INTRODUCTION

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Three major stable RNA species are thought to be involved in the replication of the hepatitis delta virus (HDV) RNA genome: (i) the genomic 1,679-nucleotide (nt) circular single-stranded RNA, (ii) its exact complement, the antigenome, and, least abundant, (iii) an 800-nt polyadenylated RNA that is presumed to be the mRNA for the delta protein (17). Using primer extension on the polyadenylated RNA extracted from cultured cells undergoing HDV genome replication, we reported a 5' end that mapped to nt 1631 ± 1 (6). For unexplained reasons, this polyadenylated species was not always detectable by Northern analyses and/or primer extension (6). Nevertheless, three subsequent studies report confirmation of a 5' end in the vicinity of nt 1631 (1, 12, 18) and a recent study has reported that nonpolyadenylated antigenomic RNAs can have a 5' end at nt 1646 (12).

The site at nt 1631, as indicated in Fig. 1, is located 9 nt from what we refer to as the top end of the predicted rodlike genomic RNA. One hypothesis is that the 5' end corresponds to a site of transcription initiation by RNA polymerase II (17). However, if initiation is at nt 1631, it will begin with UTP, which is puzzling, since transcription by Pol II, at least from the normal DNA templates, usually begins preferentially with ATP or GTP (4). A second hypothesis, which is linked to the first, is that nearby sequence elements define a promoter for Pol II (10). There are data, obtained by using cDNA constructs, which suggest that a sequence as short as 30 nt (indicated in Fig. 1) can, when expressed as a double-stranded DNA, act as a promoter for RNA polymerase II (10). There are also in vitro transcription studies with HDV genomic RNA added to nuclear extracts, which are claimed to support both initiation of transcription near nt 1631 and the concept of a nearby promoter (1).

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Predicted rodlike folding of nucleotide sequences present at one end of the genome HDV RNA. The nucleotide sequence and numbering are as in Kuo et al. (9). This end is sometimes referred to as the top of the rod (21). The three sequence features indicated by boxes are the site corresponding to the initiation codon for the small delta protein (19), the 5' end of the polyadenylated mRNA as determined by primer extension (6), and the location of a 30-nt sequence which, when expressed as a double-stranded cDNA, is reported to have promoter activity in transfected cells (10).

To test these two hypotheses further, we and others have previously investigated mutations in this region of the rodlike structure and observed some significant effects on genome replication (1, 18, 21). However, more relevant for understanding initiation are the determination of the 5' end(s) for the polyadenylated RNAs and an analysis of whether the location of this end(s) is changed by alterations in the local sequence and structure of the rodlike genomic RNA template.

The initial aim of the present study, therefore, was to apply a sensitive and specific 5' rapid amplification of cDNA ends (RACE) procedure to characterize the 5' ends of the polyadenylated HDV antigenomic RNAs, synthesized under different conditions of HDV genome replication. Primer extension involves the assumption that the elongated species are 100% identical to the template strand; it would neither detect nontemplated 5' nucleotides, as occurs naturally for influenza virus (15), nor the artifactual addition of nontemplated nucleotides by reverse transcriptase (14). In contrast, 5' RACE determines the actual sequence of the transcript. With this approach, we observed specificity for 5' ends located at nt 1630. We then went on to use this 5'-RACE method to study the 5' ends obtained during replication of various mutants of HDV, especially those with base changes near the site at nt 1630.

	MATERIALS AND METHODS

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RNA from the liver of an infected woodchuck. A woodchuck chronically infected with woodchuck hepatitis virus was superinfected with HDV. After 25 days the animal was sacrificed, and a liver sample (0.5 g) was taken and homogenized in a buffer containing guanidine isothiocyanate. Cesium chloride was then added, and after centrifugation to equilibrium, the RNA band was collected and the RNA was precipitated by the addition of salt and ethanol, all as previously described (3).

Oligo(dT) fractionation of liver RNA. The total liver RNA in 0.5 M NaCl-0.01 M Tris (pH 7.5)-0.1% sodium dodecyl sulfate, was applied to a column of 0.5 g of oligo(dT)-cellulose (Sigma). The column was washed with the same buffer and then eluted with 0.01 M Tris (pH 7.5)-0.1% sodium dodecyl sulfate. This cycle was repeated a total of six times. From 0.5 g of liver we obtained 10 to 50 µg of poly(A)-containing RNA. The quality of the HDV mRNA was confirmed by Northern analysis.

Genome mutants. Mutations were constructed in pDL553, a plasmid based on the vector pSVL (Pharmacia) and containing 1.2 cDNA copies of the HDV genome. According to the nucleotide sequence of Kuo et al. (9), the insert is from the unique NheI site (nt 430) to NheI to XbaI (nt 781). Four of the mutants used in this study were described previously (21). Additional mutants were constructed by using the strategy described by Wu et al. (21). All the mutations were made near the top of the rod, i.e., between nucleotide positions 1629 and 1648 on the genome.

In vitro RNA transcription and RNP assembly. Genomic RNA was transcribed from plasmid pTW101, which contains 1.1 copies of the HDV genome (StyI [nt 621] to StyI to XbaI [nt 781]) inserted between a T7 promoter and terminator (21). Ribonucleoprotein (RNP) was assembled by combining this RNA (500 ng) with purified recombinant small delta protein (200 ng) (provided by Zuccola and Hogle), as previously described (5).

Transfection, RNA extraction, and Northern analysis. Huh7 cells (13) in a 35-mm-diameter culture dish were grown to approximately 70% confluence before transfection with cDNA, RNA, or RNP with Lipofectamine or Lipofectamine Plus (Life Sciences) according to the manufacturer's instructions. Total RNA was harvested with Tri reagent (Molecular Research Center, Inc.).

5' RACE. 5' RACE was performed with a Clontech CapFinder kit, which is designed to allow amplification of the 5' end of messenger RNAs by reverse transcription (RT)-PCR. Our amplification strategy was based on the manufacturer's recommendations and is represented in Fig. 2. As input RNA for this procedure, we used either polyadenylated RNAs selected from infected liver tissue or total RNAs extracted from transfected cells. These RNAs (10 µg), in water, were heat denatured for 2 min at 95°C and then specifically bound in 1 M NaCl-20 mM Tris (pH 7.5)-2 mM EDTA to avidin-coated superparamagnetic beads (10 µl; Dynal) that had been preincubated in the same buffer with an excess of 5' biotin-(dT)₃₀. The immobilized RNA was added to an RT reaction mixture (10 µl) containing 50 mM Tris (pH 8.3), 6 mM MgCl₂, 75 mM KCl, 2 mM dithiothreitol, 1 mM deoxynucleoside triphosphates, and 100 U of Superscript II reverse transcriptase (Life Sciences) and then incubated at 42°C for 90 min. The reaction mixture also contained 10 pmol of Capswitch oligonucleotide 1 (CS1) (5'-TACGGCTGCGAGAAGACGACAGAAGGG-3'). This oligonucleotide is considered to function as follows. When reverse transcriptase reaches the authentic 5' end of the mRNA, its terminal transferase activity adds several nontemplated nucleotides to the 3' end of the cDNA (14). If these extra nucleotides are CCC, the sequence GGG at the 3' end of CS1 can hybridize and allow the reverse transcriptase to switch templates from the mRNA to CS1. This results in a cDNA that extends past the 5' end of the mRNA to incorporate a copy of the CS1 sequence. For the subsequent PCRs, the cDNA then has a specific primer-binding site at its 3' end.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   The 5'-RACE strategy. As described in Materials and Methods, a CapFinder kit from Clontech was adapted to determine the 5' ends of polyadenylated antigenomic HDV RNAs. Three of the numbers at the top of the diagram represent the 5' ends of the three HDV primers, using the genome numbering of Kuo et al. (9). The fourth number, 934, indicates the site of poly(A) addition (6).

	RESULTS

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HDV RNA from liver tissue and cultured cells in which the wild-type genome was replicating. An 800-nt polyadenylated HDV RNA was first observed in the liver of an infected chimpanzee by Northern analysis (2). By primer extension on polyadenylated RNA from cells transfected with HDV cDNA, the 5' end was mapped to nt 1631 ± 1 (6), using the genome numbering of Kuo et al. (9). As an independent approach to detecting these 5' ends, we used a 5'-RACE procedure, as summarized in Fig. 2 and described in Materials and Methods, and applied it to the polyadenylated RNA isolated from the liver of a woodchuck at the peak of an HDV infection. All 27 of 27 clones sequenced indicated a 5' end corresponding to nt 1630. A similar study was made with the RNAs from Huh7 cells at 6 days after transfection with a cDNA construct, pSVL(D3), which is known to initiate HDV genome replication (8). In this experiment 27 of 32 clones indicated 5' ends at nt 1630. The other five clones indicated ends at nt 1620, 1627, 1655, 1664, and 1667.

HDV RNA from cells in which mutated HDV genomes were replicating. The above-mentioned 5'-RACE studies showed that transfection of cells with cDNAs gave 5' ends largely in agreement with those detected for RNA isolated from the liver of an infected woodchuck. Therefore, we used this cDNA strategy to examine the importance of the nucleotide sequence and predicted secondary structure of the genomic HDV RNA template in determining this initiation site (Fig. 1). As will be explained, we found that the introduction of mutations could cause a decrease in the accumulation of antigenomic RNA and/or changes in the position of the 5' end. As summarized in Table 1, the locations focused on three areas: the 3-nt external bulge, which includes nt 1630 and also the top 5-nt loop, and the adjacent 6-bp stem.

View larger version (96K): [in this window] [in a new window]   FIG. 3.   Antigenomic RNA accumulation and 5' RACE for HDV RNAs produced by cells transfected with cDNAs mutated at and around a 3-nt external bulge predicted for the genomic RNA. Huh7 cells were transfected with cDNA corresponding to either the wild type or five specific mutants (as described in the text). After 6 days, the total RNA was extracted and examined by glyoxylation, agarose gel electrophoresis, and Northern analysis to detect antigenomic RNA (A) or by nested RT-PCR and nondenaturing agarose gel electrophoresis and ethidium staining of the 5'-RACE product (B). In panel A, lane M contains end-labeled 1-kb ladder (Life Sciences) and at the right is indicated the position of 1,679-nt unit-length antigenomic RNA. In panel B, lane M is unlabeled 1-kb ladder and at the right is indicated the position of the 130-bp PCR product. In lane 3, the ~60-bp product corresponds to a primer-dimer artifact.

HDV RNA from cultured cells at early times after transfection with RNP. In each of the above-mentioned studies, using RNAs from either an infected animal or transfected cells, we examined the 5' ends of those polyadenylated RNAs present at times when genome replication was well under way. The sensitivity of the 5'-RACE procedure allowed us to look at earlier times and test the possibility that during replication there was somehow a time-dependent selection for mRNAs with such specific 5' ends.

View larger version (97K): [in this window] [in a new window]   FIG. 4.   Time course for RNP transfection. Linear HDV genomic RNA was combined in vitro with purified recombinant small delta protein and then used to transfect cultures of Huh7 cells, as previously described (5). At the indicated times after transfection, total RNA was extracted and analyzed, as for Fig. 3, by Northern analysis to detect 1,679-nt unit-length antigenomic RNA (A) and by 5' RACE to detect RT-PCR products (B), with the ethidium bromide-stained band at 130 bp being a preliminary indication of a polyadenylated antigenomic RNA species with a 5' end near nt 1630.

	DISCUSSION

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This application of a 5'-RACE procedure (Fig. 2) to the polyadenylated antigenomic RNAs of HDV has provided several findings which increase our understanding of HDV genome replication. (i) The 5' site located at nt 1630 was reproducible in our studies. For example, 27 of 27 sequences from liver tissue were at nt 1630. Moreover, we obtained the same answer for RNAs from infected liver tissue and for cultured cells transfected with either a cDNA construct or an RNP assembled in vitro. (ii) The new result was different from previous primer extension studies that indicated a 5' end at nt 1631. (iii) Exploiting the sensitivity of the 5'-RACE procedure, we were able to detect 5' ends for RNAs isolated from cells as early as 24 h after transfection with RNP (Fig. 4). We thus found that these 5' ends were at nt 1630, just like those detected at much later times after the initiation of replication. (iv) Finally, when 5' RACE was applied to RNAs from cells transfected with mutated HDV genomes, we were able to detect situations in which the 5' ends were relocated, sometimes without a major inhibition of genome replication as assayed by accumulation of antigenomic RNA (Fig. 3 and Table 1). In most cases the novel sites, like nt 1630, were consistent with initiation by a purine, usually an adenosine (Table 2). However, it must be pointed out that not only Pol II but also Pol I and Pol III preferentially initiate with a purine (4, 11, 16, 20).

An obvious interpretation of the 5' ends that we have detected by 5' RACE is that they are sites for the initiation of RNA-directed RNA synthesis. Objectively, further studies are needed to establish this. Furthermore, other interpretations need to be excluded. For example, it might be that 3'-end processing by polyadenylation of antigenomic precursor RNAs is coupled with some form of 5' processing to make an mRNA that is both translatable and stable; polyadenylated RNAs with more sequence at the 5' end would be able to fold into the rodlike structure, which could make them less likely to be translated and less stable or perhaps make them substrates for RNA editing.

While our data support the interpretation that the sequence and predicted structure of the genomic RNA around nt 1630 are important, we nevertheless consider that there is not yet sufficient evidence to conclude that any RNA structure in the vicinity actually defines a promoter. Some of the mutations we have characterized here were able to change the location of the detected 5' ends, and in some cases, especially when we reduced the size of the predicted 3-nt external bulge region, they were able to reduce RNA accumulation to undetectable levels, and yet all the sites of mutagenesis considered were only adjacent to the region that has been claimed to contain a promoter element (as indicated in Fig. 1). As mentioned in the introduction, it has been claimed that a double-stranded cDNA version of the genomic sequence will act in transfected cells as a promoter for RNA polymerase II (10). However, in such studies mapping of the 5' end by primer extension either failed or yielded multiple 5' ends (10). Furthermore, in our own studies, we consider that all of the 5' ends we obtained were derived from RNA transcripts that were RNA directed rather than DNA directed. And, as an additional test to exclude the latter possibility, we studied polyadenylated RNAs expressed in cells transfected with a cDNA construct that contained a 2-nt deletion at nt 1425, about 200 nt away from most 5' ends. As previously characterized, this genome, with a mutation in the open reading frame of the delta protein, is only able to undergo genome replication when delta protein is provided from a separate plasmid (8). When this mutant alone was transfected into cells and the polyadenylated RNAs were tested by the 5'-RACE procedure, we were unable to detect any clones whose sequences indicated 5' ends at nt 1630 or anywhere in the vicinity (data not shown). Thus, we can infer, and thereby further support the interpretation, that the 5' ends detected for wild-type genomes and for the replication-competent mutants described in Table 1, were all obtained for polyadenylated RNA species that were RNA directed rather than DNA directed.