Hepatitis δ antigen is necessary for access of hepatitis s virus rna to the cell transcriptional machinery but is not part of the transcriptional complex

RNA Replication without RNA-Dependent RNA Polymerase: Surprises from Hepatitis Delta Virus

“…This study showed that HDV mRNA transcription and RNA replication could be inhibited by ␣-amanitin at 1 to 5 g/ml, consistent with the ␣-amanitin sensitivity of pol II (39,47). Most significantly, when HDV RNA replication in a cell line expressing an ␣-amanitin-resistant pol II was carried out, HDV RNA replication became resistant to ␣-amanitin (47).…”

Localization of hepatitis delta virus RNA in the nucleus of human cells

“…Visualization of HDV cDNAs integrated in the genome of Huh7 cells+ Metaphase spreads were prepared from Huh7-D12 cells and hybridized with plasmid pSVL(D3), containing the HDV cDNA+ Sites of hybridization are detected as green signals+ Total DNA is counterstained with propidium iodide (red staining)+ Arrowheads point to the viral cDNAs integrated in the chromosome+ The arrow in B indicates viral cDNAs in an interphase nucleus+ sistent with these observations, clone Huh7-D12 was shown to contain ;40 copies of full-length HDV DNA integrated per haploid genome (Monjardino et al+, unpubl+ results)+ The distribution of HDV RNA was then determined in three-dimensionally preserved Huh7-D12 cells+ The pSVL(D3) probe, which hybridizes to both genomic and antigenomic RNA, produces a nucleoplasmic staining pattern, with nucleolar exclusion+ In the majority of cells, the nucleoplasmic staining is either finely punctate or coarse-grained with additional 2-20 brighter foci (Fig+ 2A,B)+ In a small number of cells (,10%), the HDV RNA is predominantly scattered throughout the nucleoplasm and does not accumulate in brighter foci+ Staining of the nucleolus is never observed+ Similar results are obtained when cells are either denatured or nondenatured prior to hybridization+ However, the signal intensity is significantly lower and more variable when hybridization is performed under nondenaturing conditions+ This probably reflects the highly base paired structure of HDV RNA, and we therefore decided to routinely denature the cells prior to hybridization+ Previous reports have shown that, in vitro, RNA synthesis from both genomic and antigenomic HDV RNA is blocked in the presence of either a-amanitin (1 mg/mL) or a monoclonal antibody specific for RNA polymerase II, suggesting that this cellular enzyme is responsible for the RNA-directed synthesis of viral RNA (MacNaughton et al+, 1991;Fu & Taylor, 1993)+ This prompted us to analyze the distribution of HDV RNA in Huh7-D12 cells treated with a-amanitin+ Because exposure of cultured cells to 100 mg/mL a-amanitin for 4 h has been shown to trigger degradation of the largest subunit of RNA polymerase II (Nguyen et al+, 1996), Huh7-D12 cells were treated with a-amanitin at concentrations ranging from 100 to 500 mg/mL for 5 h prior FIGURE 2. Intranuclear distribution of HDV RNA+ A,B: Huh7-D12 cells were fixed with formaldehyde, permeabilized with Triton X-100, and hybridized with plasmid pSVL(D3)+ C: Cells were treated with 500 mg/mL a-amanitin for 5 h prior to fixation and hybridization+ D: Cells were treated with RNase A prior to hybridization+ E: Cells were treated with DNase I prior to hybridization+ In A and B (untreated cells), the hybridization signal is either spread throughout the nucleoplasm (arrowheads) or confined to a cluster of fine dots (double arrows); nucleoplasmic staining is finely punctate (A) or coarse-grained (B) with additional brighter foci (A, B, single arrows)+ The hybridization signal in cells treated with a-amanitin (C) is similar to that of untreated cells+ Treatment with RNase abolishes nucleoplasmic staining without affecting the clusters of fine dots (D, double arrows)+ Inversely, when cells are treated with DNase I, the fine clusters are no longer observed, whereas nucleoplasmic staining with additional concentration in foci is not affected (E, arrowheads)+ Bar, 10 mm+ to fixation and hybridization+ Interestingly, the staining pattern remained similar to that observed in nontreated cells (Fig+ 2C), indicating that both the dispersed nucleoplasmic signal and the brighter foci represent stable populations of viral RNAs+ Because cells are denatured before hybridization, the pSVL(D3) probe binds to both the HDV RNA and HDV cDNA integrated in the...…”

“…Hepatitis delta virus (HDV) was originally discovered as a cause of severe liver disease in patients infected with hepatitis B virus (HBV) (reviewed by Taylor, 1992;Monjardino & Lai, 1993)+ The clinical association of these two viruses is due to the fact that the outer coat of HDV consists of HBV surface glycoproteins+ Thus, productive infection and transmission of HDV requires either co-infection or superinfection with HBV+ The genome of HDV, the smallest animal pathogen known so far, is a single-stranded circular RNA of about 1,700 nt that is ;70% self-complementary and, as a result, forms a highly base paired rodlike structure (see Taylor, 1992)+ This genomic RNA contains a single open reading frame from which two forms of a unique protein (the delta antigen) are derived by RNA editing+ Transcription of the genome results in the synthesis of a 700-nt mRNA that encodes the small form of the delta antigen (dAg-S or HDAg-p24)+ The larger form of the delta antigen (dAg-L or HDAg-p27) is produced when RNA editing converts an amber stop codon (UAG) to a tryptophan codon (UGG), extending the open reading frame by 19 amino acids (see Taylor, 1992;Monjardino & Lai, 1993)+ The smaller form of the delta antigen is indispensable for HDV genomic replication (Kuo et al+, 1989)+ In contrast, the larger form represses replication Glenn & White, 1991) and is required for packaging of viral particles (Chang et al+, 1991;Ryu et al+, 1992)+ Upon entry into the cell, the genomic HDV ribonucleoprotein complex is transported to the nucleus, where it is replicated by an RNA-directed transcription mechanism that presumably involves cellular RNA polymerase II (MacNaughton et al+, 1991;Fu & Taylor, 1993)+ According to the current model of HDV replication, the incoming HDV genome serves as a template for rolling circle replication, resulting in the production of multimeric antigenomes+ The nascent antigenomic multimers harbor ribozymes that self-cleave at monomeric intervals+ Two self-cleavage events release an antigenomic linear monomer and ligation of its termini produces a circular molecule referred to as the antigenome+ Possibly, through a similar rolling circle mechanism, these antigenomes then serve as templates for the subsequent synthesis of genomic RNAs (reviewed by Taylor, 1992;Lazinski & Taylor, 1995)+ Editing occurs in the antigenome and is thought to involve a cellular double-stranded RNA-specific adenosine deaminase (dsRAD/DRADA/ADAR1) that converts the adenosine at the amber/W site to an inosine (Casey & Gerin, 1995;Polson et al+, 1996;Bass et al+, 1997)+ In addition, maturation of HDV RNAs requires the cellular 39 end processing apparatus for cleavage and polyadenylation …”

A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II

“…This RdRP activity of Pol II was first found to be essential for the replication of Potato spindle tuber viroid (PSTVd) (Muhlbach and Sanger, 1979) and later for transcription and replication of human hepatitis delta virus (HDV) (MacNaughton et al, 1991;Modahl et al, 2000). Recent studies showed that this RdRP activity has broader biological significance beyond pathogen infection.…”