INTRODUCTION

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Plant viroids and the human hepatitis delta virus (HDV) have much in common (47). One of the more intriguing similarities is that they both replicate via RNA-directed RNA synthesis using a host RNA polymerase (44). Since there are no known DNA intermediates (5) the most likely explanation is that these agents with their single-stranded RNA genomes are able to redirect a host polymerase that normally uses DNA templates to use RNA as a template. An alternative explanation is that the host cell contains an RNA-directed RNA polymerase that can carry out the transcription. Actually, such a polymerase was first found and purified from plants (40). cDNA cloning of this polymerase (41) revealed that all plants and some animals contain at least one copy of a related gene. However, at this time, no such gene has been detected in mammals, which can be hosts for HDV.

Several different suggestions have surfaced as to which host polymerases are involved. The plant viroids are frequently divided into two families. For one of these families there are data that transcription occurs in association with chloroplasts, using a nucleus-encoded polymerase (34). For the second viroid family, the available evidence implicates the host RNA polymerase II (pol II) (42, 49). Similarly, with HDV there are many claims for pol II as the relevant enzyme (1, 12, 13, 26), although a recent paper has suggested the possibility that more than one host polymerase may be involved (31).

For purposes of evaluation, the evidence for the polymerase(s) involved in HDV transcription may be divided into four different types: (i) circumstantial, (ii) in vivo, (iii) in vitro, and (iv) a combination of in vivo and in vitro.

(i) Consider first the circumstantial evidence. During HDV genome replication the following three RNA species are detected (5): (a) the 1,679-nucleotide (nt) circular RNA genome; (b) its exact complement, the so-called antigenome; and (c) relatively low amounts of a less-than-unit-length antigenomic RNA that contains the open reading frame for the small delta protein, a 195-amino-acid species that is essential for HDV replication. This third RNA is thought to be an mRNA. The 5' end is capped (17) and corresponds to a discrete location (16), and at the 3' end is a poly(A) tail (19). There is evidence that the genomic RNA acts as template for the transcription of multimers of antigenomic RNA, some of which are processed to become unit-length RNA circles while others are processed to become the poly(A) species. The site of this polyadenylation is directed by an AAUAAA signal (19). Overall, these features of the third RNA provide circumstantial evidence that it was transcribed and processed just as would be expected for a DNA-directed pol II transcript. Possibly another example of circumstantial evidence is that both in situ hybridization to detect HDV RNAs (9, 45) and immunomicroscopy to detect the small delta protein (3, 9) that is needed for HDV genome replication (20) indicate a nucleoplasmic location, just as is found for pol II and pol III but not for pol I (8).

(ii) Several studies have attempted to use in vivo approaches. The early reports treated cells in which HDV genome replication was occurring with the pol II-specific inhibitor -amanitin (26). These studies detected inhibition but the shortcoming was that HDV replication had been initiated via the stable transfection of the cells with a multimer of HDV cDNA; it was therefore not possible to separate transcription that was DNA directed from that which was RNA directed. A recent report by Modahl et al. used HDV RNAs transcribed in vitro to transfect cells and initiate genome replication (31). At various times after transfection -amanitin was added and the accumulation of HDV RNA was measured; the data were interpreted as evidence that pol II is needed for HDV mRNA synthesis but that some other polymerase that is more -amanitin resistant than pol II carries out the synthesis of the unit-length genomic and antigenomic RNA species (31). The major shortcoming of this study was the lack of controls for DNA-directed transcription by the host polymerases, pol I, II, and III. In addition, an intrinsic limitation of such studies is that they can only assay the accumulation of processed HDV RNAs rather than directly measure HDV transcription. That is, the accumulation detected, for example, by Northern blot analyses, reflects not only RNA transcription but also RNA processing and RNA stability.

(iii) The third experimental approach used has been in vitro, using either cell extracts or sources of purified pol II. Such studies have detected transcription of added HDV RNA that was pol II directed. However, examination of these results shows that in most cases the transcription was no more than a modest 3' end addition to a linear HDV RNA template (1, 12, 17). In one case, the RNA template somehow underwent an endonucleolytic cut which revealed a 3' end that was also used for modest 3'-end addition (12). And, except for two recent papers (17, 51), the delta protein that in vivo is essential for genome replication (20) was not even added exogenously to the in vitro transcription. In one of these studies, the exogenous delta protein was reported to have no effect (17), and in another, it was reported to modestly increase the processivity of pol II on DNA and RNA templates (51).

(iv) There is, however, a fourth experimental approach that has not been previously tried but is potentially superior. This approach has two parts. First, HDV genome replication is initiated in vivo by transfecting cells using RNA templates. Second, these cells are disrupted and examined for transcription in vitro in the presence of radioactive precursors, with and without specific polymerase inhibitors. We considered that this strategy might have several advantages relative to the other methods. It allows the use of inhibitor concentrations that could not be used in vivo because of toxic effects on the cell. The effects of a polymerase inhibitor used in this way are more likely to be direct than indirect. Another major advantage is that the HDV template is not exogenously added to the in vitro reaction but is endogenous, and moreover, if the delta protein is needed for such transcription, it is also already present, and possibly present in the appropriate stoichiometry.

The first use of approach iv is described in this manuscript. To make this possible and in parallel to assay the effects of the inhibitors of endogenous DNA-directed transcripts by pol I, II, and III, we had to make some important technical changes to the standard runoff protocols. These are described in Materials and Methods. After these changes, we were able to obtain HDV-specific transcription and to study the effects of -amanitin on runoff transcription.

As mentioned above, the recent application of approach ii by Modahl et al. offered the interpretation that for the synthesis of the unit-length genomic and antigenomic RNAs the host polymerase used, at least according to -amanitin sensitivity, was not pol II. As described in this paper, we carried out extensive studies using a very similar approach, with -amanitin and several other transcription inhibitors. In contrast to Modahl et al., we found no evidence for the involvement of a polymerase other than pol II in the transcription and ultimate accumulation of processed unit-length genomic RNA.

Overall, from an evaluation of the existing data and from our new data using approaches ii and iv, we are left with the interpretation that HDV RNA-directed RNA transcription is at least primarily via an enzyme that behaves like pol II and not like pol I or pol III.

	MATERIALS AND METHODS

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RNA for transfection. We used combinations of the three RNAs diagrammed in Fig. 1. RNA1 was an antigenomic RNA of 1.2× the unit length transcribed from plasmid pTW114 (50), which contains an HDV insert flanked by a T7 promoter and terminator, using a T7 RNA polymerase transcription kit (Promega). Similarly, RNA3 was a genomic RNA of 1.2× the unit length transcribed from plasmid pTW101 (50). RNA2 acted as an mRNA containing the open reading frame for the small delta protein. This RNA was both 5' capped and 3' polyadenylated. It was transcribed by phage T7 RNA polymerase using an expression PCR product as template and a capped RNA transcription kit (Ambion). The PCR product was such as to place (A)₂₅ at the 3' end of the T7 transcripts. The T7 polymerase reaction mixtures were treated with DNase and then extracted using Tri Reagent (Molecular Research Center). In the experiments described for Fig. 5, RNA1 was transcribed using T7 polymerase from pTW107, which had been opened at a HindIII site, to obtain transcripts with a 2-nt deletion at the unique EcoRI site to disrupt the open reading frame of the small delta protein (50).

View larger version (9K): [in this window] [in a new window]   FIG. 1.   Representation of antigenomic and genomic RNA transcripts used to initiate HDV genome replication. RNA1 is a 1.2× linear antigenomic RNA. RNA2 is a capped and polyadenylated mRNA for the small delta protein, with the open reading frame as indicated. RNA3 is a 1.2× linear genomic RNA. Note that RNA1 and RNA2 have two copies of their respective ribozymes (Rz). In the experiment for Fig. 5 we used a form of RNA1 with a 2-nt deletion at the unique EcoRI site (E). The positions of the 5' and 3' ends are indicated for RNA1 and RNA3 using the notation of Kuo et al. for the 1,679-nt RNA sequence (21).

Transfections. For RNA transfections we used two RNAs, either RNA1 and RNA2 or RNA 2 and RNA3. This two-RNA strategy was a modification of the method described by Modahl and Lai (30). For example, 1 day prior to transfection, Huh7 cells (33) were seeded at 1.7 × 10⁵ per well of a 12-well tissue culture dish (USA Scientific). Transfections were carried out with 0.5 µg of the 1.2× antigenomic RNA and 0.1 µg of the capped delta antigen mRNA, using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions.

Inhibitors and [³H]uridine labeling. The five inhibitors used were -amanitin, 5,6-dichloro-1--ribofuranosylbenzimidazole (DRB), rifampin, actinomycin D (all from Sigma), and tagetitoxin (Epicenter). The concentrations used and the treatment times are as indicated in the text and/or the figure legends. At the beginning of an in vivo inhibitor treatment, we added 20 µCi of [³H]uridine (Amersham) to label host RNA species, especially rRNA and tRNA, which are transcribed by pol I and pol III, respectively.

RNA extraction and Northern blot analyses. Total RNA was extracted from transfected cells using Tri Reagent. The RNA fraction was then treated with RQ DNase I (Promega) and reextracted. Aliquots of <5 µg were glyoxalated and assayed for HDV RNA by Northern blot analyses, as previously described (50). However, immediately after the electrotransfer and deglyoxalation, the charged nylon membrane (Zetaprobe; Bio-Rad) was air dried and the ³H-labeled RNA species were detected by placing the filter in direct contact with a special imaging screen (BAS TR2040; Fuji). After appropriate exposure the signal was quantified with a Bio-Imager (Fuji). The filter was then hybridized with strand-specific ³²P-labeled RNA probes to detect genomic HDV RNA. Radioactivity was detected with a standard imaging screen and subsequently quantitated using the Bio-Imager.

Protein extraction and immunoblot analyses. Total proteins were extracted using Tri Reagent. Samples were denatured using Laemmli buffer (22) and analyzed by our standard immunoblot procedures (32), using polyclonal rabbit antiserum against recombinant small delta protein, followed by incubation with ¹²⁵I-labeled staph A protein (DuPont). Quantitation was done with the Bio-Imager.

Preparation of nuclei and in vitro runoff assays. Each experimental point required a subconfluent monolayer of Huh7 cells on 100-mm-diameter culture dishes. One day after seeding these cells were cotransfected using Lipofectamine Plus with a combination of the two RNAs (3.2 µg of RNA3 and 0.8 µg of RNA2). HDV replication was allowed to proceed until day 6. In the 15 min prior to preparing the nuclear extracts the cells were treated with 0.2 µg of actinomycin D/ml. Prior studies showed that this dose inhibited rRNA synthesis and accumulation with no detectable effect on HDV, even when present for 1 day (see Fig. 3). Use of this as a pretreatment strategy was suggested by previous studies (35, 37) that used it to partially suppress rRNA synthesis in the subsequent runoff reactions. In our case, such suppression was additionally important because of the known ability of mammalian rRNA to cross-hybridize with HDV RNA (14).

	RESULTS

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HDV RNA-directed RNA synthesis in transfected Huh7 cells. For the two studies in which we attempted to characterize the host polymerase requirements of HDV transcription we needed cultured cells undergoing HDV genome replication. It is possible to initiate such replication by transient transfection with cDNA constructs (20) and even to select cell clones stably transfected with such constructs (6, 27). However, in such situations the HDV transcription can be both RNA and DNA directed; this could confound studies aimed at characterizing the host polymerase involved in RNA-directed transcription. Therefore, we considered initiation of replication by HDV RNA species. Several such strategies have been described (2, 11, 14, 30). For the present studies we decided that the best choice was that of Modahl and Lai (30) in which cells are transfected using a mixture of two HDV RNAs. As illustrated in Fig. 1, RNA1 is a greater-than-unit-length species of antigenomic RNA and RNA2 is a subgenomic-sized RNA with a 5' cap and a 3' poly(A) tail that can be translated to provide the initial source of the essential small delta protein. In a variation of this strategy we replaced RNA1 with RNA3, which is a greater-than-unit-length genomic RNA.

View larger version (60K): [in this window] [in a new window]   FIG. 2.   Time course of HDV RNA and protein accumulation following transfection of Huh7 cells. Replica cultures were transfected with two HDV RNAs and at the indicated times were extracted with Tri Reagent to yield RNA samples for Northern blot analysis to detect the accumulation of unit-length genomic HDV RNA (A) or protein for immunoblot to detect the two forms of the delta antigen (Ag-S and Ag-L) (B). At 1 day prior to harvest [3H]uridine was added to the cultures to provide a measure of the rate of host 18S rRNA synthesis (C).

In vivo inhibition of HDV RNA accumulation using approach ii. We tested the effects of the following five RNA transcription inhibitors. Others have shown that -amanitin, a mushroom toxin, binds specifically to a region on the large subunit of pol II and promptly inhibits transcription (10). DRB is also reported to be specific for pol II but its mechanism of inhibition is more complicated; this nucleoside analog is believed to act indirectly, causing the inhibition of a kinase whose action on the carboxyl-terminal domain of the largest subunit of pol II is essential for the continued elongation of nascent chains (36). Tagetitoxin has been reported to act on pol III transcription in some systems (43). Rifampin appears to act on the nucleus-encoded mitochondrial RNA polymerase, although there are contrary reports (15). Finally, actinomycin D is considered to be a specific inhibitor of all DNA-directed transcription. RNA-directed transcription by some viral RNA polymerases is resistant to this drug (7, 28, 38). It has been reported that HDV transcription in a stably transfected cell line was also resistant to this drug (25).

View larger version (61K): [in this window] [in a new window]   FIG. 3.   Effect of three polymerase inhibitors on accumulation of HDV genomic RNA in Huh7 cells transfected with HDV RNAs. Replica cultures were transfected as described for Fig. 2 and harvested at day 0 (lane 3), 2 (lane 4), or 3 (lanes 5 to 19), as indicated. For lanes 6 to 19, at day 2 a series of concentrations of one of three inhibitors was added to determine the effect on the accumulation of HDV unit-length genomic RNA (A). Lane 5 represents an untreated control. Also added for lanes 5 to 19, at day 2, was [3H]uridine to provide a measure of the rate of host 18S rRNA (B) and tRNA (C) synthesis. Lanes 6 to 11, -amanitin added to 0.03, 0.1, 0.3, 1, 3, and 10 µg/ml, respectively; lanes 12 to 15, DRB added to 1, 3, 10, and 30 µg/ml, respectively; lanes 16 to 19, actinomycin D added to 0.03, 0.1, 0.3, and 1 µg/ml, respectively; lane 1, size marker; lane 2, DNA standard of unit-length HDV cDNA.

View larger version (42K): [in this window] [in a new window]   FIG. 4.   Effect of -amanitin on the accumulation of HDV genomic RNA in Huh7 cells transfected with HDV RNAs. For panels A, C, and D, replica cultures were transfected as for Fig. 3. For panel B, replica cultures were transfected with pDL541, a DNA construct that expresses a 1.2-mer of a deleted form of HDV genomic RNA; the transcription is driven by pol II and the transcripts are processed by two copies of the HDV genomic ribozyme to produce 348-nt RNA circles. Lanes 1 to 5, standards as described for Fig. 3; lanes 6 to 9, at 0.2 days -amanitin was added at 0.025, 0.05, 0.1, and 0.2 µg/ml, respectively. From the gel analyses of the extracted RNAs we detected unit-length HDV genomic RNA (A), the mini-RNA transcribed from a DNA template by pol II (B), 18S rRNA transcribed by pol I (C), and tRNA transcribed by pol III (D). For panel E we used quantitation of the experiment shown in panels A to D, along with optical density measurements on total extracted RNA, to determine, relative to untreated cultures, the amount of accumulation of four different RNA species, as indicated, that was achieved in the presence of four different concentrations of -amanitin.

View larger version (39K): [in this window] [in a new window]   FIG. 5.   Effect of -amanitin on the accumulation of HDV genomic RNA in cells transfected with mutated HDV RNA. Replica cultures of Huh7 cells that had been previously stably transfected to express the small form of the delta protein were transfected with an RNA, like the RNA1 in Fig. 1, but with a 2-nt deletion at position E within the open reading frame. Data are shown for an untransfected culture (lane 1) and at days 1.5 (lane 2) and 3 (lane 3) after transfection. For certain cultures -amanitin was added at day 1.5 at 0.2 (lane 4), 0.4 (lane 5), and 0.8 (lane 6) µg/ml, and the cells were harvested at day 3. For lanes 3 to 6, [3H]uridine was also added at day 1.5 to provide a measure of the rate of host 18S rRNA (C) and tRNA (D) synthesis. Northern blot analyses were used to detect unit-length genomic RNA (A) and immunoblots were used to quantitate expression of the small delta protein (B). For panel E we obtained quantitation of the effects of -amanitin on accumulation of mutated HDV genomic RNA, just as for Fig. 4

In vitro inhibition of HDV runoff transcription using approach iv. Processed HDV RNAs can accumulate to high levels per infected or transfected cell. For example, in the average liver cell of an infected woodchuck, there can be as many as 300,000 copies of the HDV genome (5). However, the actual rate of RNA transcription can be relatively slow. Also, in transiently transfected cultures replication is not initiated in every cell (maybe 10 to 40% in our experiments). As intimated in the introduction, our aim was to initiate replication in vivo by transfection of Huh7 cells with HDV RNAs and then after 6 days to carry out an in vitro runoff reaction to detect the endogenous transcription of HDV RNAs. In a series of experiments we used the pol II-specific inhibitor -amanitin, and for controls we assayed for endogenous host transcription by 28S rRNA (pol I), total poly(A)-containing RNA (pol II), and 7S L RNA (pol III) in an attempt to determine which polymerase was needed for HDV genomic RNA transcription.

View larger version (50K): [in this window] [in a new window]   FIG. 6.   Hybridization of 32P-labeled runoff products to slot-immobilized nucleic acids. Nuclei from untransfected () or RNA transfected (+) cells were used in runoff reactions in the absence (0) or presence of -amanitin at 0.1 and 0.3 µg/ml. For panel A, the 32P-labeled RNAs so obtained were each hybridized to a filter with an array of four specific nucleic acids to assay the endogenous transcripts, as indicated. Detection of 32P was by Bio-Imaging plate, with quantitation summarized in Table 1 and deductions as presented in panel B.

	DISCUSSION

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We have described here two applications of RNA transcription inhibitors in an attempt to clarify the host polymerase requirements for HDV RNA transcription. In the first application we determined the effect of the inhibitors on the ability of cultured cells transfected with HDV RNAs to continue HDV RNA-directed RNA synthesis and to accumulate processed unit-length HDV RNA species. With low doses of actinomycin we were able to obtain evidence that processed HDV RNAs could accumulate under conditions that blocked host 18S rRNA (Fig. 3). The studies with DRB, rifampin, and tagetitoxin did not provide any evidence as to which host polymerase is involved in the transcription (Fig. 3 and data not shown). However, from the treatment of cells with closely spaced doses of -amanitin, we were able to determine that HDV RNA accumulation relative to appropriate controls for DNA-directed transcription by pols I, II, and III depended upon pol II (Fig. 3 to 5).

Our results are partially similar to those of Modahl et al. (31), who in some situations found HDV RNA accumulation was sensitive to -amanitin. However, in other situations these authors reported that HDV accumulation was resistant to as much -amanitin as 100 µg/ml. We find this additionally puzzling because in our experiments, using the same cells, exposure to more than 10 µg/ml for 24 h was sufficiently toxic that virtually all the cells detached from the monolayer. Even after exposure to 3 or 10 µg/ml for the same period of time we could detect a major inhibition of rRNA accumulation. After exposure to 1 µg/ml we observed by microsopy a rounding up of the cells and a rearrangement of 4',6'-diamidino-2-phenylindole (DAPI)-stained nuclear DNA (data not shown). Also, by immunostaining to detect either the delta antigen or pol II, we could detect nucleoplasmic rearrangement (data not shown). Others have reported similar effects for cells treated with -amanitin as well as with DRB and actinomycin (18). While it may not be unreasonable that treatment of cells with such inhibitors produces toxic effects, it raises the concern that the associated inhibition of HDV RNA accumulation might be an indirect rather than a direct effect. Actually, on a wider scale, any inhibition detected in such in vivo studies could be an indirect effect.

In order to avoid such limitations we carried out our second series of experiments. In these we applied nuclear runoff reactions, combined with RNA transcription inhibitors, in an attempt to identify the host polymerase involved in HDV RNA transcription. With -amanitin we obtained specific inhibition of both HDV RNA transcription and of DNA-directed transcription of host poly(A)-containing RNAs (Fig. 6). We interpret this as evidence that HDV RNA is transcribed by pol II.

It is reasonable to expect that the HDV transcription we detected from the runoff transcription made use of endogenous circular HDV RNA templates. The HDV genome replication was initiated in vivo by transfection of cells with greater-than-unit-length linear genomic RNAs, but it was 6 days later when replication was well under way that we isolated the nuclei for the runoff assays. Furthermore, based on Northern blot analyses of RNA extracted from such cells at day 6, we know that the majority of this de novo antigenomic HDV RNA was both unit-length in size and circular in conformation (4). We think that our experimental strategy avoided potentially artifactual transcriptional results that might arise in an alternative strategy where exogenous linear HDV RNA templates are added to in vitro transcription reactions (1, 12, 17).

We think that our ability to detect HDV runoff transcripts using nuclear extracts rather than having to use permeabilized cells excludes any requirement by HDV of a cytoplasmic polymerase, such as the nucleus-encoded mitochondrial RNA polymerase (15). We should also make two comments on the possible relevance to HDV transcription of the RNA-directed RNA polymerase that is present in all plants and also some animals (41). First, so far this polymerase has not been detected in mammals (41). And second, when this polymerase was purified from plants, it was found to be -amanitin resistant (39).

In summary, as a consequence of the two studies presented here and an evaluation of earlier studies we are now confident that in vivo HDV RNA-directed transcription at least primarily involves host RNA pol II. We can now move on and determine how this transcription is achieved. The runoff assays used here can be adapted to determine the 5' ends of the nascent transcripts. Such information will help test the hypothesis that a combination of specific nucleotide sequences and RNA folding might provide an HDV pol II promoter for transcription from an HDV RNA template (1, 16, 46). Also, it may be that the runoff assay system, in conjunction with specific antibodies (other than anti-pol II), can be used to identify the host transcription factors needed for HDV RNA-directed transcription.