Characterization of the 5′ Ends for Polyadenylated RNAs Synthesized during the Replication of Hepatitis Delta Virus

ABSTRACT The genome of hepatitis delta virus (HDV) is a 1,679-nucleotide (nt) single-stranded circular RNA that is predicted to fold into an unbranched rodlike structure. During replication, two complementary RNAs are also detected: an exact complement, referred to as the antigenome, and an 800-nt polyadenylated RNA that could act as the mRNA for the delta antigen. We used a 5′ rapid amplification of cDNA ends procedure, followed by cloning and sequencing, to determine the 5′ ends of the polyadenylated RNAs produced during HDV genome replication following initiation under different experimental conditions. The analyzed RNAs were from the liver of an infected woodchuck and from a liver cell line at 6 days after transfection with either an HDV cDNA or ribonucleoprotein (RNP) complexes assembled in vitro with HDV genomic RNA and purified recombinant small delta protein. In all three situations the 5′ ends mapped specifically to nt 1630. In relationship to what is called the top end of the unbranched rodlike structure predicted for the genomic RNA template, this site is located 10 nt from the top, and in the middle of a 3-nt external bulge. Following transfection with RNP, such specific 5′ ends could be detected as early as 24 h. We next constructed a series of mutants of this predicted bulge region and of an adjacent 6-bp stem and the top 5-nt loop. Some of these mutations decreased the ability of the genome to undergo antigenomic RNA synthesis and accumulation and/or altered the location of the detected 5′ ends. The observed end located at nt 1630, and most of the novel 5′ ends, were consistent with transcription initiation events that preferentially used a purine. The present studies do not prove that the detected 5′ ends correspond to initiation sites and do not establish the hypothesis that there is a promoter element in the vicinity, but they do show that the location of the observed 5′ ends could be controlled by nucleotide sequences at and around nt 1630.

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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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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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In vitro-synthesized hepatitis delta virus RNA initiates genome replication in cultured cells.

Journal of Virology ◽

10.1128/jvi.64.6.3104-3107.1990 ◽

1990 ◽

Vol 64 (6) ◽

pp. 3104-3107 ◽

Cited By ~ 50

Author(s):

J S Glenn ◽

J M Taylor ◽

J M White

Keyword(s):

Hepatitis Delta Virus ◽

Cultured Cells ◽

Genome Replication ◽

Hepatitis Delta ◽

Virus Rna ◽

Delta Virus

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Initiation of Hepatitis Delta Virus Genome Replication

Journal of Virology ◽

10.1128/jvi.72.6.4783-4788.1998 ◽

1998 ◽

Vol 72 (6) ◽

pp. 4783-4788 ◽

Cited By ~ 25

Author(s):

Kate Dingle ◽

Vadim Bichko ◽

Harmon Zuccola ◽

James Hogle ◽

John Taylor

Keyword(s):

Hepatitis Delta Virus ◽

Rna Synthesis ◽

De Novo ◽

Virus Genome ◽

Northern Analysis ◽

Genome Replication ◽

Hepatitis Delta ◽

Delta Virus

ABSTRACT The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (δAg-S) is essential for genome replication, i.e., for the transcription, processing, and accumulation of HDV RNAs. To better understand this requirement, we used purified recombinant δAg-S and HDV RNA synthesized in vitro to assemble high-molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs into human cells, we detected HDV genome replication, as assayed by Northern analysis or immunofluorescence microscopy. Our interpretation is that the input δAg-S is necessary for the RNA to undergo limited amounts of RNA-directed RNA synthesis, RNA processing, and mRNA formation, leading to de novo translation of δAg-S. It is this second source of δAg-S which then goes on to support genome replication. This assay made it possible to manipulate in vitro the composition of the RNP and then test in vivo the ability of the complex to initiate RNA-directed RNA synthesis and go on to achieve genome replication. For example, both genomic and antigenomic linear RNAs were acceptable. Substitution for δAg-S with truncated or modified forms of the δAg, and even with HIV nucleocapsid protein and polylysine, was unacceptable; the exception was a form of δAg-S with six histidines added at the C terminus. We expect that further in vitro modifications of these RNP complexes should help define the in vivo requirements for what we define as the initiation of HDV genome replication.

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Replication of the Human Hepatitis Delta Virus Genome Is Initiated in Mouse Hepatocytes following Intravenous Injection of Naked DNA or RNA Sequences

Journal of Virology ◽

10.1128/jvi.75.7.3469-3473.2001 ◽

2001 ◽

Vol 75 (7) ◽

pp. 3469-3473 ◽

Cited By ~ 57

Author(s):

Jinhong Chang ◽

Luis J. Sigal ◽

Anthony Lerro ◽

John Taylor

Keyword(s):

Hepatitis Delta Virus ◽

Virus Genome ◽

In Vivo Studies ◽

Genome Replication ◽

Rna Sequences ◽

Hepatitis Delta ◽

Naked Dna ◽

Delta Virus

ABSTRACT As early as 5 days after DNA copies of the hepatitis delta virus (HDV) genome or even in vitro-transcribed HDV RNA sequences were injected into the mouse tail vein using the hydrodynamics-based transfection procedure of F. Liu et al. (Gene Ther. 6:1258–1266, 1999), it was possible to detect in the liver by Northern analyses of RNA, immunoblots of protein, and immunostaining of liver sections what were considered typical features of HDV genome replication. This transfection strategy should have valuable applications for in vivo studies of HDV replication and pathogenesis and may also be useful for studies of other hepatotropic viruses.

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

Journal of Virology ◽

10.1128/jvi.00008-09 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6457-6463 ◽

Cited By ~ 15

Author(s):

Ziying Han ◽

Carolina Alves ◽

Severin Gudima ◽

John Taylor

Keyword(s):

Rna Polymerase Ii ◽

Protein Function ◽

Hepatitis Delta Virus ◽

Rna Binding ◽

Intracellular Localization ◽

Genome Replication ◽

Hepatitis Delta ◽

Pol Ii ◽

Presence And Absence ◽

Delta Virus

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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Selection in vitro of Trans -Acting Genomic Human Hepatitis Delta Virus (HDV) Ribozymes

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1996.0712p.x ◽

1996 ◽

Vol 237 (3) ◽

pp. 712-718 ◽

Cited By ~ 13

Author(s):

Fumiko Nishikawa ◽

Junji Kawakami ◽

Atsushi Chiba ◽

Makoto Shirai ◽

Penmetcha K. R. Kumar ◽

...

Keyword(s):

Hepatitis Delta Virus ◽

Hepatitis Delta ◽

Human Hepatitis ◽

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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