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

2002 ◽  
Vol 76 (8) ◽  
pp. 3709-3719 ◽  
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.

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

2009 ◽  
Vol 83 (13) ◽  
pp. 6457-6463 ◽  
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.

scholarly journals Origin of Hepatitis Delta Virus mRNA

2000 ◽  
Vol 74 (16) ◽  
pp. 7204-7210 ◽  
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.

scholarly journals Alternative Processing of Hepatitis Delta Virus Antigenomic RNA Transcripts

2004 ◽  
Vol 78 (9) ◽  
pp. 4517-4524 ◽  
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.

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

1999 ◽  
Vol 73 (8) ◽  
pp. 6533-6539 ◽  
Author(s):  
Severin Gudima ◽  
Kate Dingle ◽  
Ting-Ting Wu ◽  
Gloria Moraleda ◽  
John Taylor
Keyword(s):  
Transcription Initiation ◽  
Hepatitis Delta Virus ◽  
Circular Rna ◽  
Genome Replication ◽  
Genomic Rna ◽  
Experimental Conditions ◽  
Hepatitis Delta ◽  
Polyadenylated Rnas ◽  
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.

scholarly journals Susceptibility of Human Hepatitis Delta Virus RNAs to Small Interfering RNA Action

2003 ◽  
Vol 77 (17) ◽  
pp. 9728-9731 ◽  
Author(s):  
Jinhong Chang ◽  
John M. Taylor
Keyword(s):  
Hepatitis Delta Virus ◽  
Detectable Effect ◽  
Genome Replication ◽  
Small Interfering Rnas ◽  
Animal Cells ◽  
Rna Sequences ◽  
Hepatitis Delta ◽  
Ability To Act ◽  
Huh7 Cells ◽  
Delta Virus

ABSTRACT In animal cells, small interfering RNAs (siRNA), when exogenously provided, have been reported to be capable of inhibiting replication of several different viruses. In preliminary studies, siRNA species were designed and tested for their ability to act on the protein expressed in Huh7 cells transfected with DNA-directed mRNA constructs containing hepatitis delta virus (HDV) target sequences. The aim was to achieve siRNA specific for each of the three RNAs of HDV replication: (i) the 1,679-nucleotide circular RNA genome, (ii) its exact complement, the antigenome, and (iii) the less abundant polyadenylated mRNA for the small delta protein. Many of the 16 siRNA tested gave >80% inhibition in this assay. Next, these three classes of siRNA were tested for their ability to act during HDV genome replication. It was found that only siRNA targeted against HDV mRNA sequences could interfere with HDV genome replication. In contrast, siRNA targeted against genomic and antigenomic RNA sequences had no detectable effect on the accumulation of these RNAs. Reconstruction experiments with nonreplicating HDV RNA sequences support the interpretation that neither the potential for intramolecular rod-like RNA folding nor the presence of the delta protein conferred resistance to siRNA. In terms of replicating HDV RNAs, it is considered more likely that the genomic and antigenomic RNAs are resistant because their location within the nucleus makes them inaccessible to siRNA-mediated degradation.

scholarly journals Interactions between Hepatitis Delta Virus Proteins

2000 ◽  
Vol 74 (12) ◽  
pp. 5509-5515 ◽  
Author(s):  
Gloria Moraleda ◽  
Kate Dingle ◽  
Preetha Biswas ◽  
Jinhong Chang ◽  
Harmon Zuccola ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Hepatitis Delta Virus ◽  
Biological Activities ◽  
Dominant Negative ◽  
Genome Replication ◽  
Protein Protein Interactions ◽  
Particle Assembly ◽  
Hepatitis Delta ◽  
Delta Virus

ABSTRACT The 195- and 214-amino-acid (aa) forms of the delta protein (δAg-S and δAg-L, respectively) of hepatitis delta virus (HDV) differ only in the 19-aa C-terminal extension unique to δAg-L. δAg-S is needed for genome replication, while δAg-L is needed for particle assembly. These proteins share a region at aa 12 to 60, which mediates protein-protein interactions essential for HDV replication. H. Zuccola et al. (Structure 6:821–830, 1998) reported a crystal structure for a peptide spanning this region which demonstrates an antiparallel coiled-coil dimer interaction with the potential to form tetramers of dimers. Our studies tested whether predictions based on this structure could be extrapolated to conditions where the peptide was replaced by full-length δAg-S or δAg-L, and when the assays were not in vitro but in vivo. Nine amino acids that are conserved between several isolates of HDV and predicted to be important in multimerization were mutated to alanine on both δAg-S and δAg-L. We found that the predicted hierarchy of importance of these nine mutations correlated to a significant extent with the observed in vivo effects on the ability of these proteins to (i) support intrans the replication of the HDV genome when expressed on δAg-S and (ii) act as dominant-negative inhibitors of replication when expressed on δAg-L. We thus infer that these biological activities of δAg depend on ordered protein-protein interactions.

scholarly journals Features Affecting the Ability of Hepatitis Delta Virus RNAs To Initiate RNA-Directed RNA Synthesis

2004 ◽  
Vol 78 (11) ◽  
pp. 5737-5744 ◽  
Author(s):  
Severin O. Gudima ◽  
Jinhong Chang ◽  
John M. Taylor
Keyword(s):  
Hepatitis Delta Virus ◽  
Unit Length ◽  
Circular Rnas ◽  
Template Switching ◽  
Genome Replication ◽  
Hepatitis Delta ◽  
The Impact ◽  
Experimental Strategies ◽  
Delta Virus

ABSTRACT In models of the replication of human hepatitis delta virus (HDV) RNA, it is generally assumed that circular RNAs are the only templates. However, noncircular HDV RNAs are also produced during replication, and it is known that replication can be initiated by transfection with noncircular RNAs. Therefore, strategies were devised to determine the relative ability of different HDV RNA species to initiate RNA replication. One strategy used in vivo intermolecular competition following cotransfection into cells, between two sequence-marked HDV RNA species. Circular RNA templates were found to be at least severalfold more efficient than a dimeric linear template. Unit-length linear species, that is, equivalent to circles opened at different sites, were in most cases but not always of efficiency comparable to that of each other. Greater-than-unit-length linear species were more efficient than unit-length species, presumably because of the increased opportunities for template switching. Genomic linear RNAs were generally of initiation ability comparable to that of antigenomic RNAs. A second strategy measured the ability of initiation to occur on different regions of HDV RNAs that were twice the unit length. In summary, results from these two experimental strategies make clear that linear HDV RNA species, as well as circles, can contribute to the overall process of HDV genome replication. In addition, the results from the two experimental strategies provided information on the impact of template switching during RNA-directed transcription.

scholarly journals Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency

2015 ◽  
Vol 96 (12) ◽  
pp. 3460-3469 ◽  
Author(s):  
Mei Chao ◽  
Chia-Chi Lin ◽  
Feng-Ming Lin ◽  
Hsin-Pai Li ◽  
Shan-Bei Iang
Keyword(s):  
Hepatitis Delta Virus ◽  
Cultured Cells ◽  
Genetic Recombination ◽  
Genome Replication ◽  
Whole Genome ◽  
Rna Recombination ◽  
Whole Genome Analysis ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
Delta Virus

Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

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