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

Chang, Jinhong; Gudima, Severin O.; Tarn, Chi; Nie, Xingcao; Taylor, John M.

doi:10.1128/jvi.79.13.8182-8188.2005

Cited by 58 publications

(90 citation statements)

References 26 publications

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“…The human embryonic kidney cell line 293TRex (Invitrogen) was used to create two cell lines as previously described (4). First, we derived and cloned 293-␦Ag, which expresses a single copy of ␦Ag cDNA under TET control.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…To more comprehensively understand the mechanism of HDV replication, an in vivo, unbiased proteomic interaction screen using HDAg-S as the bait would be invaluable and has been proposed by others (Chang et al 2005). However, this requires the use of highly specific antibodies suitable for such immunoprecipitation studies.…”

Section: Introductionmentioning

confidence: 99%

Combined proteomic–RNAi screen for host factors involved in human hepatitis delta virus replication

Cao¹,

Haussecker²,

Huang³

et al. 2009

RNA

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Human hepatitis delta virus (HDV) is the only animal virus known to replicate its RNA genome using a host polymerase because its only virally encoded proteins, the small and large hepatitis delta antigens (HDAg-S and HDAg-L), lack polymerase activity. Although this makes HDV an ideal model system to study RNA-directed transcription in mammalian cells, little is known about the host factors involved in its replication. To comprehensively identify such host factors, we created a stable cell line carrying a functional FLAG-HDAg-S. Anti-Flag immunopurification and mass spectrometry identified >100 proteins associated with FLAG-HDAg-S, many of which had predicted roles in RNA metabolism. The biological relevance of this screen was strongly supported by the identification of nine out of the 12 subunits of the RNA polymerase II complex thought to mediate HDV replication. To further investigate the significance of these factors for HDV replication, we selected 65 proteins to look for factors that would also affect the accumulation of HDV RNA following siRNA knockdown. Fifteen and three factors were found to regulate HDV RNA accumulation negatively and positively, respectively, upon RNAi knockdown. Our results provide a valuable resource for future research to advance our mechanistic understanding of HDV replication and RNA-directed transcription in mammalian cells.

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“…The genome of this 293-derived cell line contains stable integrations of a single Flp recombination target (FRT) site and a gene that expresses a TET repressor. Cotransfection of such cells with an FRT sitecontaining plasmid (pcDNA5/FRT/hcr) that encodes an miR-122 sequence and a plasmid (pOG44) that expresses Flp IN recombinase results in site-specific integration of the miR-122-containing cDNA through the FRT site with its expression under the control of the TET-on promoter (9). Two days after transfection, cells were trypsinized and reseeded at less than 25% confluence.…”

mentioning

confidence: 99%

Liver-Specific MicroRNA miR-122 Enhances the Replication of Hepatitis C Virus in Nonhepatic Cells

Chang

Guo

Jiang

et al. 2008

J Virol

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134

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The liver-specific microRNA miR-122 has been shown to be required for the replication of hepatitis C virus (HCV) in the hepatoma cell line Huh7. The aim of this study was to test if HCV replication can be modulated by exogenously expressed miR-122 in human embryonic kidney epithelial cells (HEK-293). Our results demonstrate that miR-122 enhances the colony formation efficiency of the HCV replicon and increases the steady-state level of HCV RNA in HEK-293 cells. Therefore, we conclude that although miR-122 is not absolutely required, it greatly enhances HCV replication in nonhepatic cells.

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

Cited by 58 publications

References 26 publications

Action of Inhibitors on Accumulation of Processed Hepatitis Delta Virus RNAs

Action of Inhibitors on Accumulation of Processed Hepatitis Delta Virus RNAs

Combined proteomic–RNAi screen for host factors involved in human hepatitis delta virus replication

Liver-Specific MicroRNA miR-122 Enhances the Replication of Hepatitis C Virus in Nonhepatic Cells

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