Interactions between Hepatitis Delta Virus Proteins

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.

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

Journal of Virology ◽

10.1128/jvi.78.11.5737-5744.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5737-5744 ◽

Cited By ~ 12

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.

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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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Unique Properties of the Large Antigen of Hepatitis Delta Virus

Journal of Virology ◽

10.1128/jvi.73.9.7147-7152.1999 ◽

1999 ◽

Vol 73 (9) ◽

pp. 7147-7152 ◽

Cited By ~ 10

Author(s):

Gloria Moraleda ◽

Steven Seeholzer ◽

Vadim Bichko ◽

Roland Dunbrack ◽

James Otto ◽

...

Keyword(s):

Hepatitis Delta Virus ◽

Surface Proteins ◽

Translation System ◽

Dimerization Domain ◽

Particle Assembly ◽

Hepatitis Delta ◽

C Terminus ◽

Delta Virus

ABSTRACT The large form of the hepatitis delta virus (HDV) protein (L) can be isoprenylated near its C terminus, and this modification is considered essential for particle assembly. Using gel electrophoresis, we separated L into two species of similar mobilities. The slower species could be labeled by the incorporation of [14C]mevalonolactone and is interpreted to be isoprenylated L (Li). In serum particles, infected liver, transfected cells, and assembled particles, 25 to 85% of L was isoprenylated. Isoprenylation was also demonstrated by 14C incorporation in vitro with a rabbit reticulocyte coupled transcription-translation system. However, the species obtained migrated even slower than that detected by labeling in vivo. Next, in studies of HDV particle assembly in the presence of the surface proteins of human hepatitis B virus, we observed the following. (i) Relative to L, Li was preferentially assembled into virus-like particles. (ii) Li could coassemble the unmodified L and the small delta protein, S. (iii) In contrast, a form of L with a deletion in the dimerization domain was both isoprenylated and assembled, but it could not support the coassembly of S. Finally, to test the expectation that the isoprenylation of L would increase its hydrophobicity, we applied a phase separation strategy based on micelle formation with the nonionic detergent Triton X-114. We showed the following. (i) The unique C-terminal 19 amino acids present on L relative to S caused a significant increase in the hydrophobicity. (ii) This increase was independent of isoprenylation. (iii) In contrast, other, artificial modifications at either the N or C terminus of S did not increase the hydrophobicity. (iv) The increased hydrophobicity was not sufficient for particle assembly; nevertheless, we speculate that it might facilitate virion assembly.

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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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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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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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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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