Reconstitution in cultured cells of replicating HDV RNA from pairs of less than full-length RNAs

Gudima, Severin O.; Chang, Jinhong; Taylor, John M.

doi:10.1261/rna.7164905

Cited by 25 publications

(20 citation statements)

References 47 publications

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“…The procedure was based on a method described previously (1,14). As primary antibodies, for ␦Ag, we used a 1:1,000 dilution of a rabbit polyclonal antibody; for SC35, we used a 1:2,000 dilution of a mouse monoclonal antibody (Abcam); and for fibrillarin, we used a 1:500 dilution of a mouse monoclonal antibody (Abcam).…”

Section: Methodsmentioning

confidence: 99%

Development of a Novel System To Study Hepatitis Delta Virus Genome Replication

Chang

Gudima

Tarn

et al. 2005

J Virol

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Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (␦Ag).During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of ␦Ag species that are nonfunctional or even inhibitory. A replication system was devised where all ␦Ag was conditionally provided from a separate and unchanging source. A line of human embryonic kidney cells was stably transfected with a single copy of cDNA encoding small ␦Ag, with expression under tetracycline (TET) control. Next, HDV genome replication was initiated in these cells by transfection with a mutated RNA unable to express ␦Ag. Thus, replication of this RNA was under control of the TET-inducible ␦Ag. In the absence of TET, there was sufficient ␦Ag to allow a low level of HDV replication that could be maintained for at least 1 year. When TET was added, both ␦Ag and genomic RNA increased dramatically within 2 days. With clones of such cells, designated 293-HDV, the burst of HDV RNA replication interfered with cell cycling. Within 2 days, there was a fivefold enhancement of G 1 /G 0 cells relative to both S and G 2 /M cells, and by 6 days, there was extensive cell detachment and death. These findings and those of other studies that are under way demonstrate the potential applications of this experimental system.

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Section: Methodsmentioning

confidence: 99%

Development of a Novel System To Study Hepatitis Delta Virus Genome Replication

Chang

Gudima

Tarn

et al. 2005

J Virol

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“…41 The possibility that the complex formed by a host RNA polymerase and a host RNA being transcribed from a DNA template could detach, jump and resume transcription on a HDV RNA, has experimental support because template switching appears particularly feasible in HDV replication. Actually, co-transfected less-than-unit HDV RNAs can reconstitute the full-length replicating form, 54 a finding that must be explained by template switching or by invoking an unknown, homology-based RNA trans-splicing mechanism. Template switching in this case may be stimulated by the low processivity of host RNA polymerases transcribing RNA templates instead of their physiological DNA counterparts.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: Variations on a theme

Flores¹,

Grubb²,

Elleuch³

et al. 2011

RNA Biology

114

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“…5D to F. In this second strategy, we used 293-␦Ag cells that contain the TET-inducible ␦Ag but do not contain replicating HDV RNA. These cells were induced with TET for 24 h to allow expression of ␦Ag, and then TET was removed and HDV replication was initiated by transfecting these cells with total RNA that had been extracted from 293-HDV cells undergoing HDV replication (19)…”

Section: Vol 80 2006 Accumulation Of Processed Hdv Rnas 3207mentioning

confidence: 99%

Action of Inhibitors on Accumulation of Processed Hepatitis Delta Virus RNAs

Chang

Nie

Gudima

et al. 2006

J Virol

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Hepatitis delta virus (HDV) replication involves processing and accumulation of three RNA species: the genome, its exact complement (the antigenome), and a polyadenylated mRNA that acts as a template for the small delta antigen (␦Ag), the only protein of HDV and essential for genome replication. In a recently reported experimental system, addition of tetracycline induced synthesis of a DNA-directed source of ␦Ag, producing within 24 h a significant increase in accumulation of newly transcribed and processed HDV RNAs. This induction was used here to study the action of various inhibitors on accumulation. For example, potent and HDV-specific inhibition, in the absence of detected host toxicity, could be obtained with ribavirin, mycophenolic acid, and viramidine. An interpretation is that these inhibitors reduced the available GTP pool, leading to a specific inhibition of the synthesis and accumulation of HDV RNA-directed RNA species. In contrast, no inhibition was observed with L-FMAU (2-fluoro-5-methyl-␤-L-arabinofuranosyl-uridine), alpha interferon, or pegylated alpha interferon. After modifications to the experimental system, it was also possible to examine the effects of three known host RNA polymerase inhibitors on HDV genome replication: amanitin, 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB), and actinomycin. Of most interest, amanitin at low doses blocked accumulation of HDV RNA-directed mRNA but had less effect on HDV genomic and antigenomic RNAs. Additional experiments indicated that this apparent resistance to amanitin inhibition of genomic and antigenomic RNA relative to mRNA may not reflect a difference in the transcribing polymerase but rather relative differences in the processing and stabilization of nascent RNA transcripts.The 1,679-nucleotide, single-stranded, circular RNA genome of hepatitis delta virus (HDV) is replicated by RNAdirected RNA transcription mediated by a host polymerase (7). During replication, nascent RNA transcripts are processed to produce three different RNAs. The RNA genome and its exact complement, the antigenome, both arise from greaterthan-unit-length RNA linear transcripts that are reduced to unit length by ribozyme processing and then converted to a circular conformation by RNA ligation. An alternative processing of antigenomic RNA transcripts involves 5Ј capping and 3Ј polyadenylation to produce an mRNA of about 800 nucleotides in length. It is translated to produce a 195-aminoacid species, the small delta antigen (␦Ag), which is known to be essential for HDV replication (6). The features of HDV RNA transcription and processing have been incorporated into what is referred to as a double rolling-circle model (39).In order to study HDV replication experimentally, it is possible to infect primary hepatocyte cultures (2, 38). Such culture systems are somewhat inconvenient, but, as yet, infection of established cell lines has not been reported. The replication of the HDV genome can however be studied with cell lines following transfection with HDV RNAs or cDNA (39). Re...

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Reconstitution in cultured cells of replicating HDV RNA from pairs of less than full-length RNAs

Cited by 25 publications

References 47 publications

Development of a Novel System To Study Hepatitis Delta Virus Genome Replication

Development of a Novel System To Study Hepatitis Delta Virus Genome Replication

Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: Variations on a theme

Action of Inhibitors on Accumulation of Processed Hepatitis Delta Virus RNAs

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