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

2001 ◽  
Vol 75 (7) ◽  
pp. 3469-3473 ◽  
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.

scholarly journals Initiation of Hepatitis Delta Virus Genome Replication

1998 ◽  
Vol 72 (6) ◽  
pp. 4783-4788 ◽  
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.

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 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 Hepatitis delta virus facilitates the selection of hepatitis B virus mutants in vivo and functionally impacts on their replicative capacity in vitro

2016 ◽  
Vol 22 (1) ◽  
pp. 98.e1-98.e6 ◽  
Author(s):  
E. Shirvani-Dastgerdi ◽  
M.R. Pourkarim ◽  
U. Herbers ◽  
S. Amini-Bavil-Olyaee ◽  
E. Yagmur ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hepatitis Delta Virus ◽  
Replicative Capacity ◽  
Hepatitis Delta ◽  
B Virus ◽  
Selection Of ◽  
Delta Virus

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.

Hepatitis Delta Virus can persist and propagate through human cell division both in vitro and in vivo

2015 ◽  
Vol 53 (01) ◽  
Author(s):  
OD Bhadra ◽  
K Giersch ◽  
T Volz ◽  
L Allweiss ◽  
AW Lohse ◽  
...  
Keyword(s):  
Cell Division ◽  
Human Cell ◽  
Hepatitis Delta Virus ◽  
Hepatitis Delta ◽  
Delta Virus
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