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Journal of Virology, May 2004, p. 4517-4524, Vol. 78, No. 9
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.9.4517-4524.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Alternative Processing of Hepatitis Delta Virus Antigenomic RNA Transcripts

Xingcao Nie, Jinhong Chang, and John M. Taylor*

Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111-2497

Received 12 November 2003/ Accepted 29 December 2003


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ABSTRACT
 
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 {delta}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.


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INTRODUCTION
 
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-length RNA of the same polarity as the antigenome. It is a 5'-capped, 3'-polyadenylated species that acts as the mRNA for the one protein encoded by HDV, the small delta protein, {delta}Ag-S (10). This 195-amino-acid protein is essential for HDV replication (6). During replication, the termination codon on the mRNA for {delta}Ag-S is changed as a result of posttranscriptional RNA editing by an adenosine deaminase (18), leading to the translation of a longer protein, large delta antigen, {delta}Ag-L, which may or may not act as an inhibitor of HDV replication (6, 19) but which is certainly needed for particle assembly (3).

From this account, it should be clear that multiple posttranscriptional RNA-processing events must occur during the replication of HDV RNA. Previous studies have focused on these processing events (13, 21). Also, detailed double-rolling-circle models have been formulated to explain what might be the coordination of these processing events (9, 20, 21, 25). However, in parallel with consideration of such models, some attention must be given to the mechanism of RNA-directed transcription, especially since we know that some forms of RNA processing are associated with transcripts made by a particular RNA polymerase. Specifically, poly(A) processing is known to occur only for polymerase II (Pol II) transcripts.

In one rolling-circle model of HDV replication, it is presumed that all RNA transcription is carried out by the same RNA polymerase (9, 25). Two independent studies agree with the interpretation that both the mRNA and the genomic RNA arise via transcription using the host RNA Pol II (11, 20, 22). However, one study using the inhibitor {alpha}-amanitin has been interpreted as evidence that the antigenomic RNA is transcribed by a polymerase other than Pol II (20). This interpretation, which we consider not to be justified by the data, has become the basis of a model in which the genomic RNA is somehow used as a template by two different polymerases: by Pol II, to make an RNA that is processed by 5' capping and 3' polyadenylation to produce the mRNA, and in addition by Pol I, to make an RNA that is ribozyme processed to produce the unit-length antigenomic RNA (20, 21).

Figure 1 presents the relevant part of a detailed rolling-circle model of HDV genome replication, in which all antigenomic RNA transcripts were assumed to be initiated by the same polymerase, presumably Pol II, and predominantly at a single site, namely, that corresponding to the 5' end of the what becomes a 5'-capped and 3'-polyadenylated mRNA (9-11). This model implies that such nascent transcripts could "continue" to be transcribed long distances beyond the poly(A) signals, so as to become greater-than-unit-length antigenomic species. These multimers could be processed by the antigenomic ribozyme to become unit-length linear RNAs which, in turn, would be ligated to produce unit-length circles. The studies described here were undertaken to test some of the predictions and speculations associated with this model. One of our findings is that RNA processing by polyadenylation and ribozyme cleavage should be considered events that in most cases act on two separate RNA transcripts. That is, a nascent RNA transcript may undergo either polyadenylation or ribozyme cleavage, but not both.



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  		FIG. 1. Model for transcription and processing of nascent antigenomic HDV RNAs. This diagram represents an abbreviated form of an earlier model (25). It does not incorporate the results of the present study. The circular genomic RNA template folded into an unbranched rod-like structure is shown in gray, and the nascent antigenomic RNA is shown in black. Posttranscriptional processing on antigenomic RNA is indicated by a box for the poly(A) signal and a circle for the ribozyme. Transcription of antigenomic RNA is considered to start at position 1630 (11), according to the sequence and numbering of Kuo et al. (14) for the 1,679-nt RNA. After step 1, the nascent transcript is about half of unit length. In step 2, the 5' end of the transcript undergoes ribozyme cleavage and then polyadenylation, while the 3' end of the transcript is proposed to continue. In steps 3 and 4 the transcript continues to extend, until in steps 5 and 8 a second ribozyme cleavage releases a unit-length linear RNA, which in step 9 folds into a rod-like structure and in step 10 is ligated to form a new antigenomic RNA circle. In steps 6 and 7, the continuing transcript is an additional source of unit-length RNA. Somehow poly(A) processing is suppressed on the unit-length antigenomic RNAs. The impact of new findings on this model is presented in the Discussion.

Two experimental approaches were used in the present studies. The first represented an examination under more controlled conditions of mutations in the poly(A) signal, AAUAAA, which is essential for this processing (13). The second approach involved an examination of the processing in transfected cells of specific Pol II DNA-directed transcripts of HDV antigenomic sequences. As a consequence of using these two different approaches, we obtained a better understanding of how the processing of antigenomic HDV RNA transcripts is coordinated. To take these findings into account, we propose several revisions to the model of HDV genome replication.


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MATERIALS AND METHODS
 
Cell culture and transfection. Cells of the human hepatocellular carcinoma line Huh7 (23) were cultured in Dulbecco's modifed Eagle medium supplemented with 10% fetal bovine serum. A line of human embryonic kidney cells (Flp-In T-REx-293 cells; Invitrogen) was used to establish a tetracycline-inducible line, {delta}-293, that expresses the small form of delta antigen, {delta}Ag-S. Transfections with either DNA or RNA were performed by using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen).

Plasmids. For the studies for which results are shown in Fig. 2, we used RNAs transcribed from plasmids pTW108 and pXN102. As previously described, construct pTW108, when opened with HindIII and transcribed in vitro with T7 polymerase, yields a genomic HDV RNA (29). This RNA is 1.2 times the unit length and has a 2-nt deletion in the open reading frame (ORF) for {delta}Ag-S; therefore, it is unable to make a functional protein. The RNA transcribed from pXN102 was identical to that from pTW108 except for the modification of the poly(A) signal from AAUAAA to UUUAAA.



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  		FIG. 2. Effect of a poly(A) signal mutation on HDV RNA accumulation. These experiments were carried out by using a line of {delta}-293 cells that stably expressed large amounts of {delta}Ag-S. These cells were transfected with 1.2-times-unit-length genomic RNAs as transcribed in vitro. These RNAs each had a 2-nt deletion in the ORF for {delta}Ag-S. One RNA had an additional mutation in the poly(A) signal. At 3 days after transfection of the cells, total RNA was extracted and assayed by Northern blotting to detect antigenomic RNA sequences. (A) Results of Northern blot analysis. Lanes p and q, HDV RNAs detected in cells transfected with wild-type and poly(A)-mutated RNAs, respectively. By reference to unit-length HDV cDNA, used as a standard (lane r), we identify the predominant bands in lanes p and q as unit-length antigenomic RNA. From separate studies, we identify the two mRNA species as that stably produced by {delta}-293 cells and that produced by HDV genome replication. (B) Quantitation of the data for the three HDV-specific bands detected in panel A, lanes p and q. (C) Total RNA isolated from {delta}-293 cultures, as in panel A, lane p, but obtained from a separate transfection, was applied to a column of protein A-Sepharose (Sigma) to which had been complexed a monoclonal antibody that binds the 7-methylguanosine of capped RNA species (2). Bound (C+) and unbound (C–) fractions were obtained and subjected to a Northern blot assay to detect antigenomic HDV RNA species. Electrophoretic separation was increased relative to that for panel A in order to better separate {delta}-293-mRNA from HDV-mRNA.

For the studies for which results are shown in Fig. 3 and 4, eight HDV constructs were assembled in the pSVL expression vector (Pharmacia) to produce RNA transcripts a through h, as diagrammed in Fig. 3B. This vector uses the simian virus 40 (SV40) late promoter for Pol II-directed transcription. We modified the vector and removed the SV40 poly(A) signals at the 3' end and, in most cases (except for constructs which transcribe b and f), the SV40 intron at the 5' end. The inserted HDV cDNA sequences with the in-frame deletion in the {delta}Ag-S ORF were derived from plasmid pDL448, as previously described (15). Those with the frameshift in the ORF contained a 2-nt deletion at the unique EcoRI site, as previously described (6). The mutation of the poly(A) signal has also been described previously (13). These eight cDNA constructs were cotransfected into Huh7 cells along with pSVTVA, a plasmid which expresses both T-antigen and VA genes and enhances in vivo transcription from SV40-based vectors 16-fold (1).



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  		FIG. 3. RNA species obtained by processing of RNA transcripts arising from in vivo DNA-directed antigenomic HDV RNA transcripts. (A) (Top) Diagram of RNA-processing features present on a greater-than-unit-length antigenomic RNA. This RNA begins at nt 1630 (numbering according to the HDV nucleotide sequence of Kuo et al. [14]), which has been separately determined to be the 5'-cap site for HDV mRNA (11). Other features shown, from left to right, are the ORF for {delta}Ag-S, followed by the known poly(A) signal and poly(A) acceptor site; the site of cleavage by the antigenomic ribozyme, at positions 901 and 900; and a complete antigenomic RNA sequence with a repetition of all the RNA-processing signals, followed by non-HDV sequences (dashed line). (Bottom) Six theoretical possibilities for RNAs that may be derived by alternative RNA processing of the nascent HDV RNA transcript that would be produced in vivo from a multimeric HDV cDNA construct driven by Pol II from a promoter just upstream of the known 5' end of the mRNA. These six processed RNAs fall into three main size classes: RNAs 1 to 3. Within each size class, there is a pair of species that differ only with regard to their 3' processing, by polyadenylation (pA) or ribozyme cleavage (Rz). (B) Unprocessed RNAs that could be transcribed from a series of eight HDV cDNA constructs. Transcript e is essentially the same as the transcript proposed in panel A. Note that the first and second ORFs have been changed; the first has an in-frame deletion of 39 nt, to produce a protein that does not support replication, while the second has a 2-nt deletion, to produce a small, unstable protein fragment. Otherwise, constructs a through h are as described in the text.



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  		FIG. 4. (A) Northern blot analysis of processed HDV RNAs. Huh7 cells were transfected with constructs to express four of the primary RNA transcripts, a through d, as diagrammed in Fig. 3B. At day 3, total RNA was extracted and subjected to Northern blot analysis to detect antigenomic HDV RNA species. The main HDV-specific band is indicated as RNA-1. Also indicated is the cross-hybridizing band of 28S rRNA, which was used as an internal control for the amount of total-cell RNA loaded. We note that in lanes a through d, there are other, less intense cross-hybridizing bands of about 2 kb and larger. Not all of these are detected in RNA from untransfected cells (lane u) or even consistently. Therefore, we consider these extra bands to represent endonucleolytic fragmentation of an abundant host RNA, probably rRNA, that occurs in some of the transfected cells. Lane r, unit-length HDV cDNA used as a control. (B) Quantitation of the data for HDV-specific bands detected in panel A, lanes a through d; data for transcript a are set to 100%.

In vitro RNA transcription. Most in vitro RNA transcriptions were performed by using a RiboMAX large-scale RNA production system with T7 polymerase (Promega).

Northern blot analysis. Total RNA was extracted with Tri Reagent (Molecular Research Center), glyoxalated, and typically loaded onto 1.5% agarose gels. Electrophoresis and electrotransfer were performed as described previously (5). HDV RNA probes were prepared by T7 transcription with [{alpha}-32P]UTP (Perkin-Elmer). Hybridization was performed at 65°C in Ekono Hybridization Solution (Research Products International). Radioactivity was detected and quantitated with a bio-imager (Fuji).

Selection of 5'-capped RNAs. For Fig. 2C, total RNA from transfected cells was fractionated to separate capped and noncapped species by using an affinity column to which was bound a monoclonal antibody that binds cap structures (2). First, this antibody was complexed to protein A-coated Sepharose (Sigma); then 50 µg of total RNA was applied. Bound RNAs were extracted with Tri Reagent. Bound and unbound RNAs were collected by ethanol precipitation prior to Northern blot analyses.

Selection of 3'-polyadenylated RNAs. For Fig. 4B, total RNA from transfected cells was fractionated to separate polyadenylated and nonpolyadenylated species. The strategy was essentially that described for the Dynabeads mRNA purification kit (Dynal). (dT)25 with a 5' biotin was first complexed to avidin-coated superparamagnetic beads. Then 8 µg of total RNA was subjected to binding and elution and was subsequently collected by ethanol precipitation prior to Northern blot analysis.


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RESULTS
 
Effects on HDV genome replication of mutating the poly(A) signal. In a previous study, Hsieh and Taylor reported that mutation of the predicted HDV poly(A) signal from AAUAAA to UUUAAA was sufficient to block the ability of a transfected HDV cDNA construct to initiate HDV genome replication (13). The same study also showed that when a second plasmid, which transiently expressed the small form of the delta protein, was cotransfected with a cDNA carrying the poly(A) mutation, there was some recovery of HDV genome replication. In light of recent findings, we have reassessed these earlier data and made the following observations. (i) The original study used a cDNA construct to initiate replication, and thus it was possible that the inhibition caused by the poly(A) mutation was unique to the processing of an RNA transcript that was DNA directed. (ii) It was also possible that the rescue, achieved with a second cDNA construct that expressed small delta antigen, might have involved DNA recombination. Based on these two possibilities, we therefore wanted to know whether HDV genome replication and RNA accumulation could occur in the absence of polyadenylation but in the presence of a separate stable source of delta protein.

Therefore, we initiated HDV genome replication by using, instead of DNA constructs, greater-than-unit-length genomic RNAs, with and without the poly(A) signal mutation. These RNAs were transcribed in vitro. Both were first modified to contain a frameshift mutation in the ORF for the small delta protein. To provide a stable source of small delta protein, we carried out the transfection into a line of {delta}-293 cells that were able to express about 2.5 x 106 copies of this protein per cell in the presence of tetracycline. At day 2 after transfection, total RNA was extracted and examined by Northern blot analysis to detect antigenomic RNAs. Results are shown in Fig. 2A. We observed that for both wild-type and poly(A)-mutated genomic RNAs (Fig. 2A, lanes p and q, respectively), transfection led to significant accumulation of newly synthesized unit-length antigenomic RNA, which could be identified relative to a unit-length DNA standard in lane r. We also detected two polyadenylated mRNA species. As indicated in Fig. 2A, one was that stably produced by the {delta}-293 cells and the other was that produced by the replicating HDV genome. Note that for the poly(A)-mutated genome, much less of this mRNA was produced during HDV genome replication.

Figure 2B provides quantitation of the Northern blot data in Fig. 2A. The amount of each of the three RNA species in Fig. 2B, lanes q, is expressed relative to the amount in the corresponding lane p, taken as 100%. Note first that under these conditions the amount of unit-length RNA produced by the poly(A) mutant was 75% that of the wild type. In contrast, the amount of mRNA produced by this replication was only 14%, consistent with the mutation interfering with polyadenylation. As expected, the amount of mRNA from {delta}-293 cells was not changed. Similar results were obtained for RNAs harvested at days 3 and 4 after transfection (data not shown).

These results support the interpretation that as long as {delta}Ag-S was provided separately, the poly(A) mutation did not have an effect on unit-length viral RNA accumulation. Specifically, it did not have a major effect on the accumulation of these ribozyme-processed RNA transcripts. This finding is important, because Pol II is needed both for transcription and for several different aspects of RNA processing, and thus the poly(A) mutation could have had an effect on RNA transcription (27).

The studies for which results are shown in Fig. 2A provided a unique opportunity to compare and contrast the mRNA species that arose via RNA-directed versus DNA-directed transcription. Therefore, in the experiment for which results are shown in Fig. 2C, we tested the abilities of these two RNAs, present in total RNA isolated from a transfected culture as in Fig. 2A, lane p, to be selected for a 5'-cap structure. We used an affinity column with an antibody specific for the cap structure (2). As shown in Fig. 2C, we found that these two mRNAs were selected with significant and equal efficiency. In contrast, unit-length HDV RNA, used as a negative control, was less efficiently selected. This study thus provided the first direct evidence that the 5' end of RNA-directed HDV mRNA, like that of DNA-directed mRNA, is posttranscriptionally modified to contain a similar 5'-cap structure. The results confirm the previous evidence obtained by an indirect method (10).

Analysis of processing of DNA-directed antigenomic HDV RNA sequences. For the following studies, we devised a strategy to monitor the processing of nascent Pol II-directed transcripts of antigenomic RNA. As indicated in Fig. 3A, our aim was to monitor the processing events for RNAs that started at what we consider might be the 5' end for HDV RNA transcripts (10). Also, we wished to consider RNAs that could extend beyond unit length and might mimic the antigenomic transcripts achievable in rolling-circle replication. We realized that amounts of such transcripts that would allow characterization of the subsequent RNA processing could not be obtained in vivo from RNA-directed transcription. Furthermore, for these studies we actually wanted to study RNA processing in the absence of any RNA-directed replication. And because we wanted to consider the processing of HDV RNA transcripts of different lengths, with and without specific prior mutations, we could do this only in the context of DNA-directed transcription. With this in mind, we designed a multimeric HDV cDNA insert for an expression vector. This prototypic construct was such that when it was transiently transfected into cells, Pol II-directed RNA transcription would begin very close to the site corresponding to the 5' end of the HDV mRNA and then, as indicated in Fig. 3A, proceed through the ORF for the delta protein, the poly(A) signal, AAUAAA, the poly(A) acceptor site, CA, and the almost adjacent antigenomic ribozyme sequence. As indicated, this construct would next direct the transcription of a complete unit length of HDV antigenomic RNA, which again contained the ORF, the poly(A) signals, and a ribozyme.

Also shown in Fig. 3A are six HDV-specific RNAs that might possibly be derived by RNA processing from this multimeric antigenomic RNA transcript. These species are arranged in order of increasing size. Actually, they are organized as three pairs of transcripts; for each pair, the 3' end of one transcript is produced by polyadenylation (pA) while that of the other is produced by ribozyme cleavage (Rz). Note that species 1-pA and 2-Rz correspond to the natural HDV mRNA and unit-length antigenomic RNA, respectively.

Next, as shown in Fig. 3B, we considered the processing of RNA transcripts made not just from one but from a total of eight different constructs. The strategy was to compare and contrast the processing of different HDV RNA species. The first four constructs, which acted as templates for transcripts a through d, contained less-than-unit-length antigenomic RNA, while the last four, e through h, acted as templates for transcripts that were greater than unit length. In addition to changing the lengths of the transcripts, we employed mutagenesis of the poly(A) signals (transcripts c, g, and h) or the ribozyme domain (d). For transcripts b and f, we added an intron to the 5' sequences in order to determine whether this would have an effect on the processing of the HDV sequences.

In all of the eight processing studies that will now be described, we did not want the presence or absence of HDV RNA-directed replication to be a variable. We also wanted to exclude any effects on RNA processing that might be mediated by the presence or absence of the small delta protein. Therefore, we mutated the ORFs present in the RNA transcripts such that functional small delta protein was not expressed and there was no HDV replication.

In all of the following experiments, Huh7 cells were transfected with expression constructs, and at day 3, total RNA was extracted and subjected to Northern blot analyses to detect the processed HDV-specific sequences.

Consider first the analyses of the processing of transcripts a through d, which were less than unit length. Because of this short length, we could expect processed RNAs of class RNA-1 only, as indicated in Fig. 3A. The Northern blotting results are presented in Fig. 4A, and the position of RNA-1 is indicated. Note that in all lanes we detected a level of cross-hybridization to abundant host cell RNAs, especially 28S rRNA, as indicated for RNA from untransfected cells (Fig. 4A, lane u). Quantitation of the RNA-1 signal, after normalization to the 28S rRNA signal, is presented in Fig. 4B. The signals for transcripts b through d are expressed relative to that for transcript a, taken as 100%.

For transcript a, the band indicated as RNA-1 was heterogeneous and had an average size of about 800 nt, as expected for HDV mRNA (12). For these and other reasons, including poly(A) selection (data not shown), we identified this band as the polyadenylated species RNA-1-pA from Fig. 3A, and we believe that there was not a significant amount of RNA-1-Rz.

For RNA transcript b, a sequence containing an intron was added to the 5' noncoding sequence. Our intent with this construct was to determine whether the presence of an intron could change the nature or extent of RNA processing. Others have reported that poly(A) processing of host RNAs can be facilitated by the presence within the RNA precursor of intronic sequences (17). As shown in Fig. 4A, the band detected was less heterogeneous and also migrated somewhat more slowly. While the reduced heterogeneity may be due in part to a more uniform length of polyadenylation, we consider the increased size to be due largely to the 276 nt of added non-HDV sequences remaining after the splicing out of the 973-nt intron. Interestingly, from quantitation of the Northern blot data, we found that the amount of HDV-specific signal was not detectably different from that for transcript a. Thus, we conclude that processing of the normal HDV mRNA is not at any significant disadvantage relative to that for an RNA with an intronic sequence.

RNA transcript c had the mutant poly(A) signal, UUUAAA. As expected, the amount of RNA-1-pA was reduced significantly, to 14% that of transcript a.

In transcript d, the ribozyme located 3' of the poly(A) signals was deleted. Nevertheless, the amount of RNA-1 was only changed 30% from that detected for transcript a. This result indicates that the presence of the ribozyme domain is not needed for polyadenylation. It also shows that the bands indicated here as RNA-1 are fully RNA-1-pA and do not contain any species that could be RNA-1-Rz.

Next consider the processing of RNA transcripts e through h, which differ from transcripts a through d in that a complete unit-length HDV sequence has been added 3' of the poly(A) signals and the first ribozyme. Therefore, we might now be able to detect some or all of the six processed RNA species diagrammed in Fig. 3A. In the Northern blot results (Fig. 5A), we detect the mass distribution of species RNA-1 to RNA-3. In order to determine the relative molar distribution, we first normalized the hybridization signals to the amount of the cross-hybridizing band of 28S rRNA and then corrected for the length of the HDV-specific sequence present in each species. We thus obtained the relative molar distribution of the three pairs of processed RNA species for each of RNA transcripts e through h (Fig. 5D).



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  		FIG. 5. Northern blot analysis of processed HDV RNAs. (A) Huh7 cells were transfected with constructs to express four of the primary RNA transcripts, e through h, as diagrammed in Fig. 3B. At day 3, total RNA was extracted and subjected to Northern blot analysis to detect antigenomic HDV RNA species. Lane r, unit-length HDV cDNA, used as a control. The designation of RNA species 1 to 3 uses the theoretical possibilities of Fig. 3A. A cross-hybridizing band of 28S rRNA, which was used as an internal control for the amount of total-cell RNA loaded, is indicated. In lanes e and h, migrating somewhat faster than this 28S rRNA band, are minor amounts of bands, as explained in the legend to Fig. 4. (B) Lane e, total RNA from a transfected culture (as in panel A, lane e) was subjected to glyoxalation and separation on a 3% agarose gel with a longer period of electrophoresis (4). Lane s, parallel sample of total RNA from the liver of an HDV-infected woodchuck, examined as a positive control. We were able to identify unit-length antigenomic RNA in linear and circular conformations in both samples. In lane e, 74% of the unit-length RNA was circular. (C) Poly(A) fractionation of the RNA from a sample similar to that in panel A, lane f, but obtained from a separate transfection, into total (T), poly(A)-deficient (A–), and poly(A)-containing (A+) RNAs. The electrophoresis was more extensive than that used for panel A. (D) Quantitation of the data from panel A for species e through h. Note that the data for each of the processed transcripts have been corrected to express the molar amounts rather than the mass amounts of the three main RNA species. Furthermore, they are normalized so that the total of RNAs 1 to 3 is 100%.

For RNA transcript e, we detected three main HDV-specific bands. The fastest-migrating band is further identified as RNA-1-pA. The band indicated as RNA-2 has the same mobility as a unit-length standard (Fig. 5A, lane r). Furthermore, from this and other analyses, we conclude that this RNA-2 is not detectably polyadenylated and in fact is RNA-2-Rz. By using glyoxalation and extensive electrophoresis on a 3% agarose gel (4), we found that 74% of this RNA-2 was in a circular conformation (Fig. 5B). Also detected for the processing of transcript e is the band indicated as RNA-3. While the composition of the RNA-3 band in Fig. 5A, lane e, is not obvious, small electrophoretic mobility differences and prior poly(A) fractionation data (not shown) led us to conclude that this band contains comparable amounts of RNA-3-pA and RNA-3-Rz.

For transcript f, there was only one change relative to transcript e, namely, the presence of the 5' intron. In terms of the resultant RNA processing, the only obvious consequence was the reduced mobility of RNA-1. We conclude that just as for transcript b relative to transcript a, the presence of the intron in the nascent transcript led to a processed polyadenylated species, RNA-1-pA, that was less heterogeneous and also somewhat larger. A second but less obvious change relative to transcript e was that the slowest-migrating band, indicated as RNA-3, was less heterogeneous in size. In order to better characterize this species, the total RNA was fractionated by poly(A) selection. As shown in Fig. 5C, we fractionated the total RNA into nonpolyadenylated and polyadenylated RNAs. It can now be seen that RNA-3 was predominantly polyadenylated and can thus be identified as RNA-3-pA rather than RNA-3-Rz. Likewise, the RNA-1 band can be identified as predominantly RNA-1-pA. The RNA-2 band is identified as RNA-2-Rz.

For transcript g, the first poly(A) signal has been removed. As expected, this led to a reduced amount of RNA-1-pA. In addition, it increased the amount of RNA-3. However, after quantitation and normalization of the data, as shown in Fig. 5D, there was no significant change in the amount of RNA-2-Rz.

In transcript h the second poly(A) signal was mutated. The only obvious consequence for RNA processing was a major reduction in the amount of RNA-3. This is expressed clearly in Fig. 5D. The residual RNA-3 was not polyadenylated (data not shown) and is identified as RNA-3-Rz. It is important that this mutation of the second poly(A) signal had no detectable effect on the levels of RNA-1 and RNA-2.

As considered further in the Discussion, the results of this examination of RNA processing for the eight different RNA transcripts led us to propose the interpretation that poly(A) addition and ribozyme cleavage to a large extent are alternative processing outcomes for separate antigenomic HDV RNA transcripts.

While {delta}Ag expression is not directly related to the question of RNA processing, we performed, in parallel with the Northern blot analyses for which results are shown in Fig. 4A and 5A, immunoblot assays to detect {delta}Ag. Two points were revealed from this analysis (data not shown). First, for both transcripts c and g, mutation of the first poly(A) signal led to a 50 to 60% reduction in the accumulation of {delta}Ag. Second, for both transcripts b and f, insertion of the intron in the 5' untranslated region produced a 70 to 100% increase in {delta}Ag levels. Since the amounts of RNA-1 produced by transcripts b and f were unchanged, it may be that the involvement of RNA splicing led to more efficient mRNA transport from the nucleus and subsequent translation in the cytoplasm. Such results are consistent with the reports of others that exon junction complexes formed on an RNA can enhance the expression of protein from the mRNA (16, 17, 28).


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DISCUSSION
 
The present studies were undertaken to obtain a better understanding of the posttranscriptional processing of antigenomic transcripts of HDV RNA, leading to the production of both a polyadenylated mRNA and a ribozyme-cleaved unit-length antigenomic RNA. At the same time, we tested some of the relevant speculations implicit in the double-rolling-circle model of HDV genome replication (Fig. 1) (9, 25). In the first half of our study, we extended the previous report of Hsieh and Taylor that mutagenesis of the poly(A) signal on the HDV RNA inhibited HDV genome replication (13). We showed here that when such a mutated RNA was transfected into cells stably expressing the small form of the delta protein, HDV genome replication was no longer inhibited. In the second half of our study, we examined the various forms of antigenomic RNA processing that occur when antigenomic RNAs are transcribed by Pol II from a transfected DNA template. As will now be explained, the data obtained in both studies support the interpretation that processing of antigenomic HDV RNAs by polyadenylation and by ribozyme cleavage are alternative events and furthermore can be mutually exclusive events.

The double-rolling-circle model of replication shown in Fig. 1 suggests that antigenomic transcripts, once initiated, could first be processed to yield capped and polyadenylated mRNA, and then the "continuing transcript," following further elongation, could be processed by two ribozyme cleavages to release unit-length RNAs, which in turn could be processed by ligation to become unit-length circular antigenomic RNAs (13, 25). In a contrasting model, Modahl and Lai (21) have asserted that poly(A) processing and ribozyme cleavage are completely separate processing events, because the substrate for each is transcribed by a different RNA polymerase. While they agreed that Pol II transcribes the species that becomes the mRNA, they asserted that a different polymerase, suggested to be Pol I, is used for those RNAs which become the unit-length antigenomic RNA (20). The present study clearly shows that of RNAs synthesized in vivo using the same template sequence and directed by the same Pol II promoter, some are processed by polyadenylation and others are processed to become unit-length RNA circles.

In agreement with the concept of mutually exclusive processing, we found that mutating the poly(A) signal had no effect on the level of ribozyme processing (Fig. 5A and D, lanes g and h). Conversely, removal of a ribozyme had no effect on the level of polyadenylation (Fig. 4, lanes d).

It is relevant that since the earlier rolling-circle model was first formulated, a greater understanding of the process of polyadenylation has been achieved. Dye and Proudfoot have shown that nascent RNAs often proceed 500 to 3,000 nt beyond the poly(A) signal before transcriptional termination can occur, followed by disengagement of the polymerase from the template, formation of a complex of polyadenylation factors at and around the poly(A) signal, cleavage at the poly(A) acceptor site, and finally addition of poly(A) to that site (8). Therefore, we now suggest that for HDV it is more likely that the "choice" between poly(A) processing and ribozyme cleavage not only is stochastic but also is made after nascent transcripts have achieved sizes much greater than unit length.

This interpretation of mutually exclusive processing then leads to the question of what factors determine this choice. It seems that there should be no difference in the initiation of transcription. It could well be that at some point(s) after transcription is initiated, differences in the recruitment of host factors needed for the two processing events lead to the alternatives in processing.

In the earlier rolling-circle model of HDV replication, it was proposed that a viral protein, {delta}Ag-S, could bind to nascent RNAs that can fold into the rod-like structure and preferentially suppress processing by polyadenylation. This was asserted largely because Hsieh and Taylor had shown that {delta}Ag-S could inhibit polyadenylation (13). However, in contrast to the present studies, Hsieh and Taylor did not have a simultaneous measure of polyadenylated RNAs and RNAs that were processed to become unit-length antigenomic RNAs. Furthermore, the present studies show that the added presence during transcription of either {delta}Ag-S or {delta}Ag-L was not able to decrease the level of poly(A) processing (data not shown).

During HDV replication either in the liver of an infected animal or in cultured cells, the molar amount of polyadenylated mRNA is often 50 times less than that of the unit-length antigenomic RNA species produced by ribozyme cleavage (7, 10, 12). In contrast, when we determined the molar amounts of the main forms of processed DNA-directed RNA transcripts, we observed that the short polyadenylated mRNA species was three times more abundant than the unit-length RNA (Fig. 5D). This ratio was not significantly changed when either {delta}Ag-S or {delta}Ag-L was transiently expressed, and it was not changed in {delta}-293 cells when {delta}Ag-S was provided by stable transfection (data not shown). One possible interpretation is that somehow the nature of our transient expression studies offered less opportunity for the decay of mRNA than for that of RNA circles. Consistent with this explanation was the fact that even species polyadenylated at a second site were detected in these experiments, and yet such RNAs have never been reported during HDV genome replication in the liver or in cultured cells.

In summary, the present studies demand that significant changes be made to the rolling-circle models of HDV genome replication (Fig. 1) (9, 20), especially as they might apply to the processing of antigenomic RNA transcripts. First, according to these models, polyadenylation is needed to make the mRNA for the small delta protein, but the new data make clear that if the protein itself is provided separately, neither the presence nor the functioning of the polyadenylation signals is needed for HDV replication. Second, contrary to the model, ribozyme cleavage of antigenomic RNAs neither precedes nor is necessary for the ability of such RNAs to undergo poly(A) processing. Third, the present studies show that transcripts starting at the same location and using the same polymerase (Pol II) can be processed to yield either mRNA or unit-length RNA circles. (Incidentally, contrary to one published model [20], two different polymerases are not needed.) A concept of the old model is that a nascent antigenomic RNA undergoes first poly(A) processing and then ribozyme processing. However, this concept is not necessary and probably is incorrect, because in the present study, the two forms of processing appear to be largely mutually exclusive. That is, it is more likely that a given nascent HDV transcript will undergo either one form of processing or the other, but not both. To some extent such alternative possibilities are analogous to what we realize happens for most mRNA precursors that contain multiple introns; such RNAs can undergo multiple alternatives in terms of which introns are spliced (24).

In conclusion, the results presented here, along with other data recently reviewed (26), make clear that HDV genome replication is much more complex than the rolling-circle models have led us to believe.


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ACKNOWLEDGMENTS
 
Constructive comments on the manuscript were given by Glenn Rall, Severin Gudima, Chi Tarn, William Mason, and Rich Katz. We thank Sven Behrens and Martina Behrens for guidance in the cap-affinity chromatography and Reinhard Lührmann for generously providing the cap-specific antibody.

This work was supported by grants AI-26522 and CA-06927 from the NIH and by an appropriation from the Commonwealth of Pennsylvania.


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FOOTNOTES
 
* Corresponding author. Mailing address: Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111-2497. Phone: (215) 728-2436. Fax: (215) 728-3105. E-mail: John.Taylor{at}FCCC.edu. Back


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Journal of Virology, May 2004, p. 4517-4524, Vol. 78, No. 9
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.9.4517-4524.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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