virus ◽

Hepatitis Delta ◽

Double Stranded Rna ◽

Ribonucleoprotein Complex ◽

Delta Antigen ◽

Virus Rna ◽

Hepatitis Delta Antigen ◽

Delta Virus

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Hepatitis Delta Antigen Requires a Minimum Length of the Hepatitis Delta Virus Unbranched Rod RNA Structure for Binding

Journal of Virology ◽

10.1128/jvi.02467-08 ◽

2009 ◽

Vol 83 (9) ◽

pp. 4548-4556 ◽

Cited By ~ 16

Author(s):

Dawn A. Defenbaugh ◽

Matthew Johnson ◽

Renxiang Chen ◽

Ying Yi Zheng ◽

John L. Casey

Keyword(s):

Hepatitis Delta Virus ◽

Rna Binding ◽

Specific Binding ◽

Micrococcal Nuclease ◽

Minimum Length ◽

Hepatitis Delta ◽

Ribonucleoprotein Complexes ◽

Delta Antigen ◽

Hepatitis Delta Antigen ◽

Delta Virus

ABSTRACT Hepatitis delta virus (HDV) is a subviral pathogen that increases the severity of liver disease caused by hepatitis B virus. Both the small circular RNA genome and its complement, the antigenome, form a characteristic unbranched rod structure in which approximately 70% of the nucleotides are base paired. These RNAs are associated with the sole virally encoded protein, hepatitis delta antigen (HDAg), in infected cells; however, the nature of the ribonucleoprotein complexes (RNPs) is not well understood. Previous analyses of binding in vitro using native, bacterially expressed HDAg have been hampered by a lack of specificity for HDV RNA. Here, we show that removal of the C-terminal 35 amino acids of HDAg yields a native, bacterially expressed protein, HDAg-160, that specifically binds HDV unbranched rod RNA with high affinity. In an electrophoretic mobility shift assay, this protein produced a discrete, micrococcal nuclease-resistant complex with an ∼400-nucleotide (nt) segment of HDV unbranched rod RNA. Binding occurred with several segments of HDV RNA, although with various affinities and efficiencies. Analysis of the effects of deleting segments of the unbranched rod indicated that binding did not require one or two specific binding sites within these RNA segments. Rather, a minimum-length HDV RNA unbranched rod approximately 311 nt was essential for RNP formation. The results are consistent with a model in which HDAg binds HDV unbranched rod RNA as multimers of fixed size rather than as individual subunits.

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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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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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Hepatitis Delta Antigen Regulates mRNA and Antigenome RNA Levels during Hepatitis Delta Virus Replication

Journal of Virology ◽

10.1128/jvi.01989-18 ◽

2019 ◽

Vol 93 (8) ◽

Cited By ~ 3

Author(s):

Kaneemozhe Harichandran ◽

Yiran Shen ◽

Susannah Stephenson Tsoris ◽

See-Chi Lee ◽

John L. Casey

Keyword(s):

Liver Disease ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Hepatitis Delta Virus ◽

Hepatitis Delta ◽

Delta Antigen ◽

B Virus ◽

Hepatitis Delta Antigen ◽

Infected Cells ◽

Delta Virus

ABSTRACTHepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of acute and chronic liver disease. HDV produces three processed RNAs that accumulate in infected cells: the circular genome; the circular antigenome, which serves as a replication intermediate; and lesser amounts of the mRNA, which encodes the sole viral protein, hepatitis delta antigen (HDAg). The HDV genome and antigenome RNAs form ribonucleoprotein complexes with HDAg. Although HDAg is required for HDV replication, it is not known how the relative amounts of HDAg and HDV RNA affect replication, or whether HDAg synthesis is regulated by the virus. Using a novel transfection system in which HDV replication is initiated usingin vitro-synthesized circular HDV RNAs, HDV replication was found to depend strongly on the relative amounts of HDV RNA and HDAg. HDV controls these relative amounts via differential effects of HDAg on the production of HDV mRNA and antigenome RNA, both of which are synthesized from the genome RNA template. mRNA synthesis is favored at low HDAg levels but becomes saturated at high HDAg concentrations. Antigenome RNA accumulation increases linearly with HDAg and dominates at high HDAg levels. These results provide a conceptual model for how HDV antigenome RNA production and mRNA transcription are controlled from the earliest stage of infection onward and also demonstrate that, in this control, HDV behaves similarly to other negative-strand RNA viruses, even though there is no genetic similarity between them.IMPORTANCEHepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of liver disease; approximately 15 million people are chronically infected worldwide. There are no licensed therapies available. HDV is not related to any known virus, and few details regarding its replication cycle are known. One key question is whether and how HDV regulates the relative amounts of viral RNA and protein in infected cells. Such regulation might be important because the HDV RNA and protein form complexes that are essential for HDV replication, and the proper stoichiometry of these complexes could be critical for their function. Our results show that the relative amounts of HDV RNA and protein in cells are indeed important for HDV replication and that the virus does control them. These observations indicate that further study of these regulatory mechanisms is required to better understand replication of this serious human pathogen.

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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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