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.

Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.During the replication of human hepatitis delta virus (HDV), three RNAs are generated by RNA-directed RNA transcription and posttranscriptional processing. The genome and its exact complement, the antigenome, are 1,679-nucleotide circular RNAs that fold into a rod-like structure with 74% of their nucleotides base paired (20,38). It is considered that these RNAs are derived from longer than unit length primary transcripts that are processed to unit length by the HDV ribozymes and then ligated into circles (35). The third RNA is of the same polarity as the antigenome but only about 800 nucleotides in length. It has a defined 5Ј end that is capped and a defined 3Ј end that is polyadenylated (14). This mRNA contains the open reading frame for the only protein of HDV, the small delta antigen (␦Ag). This protein is essential for HDV replication but at 195 amino acids in length is too small to have polymerase activity (7).Several lines of evidence implicate the host RNA polymerase II (Pol II) as being required for the transcription of HDV RNAs (reviewed in references 21, 35, and 36). However, data from Lai and coworkers has been interpreted as evidence that the synthesis of antigenomic RNA is resistant to high doses of amanitin and therefore may be more consistent with being directed by RNA Pol I, the enzyme involved in the transcription of rRNAs (23,25,27). In order to resolve this controversy and also to obtain more information about HDV RNA-directed transcription, we have made use of the following experimental system.As previously described, we first established 293-␦Ag cells, a line of 293 cells in which the essential ␦Ag is provided by an integrated cDNA, with expression inducible by tetracycline (TET). These cells w...

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 ␦Ag-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.During the replication of the RNA genome of human hepatitis delta virus (HDV), three RNA species of discrete sizes accumulate (7). (i) First, there is the genome itself, a 1,679-nucleotide (nt) single-stranded RNA with a circular conformation. (ii) Next, there is an exact complement of the genome, the antigenome. Both the genomic and antigenomic RNAs contain a ribozyme domain. These circular RNAs arise via posttranscriptional RNA processing. It is thought that RNA-directed RNA transcripts of greater than unit length and containing at least two ribozyme domains undergo site-specific cleavage to release unit-length linear RNAs which are then ligated to produce circular species. (iii) Finally, there is a less-than-full-

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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