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

ObjectiveHepatitis delta virus (HDV) was shown to persist for weeks in the absence of HBV and for months after liver transplantation, demonstrating the ability of HDV to persevere in quiescent hepatocytes. The aim of the study was to evaluate the impact of cell proliferation on HDV persistence in vitro and in vivo.DesignGenetically labelled human sodium taurocholate cotransporting polypeptide (hNTCP)-transduced human hepatoma(HepG2) cells were infected with HBV/HDV and passaged every 7 days for 100 days in the presence of the entry inhibitor Myrcludex-B. In vivo, cell proliferation was triggered by transplanting primary human hepatocytes (PHHs) isolated from HBV/HDV-infected humanised mice into naïve recipients. Virological parameters were measured by quantitative real time polymerase chain reaction (qRT-PCR). Hepatitis delta antigen (HDAg), hepatitis B core antigen (HBcAg) and cell proliferation were determined by immunofluorescence.ResultsDespite 15 in vitro cell passages and block of viral spreading by Myrcludex-B, clonal cell expansion permitted amplification of HDV infection. In vivo, expansion of PHHs isolated from HBV/HDV-infected humanised mice was confirmed 3 days, 2, 4 and 8 weeks after transplantation. While HBV markers rapidly dropped in proliferating PHHs, HDAg-positive hepatocytes were observed among dividing cells at all time points. Notably, HDAg-positive cells appeared in clusters, indicating that HDV was transmitted to daughter cells during liver regeneration even in the absence of de novo infection.ConclusionThis study demonstrates that HDV persists during liver regeneration by transmitting HDV RNA to dividing cells even in the absence of HBV coinfection. The strong persistence capacities of HDV may also explain why HDV clearance is difficult to achieve in HBV/HDV chronically infected patients.

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Resistance of Human Hepatitis Delta Virus RNAs to Dicer Activity

Journal of Virology ◽

10.1128/jvi.77.22.11910-11917.2003 ◽

2003 ◽

Vol 77 (22) ◽

pp. 11910-11917 ◽

Cited By ~ 41

Author(s):

Jinhong Chang ◽

Patrick Provost ◽

John M. Taylor

Keyword(s):

Hepatitis Delta Virus ◽

Unit Length ◽

Potato Spindle Tuber Viroid ◽

Micro Rna ◽

Hepatitis Delta ◽

Human Hepatitis ◽

Putative Precursor ◽

Delta Virus

ABSTRACT The endonuclease dicer cleaves RNAs that are 100% double stranded and certain RNAs with extensive but <100% pairing to release ∼21-nucleotide (nt) fragments. Circular 1,679-nt genomic and antigenomic RNAs of human hepatitis delta virus (HDV) can fold into a rod-like structure with 74% pairing. However, during HDV replication in hepatocytes of human, woodchuck, and mouse origin, no ∼21-nt RNAs were detected. Likewise, in vitro, purified recombinant dicer gave <0.2% cleavage of unit-length HDV RNAs. Similarly, rod-like RNAs of potato spindle tuber viroid (PSTVd) and avocado sunblotch viroid (ASBVd) were only 0.5% cleaved. Furthermore, when a 66-nt hairpin RNA with 79% pairing, the putative precursor to miR-122, which is an abundant liver micro-RNA, replaced one end of HDV genomic RNA, it was poorly cleaved, both in vivo and in vitro. In contrast, this 66-nt hairpin, in the absence of appended HDV sequences, was >80% cleaved in vitro. Other 66-nt hairpins derived from one end of genomic HDV, PSTVd, or ASBVd RNAs were also cleaved. Apparently, for unit-length RNAs of HDV, PSTVd, and ASBVd, it is the extended structure with <100% base pairing that confers significant resistance to dicer action.

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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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In vitro and in vivo comparison of hammerhead, hairpin, and hepatitis delta virus self-processing ribozyme cassettes.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)47325-5 ◽

1994 ◽

Vol 269 (41) ◽

pp. 25856-25864

Author(s):

B M Chowrira ◽

P A Pavco ◽

J A McSwiggen

Keyword(s):

Hepatitis Delta Virus ◽

Hepatitis Delta ◽

Delta Virus

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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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