Intracellular Localization of Hepatitis Delta Virus Proteins in the Presence and Absence of Viral RNA Accumulation

ABSTRACT Hepatitis delta virus (HDV) encodes one protein, hepatitis delta antigen (δAg), a 195-amino-acid RNA binding protein essential for the accumulation of HDV RNA-directed RNA transcripts. It has been accepted that δAg localizes predominantly to the nucleolus in the absence of HDV genome replication while in the presence of replication, δAg facilitates HDV RNA transport to the nucleoplasm and helps redirect host RNA polymerase II (Pol II) to achieve transcription and accumulation of processed HDV RNA species. This study used immunostaining and confocal microscopy to evaluate factors controlling the localization of δAg in the presence and absence of replicating and nonreplicating HDV RNAs. When δAg was expressed in the absence of full-length HDV RNAs, it colocalized with nucleolin, a predominant nucleolar protein. With time, or more quickly after induced cell stress, there was a redistribution of both δAg and nucleolin to the nucleoplasm. Following expression of nonreplicating HDV RNAs, δAg moved to the nucleoplasm, but nucleolin was unchanged. When δAg was expressed along with replicating HDV RNA, it was found predominantly in the nucleoplasm along with Pol II. This localization was insensitive to inhibitors of HDV replication, suggesting that the majority of δAg in the nucleoplasm reflects ribonucleoprotein accumulation rather than ongoing transcription. An additional approach was to reevaluate several forms of δAg altered at specific locations considered to be essential for protein function. These studies provide evidence that δAg does not interact directly with either Pol II or nucleolin and that forms of δAg which support replication are also capable of prior nucleolar transit.

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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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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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RNA-Dependent Replication and Transcription of Hepatitis Delta Virus RNA Involve Distinct Cellular RNA Polymerases

Molecular and Cellular Biology ◽

10.1128/mcb.20.16.6030-6039.2000 ◽

2000 ◽

Vol 20 (16) ◽

pp. 6030-6039 ◽

Cited By ~ 80

Author(s):

Lucy E. Modahl ◽

Thomas B. Macnaughton ◽

Nongliao Zhu ◽

Deborah L. Johnson ◽

Michael M. C. Lai

Keyword(s):

Rna Polymerase Ii ◽

Hepatitis Delta Virus ◽

Molecular Mechanisms ◽

De Novo ◽

Full Length ◽

Supporting Evidence ◽

Mrna Transcription ◽

Hepatitis Delta ◽

Pol Ii ◽

Delta Virus

ABSTRACT Cellular DNA-dependent RNA polymerase II (pol II) has been postulated to carry out RNA-dependent RNA replication and transcription of hepatitis delta virus (HDV) RNA, generating a full-length (1.7-kb) RNA genome and a subgenomic-length (0.8-kb) mRNA. However, the supporting evidence for this hypothesis was ambiguous because the previous experiments relied on DNA-templated transcription to initiate HDV RNA synthesis. Furthermore, there is no evidence that the same cellular enzyme is involved in the synthesis of both RNA species. In this study, we used a novel HDV RNA-based transfection approach, devoid of any artificial HDV cDNA intermediates, to determine the enzymatic and metabolic requirements for the synthesis of these two RNA species. We showed that HDV subgenomic mRNA transcription was inhibited by a low concentration of α-amanitin (<3 μg/ml) and could be partially restored by an α-amanitin-resistant mutant pol II; however, surprisingly, the synthesis of the full-length (1.7-kb) antigenomic RNA was not affected by α-amanitin to a concentration higher than 25 μg/ml. By several other criteria, such as the differing requirement for the de novo-synthesized hepatitis delta antigen and temperature dependence, we further showed that the metabolic requirements of subgenomic HDV mRNA synthesis are different from those for the synthesis of genomic-length HDV RNA and cellular pol II transcripts. The synthesis of the two HDV RNA species could also be uncoupled under several different conditions. These findings provide strong evidence that pol II, or proteins derived from pol II transcripts, is involved in mRNA transcription from the HDV RNA template. In contrast, the synthesis of the 1.7-kb HDV antigenomic RNA appears not to be dependent on pol II. These results reveal that there are distinct molecular mechanisms for the synthesis of these two RNA species.

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Potato Spindle Tuber Viroid RNA-Templated Transcription: Factors and Regulation

Viruses ◽

10.3390/v10090503 ◽

2018 ◽

Vol 10 (9) ◽

pp. 503 ◽

Cited By ~ 10

Author(s):

Shachinthaka Dissanayaka Mudiyanselage ◽

Jie Qu ◽

Nancy Tian ◽

Jian Jiang ◽

Ying Wang

Keyword(s):

Rna Polymerase Ii ◽

Hepatitis Delta Virus ◽

Regulatory Mechanism ◽

Noncoding Rnas ◽

Direct Interaction ◽

Potato Spindle Tuber Viroid ◽

Pol Ii ◽

Transcription Factor Iiia ◽

Transcription System ◽

Delta Virus

Viroids are circular noncoding RNAs that infect plants. Without encoding any protein, these noncoding RNAs contain the necessary genetic information for propagation in hosts. Nuclear-replicating viroids employ DNA-dependent RNA polymerase II (Pol II) for replication, a process that makes a DNA-dependent enzyme recognize RNA templates. Recently, a splicing variant of transcription factor IIIA (TFIIIA-7ZF) was identified as essential for Pol II to replicate potato spindle tuber viroid (PSTVd). The expression of TFIIIA-7ZF, particularly the splicing event, is regulated by a ribosomal protein (RPL5). PSTVd modulates its expression through a direct interaction with RPL5 resulting in optimized expression of TFIIIA-7ZF. This review summarizes the recent discoveries of host factors and regulatory mechanisms underlying PSTVd-templated transcription processes and raises new questions that may help future exploration in this direction. In addition, it briefly compares the machinery and the regulatory mechanism for PSTVd with the replication/transcription system of human hepatitis delta virus.

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Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module 1 1Edited by D. C. Rees

Journal of Molecular Biology ◽

10.1006/jmbi.1999.3398 ◽

2000 ◽

Vol 295 (3) ◽

pp. 541-556 ◽

Cited By ~ 88

Author(s):

Adrian R. Ferré-D’Amaré ◽

Jennifer A. Doudna

Keyword(s):

Structure Determination ◽

Hepatitis Delta Virus ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Hepatitis Delta ◽

Hepatitis Delta Virus Ribozyme ◽

Delta Virus

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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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Inhibition of Cellular RNA Polymerase II Transcription by Delta Antigen of Hepatitis Delta Virus

Virology ◽

10.1006/viro.1998.9253 ◽

1998 ◽

Vol 247 (2) ◽

pp. 178-188 ◽

Cited By ~ 27

Author(s):

Kiersten Lo ◽

Gwo-Tarng Sheu ◽

Michael M.C. Lai

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Hepatitis Delta Virus ◽

Hepatitis Delta ◽

Delta Antigen ◽

Rna Polymerase Ii Transcription ◽

Delta Virus

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Parameters of Human Hepatitis Delta Virus Genome Replication: the Quantity, Quality, and Intracellular Distribution of Viral Proteins and RNA

Journal of Virology ◽

10.1128/jvi.76.8.3709-3719.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 3709-3719 ◽

Cited By ~ 61

Author(s):

Severin Gudima ◽

Jinhong Chang ◽

Gloria Moraleda ◽

Anna Azvolinsky ◽

John Taylor

Keyword(s):

Hepatitis Delta Virus ◽

Virus Assembly ◽

Intracellular Distribution ◽

Molar Ratio ◽

Genome Replication ◽

Cell Fractionation ◽

Pcr Cloning ◽

Hepatitis Delta ◽

Initiation Of Replication ◽

Delta Virus

ABSTRACT Assembly of hepatitis delta virus (HDV) in infected human hepatocytes involves association of the 1,679- nucleotide single-stranded genomic RNA (δRNA) with multiple copies of both small and large forms of the delta protein (δAg) to form a ribonucleoprotein particle which in turn interacts with envelope proteins of the natural helper virus, hepatitis B virus. Subsequently, for initiation of a new round of replication, the amount of small δAg within the assembled HDV particle is both necessary and sufficient. Quantitative assays were used in order to better understand just how much δAg is needed. The molar ratio of δAg species to genomic δRNA in assembled HDV particles was approximately 200. Next, this ratio was determined for cells under several different experimental situations in which HDV genome replication was occurring. These included replication in woodchuck liver and also in mouse liver and skeletal muscle, as well as replication in stably and transiently transfected cultured human hepatoblastoma cells. Surprisingly, in almost all these situations the molar ratios were comparable to that observed for HDV particles. This was true for different times after the initiation of replication and was independent of whether or not virus assembly was occurring. Cell fractionation combined with quantitative assays was used to test whether the majority of δAg and δRNA were colocalized during HDV replication in transfected cells. The cytoplasmic fraction contained the majority of δAg and genomic δRNA. Finally, the quality of δAg and δRNA, especially at relatively late times after the initiation of replication, was examined by using reverse transcription-PCR, cloning, and sequencing through the entire δAg open reading frame. When virus assembly and spread were not possible, 20% or less of the predicted δAg would have been able to support HDV replication. In summary, an examination of the quantity, quality and intracellular distribution of δAg and δRNA in several different experimental systems has provided a better understanding of the parameters associated with the initiation, maintenance, and ultimate decline of HDV genome replication.

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Alternative Processing of Hepatitis Delta Virus Antigenomic RNA Transcripts

Journal of Virology ◽

10.1128/jvi.78.9.4517-4524.2004 ◽

2004 ◽

Vol 78 (9) ◽

pp. 4517-4524 ◽

Cited By ~ 16

Author(s):

Xingcao Nie ◽

Jinhong Chang ◽

John M. Taylor

Keyword(s):

Hepatitis Delta Virus ◽

Genome Replication ◽

Hepatitis Delta ◽

Rolling Circle ◽

Rna Transcripts ◽

Alternative Processing ◽

Experimental Approaches ◽

Rna Ligation ◽

Stable Source ◽

Delta Virus

ABSTRACT Intrinsic to the life cycle of hepatitis delta virus (HDV) is the fact that its RNAs undergo different forms of posttranscriptional RNA processing. Transcripts of both the genomic RNA and its exact complement, the antigenomic RNA, undergo ribozyme cleavage and RNA ligation. In addition, antigenomic RNA transcripts can undergo 5′ capping, 3′ polyadenylation, and even RNA editing by an adenosine deaminase. This study focused on the processing of antigenomic RNA transcripts. Two approaches were used to study the relationship between the events of polyadenylation, ribozyme cleavage, and RNA ligation. The first represented an examination under more controlled conditions of mutations in the poly(A) signal, AAUAAA, which is essential for this processing. We found that when a separate stable source of δAg-S, the small delta protein, was provided, the replication ability of the mutated RNA was restored. The second approach involved an examination of the processing in transfected cells of specific Pol II DNA-directed transcripts of HDV antigenomic sequences. The DNA constructs used were such that the RNA transcripts were antigenomic and began at the same 5′ site as the mRNA produced during RNA-directed HDV genome replication. A series of such constructs was assembled in order to test the relative abilities of the transcripts to undergo processing by polyadenylation or ribozyme cleavage at sites further 3′ on a multimer of HDV sequences. The findings from the two experimental approaches led to significant modifications in the rolling-circle model of HDV genome replication.

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