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The Large Delta Antigen of Hepatitis Delta Virus Potently Inhibits Genomic but Not Antigenomic RNA Synthesis: a Mechanism Enabling Initiation of Viral Replication

Abstract

Hepatitis delta virus (HDV) contains two types of hepatitis delta antigens (HDAg) in the virion. The small form (S-HDAg) is required for HDV RNA replication, whereas the large form (L-HDAg) potently inhibits it by a dominant-negative inhibitory mechanism. The sequential appearance of these two forms in the infected cells regulates HDV RNA synthesis during the viral life cycle. However, the presence of almost equal amounts of S-HDAg and L-HDAg in the virion raised a puzzling question concerning how HDV can escape the inhibitory effects of L-HDAg and initiate RNA replication after infection. In this study, we examined the inhibitory effects of L-HDAg on the synthesis of various HDV RNA species. Using an HDV RNA-based transfection approach devoid of any artificial DNA intermediates, we showed that a small amount of L-HDAg is sufficient to inhibit HDV genomic RNA synthesis from the antigenomic RNA template. However, the synthesis of antigenomic RNA, including both the 1.7-kb HDV RNA and the 0.8-kb HDAg mRNA, from the genomic-sense RNA was surprisingly resistant to inhibition by L-HDAg. The synthesis of these RNAs was inhibited only when L-HDAg was in vast excess over S-HDAg. These results explain why HDV genomic RNA can initiate replication after infection even though the incoming viral genome is complexed with equal amounts of L-HDAg and S-HDAg. These results also suggest that the mechanisms of synthesis of genomic versus antigenomic RNA are different. This study thus resolves a puzzling question about the early events of the HDV life cycle.

Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus, contains a circular RNA genome of 1.7 kb which encodes a single protein, hepatitis delta antigen (HDAg) (18). In virus-infected cells, HDV RNA undergoes RNA-dependent RNA synthesis to generate three RNA species, a 1.7-kb genomic RNA, a 1.7-kb antigenomic RNA, and a 0.8-kb antigenomic-sense, polyadenylated mRNA, which is the mRNA for translation of HDAg (18). Two forms of HDAg are detected in the infected cells as well as in the virion: the small form (S-HDAg) of 24 kDa is 195 amino acids in length, and the large form (L-HDAg) of 27 kDa is 214 amino acids in length (1, 2, 26). HDV depends almost entirely on cellular machineries to carry out its RNA synthesis, which is, however, regulated by the relative abundance of the two forms of HDAg: the S-HDAg trans-activates HDV replication in vivo and is strictly required for HDV RNA replication (17), whereas the L-HDAg potently inhibits HDV RNA replication (5, 9) and is required for virus assembly (4, 29). In accordance with these two functions, the two forms of HDAg are synthesized in a temporally regulated manner during the course of the viral life cycle (22): S-HDAg appears early in the replication cycle, when HDV RNA replication occurs, while L-HDAg appears later, when HDV virions are assembled. The sequential appearance of S- and L-HDAg is achieved via an RNA editing event in which the amber stop codon for the S-HDAg open reading frame (ORF) is converted to a tryptophan codon by a double-stranded RNA adenosine deaminase (27, 28). This conversion extends the ORF and enables the translation of L-HDAg, which possesses an additional 19 amino acids at its C terminus compared to S-HDAg.

The ability of L-HDAg to inhibit HDV RNA synthesis is thought to be a key property that allows HDAg to regulate HDV replication. Using HDV cDNA-based transfection approaches, various laboratories have shown that L-HDAg inhibits HDV replication in a dominant-negative inhibitory manner (3, 5, 9, 13, 36). It was shown that a ratio of L-HDAg to S-HDAg as low as 1:10 almost completely abolished the synthesis of both genomic- and antigenomic-sense HDV RNA (5, 9). Thus, it is thought that the appearance of L-HDAg, as a result of RNA editing, signals the cells to stop viral RNA synthesis and to trigger the assembly of virus particles (18). Therefore, the appearance of L-HDAg plays a key role in the switching of molecular events in HDV replication. However, this observation creates a quandary in understanding HDV replication; namely, it is difficult to conceptualize how HDV RNA replication can be initiated during natural infection, where the infecting virion contains both S-HDAg and L-HDAg. While the ratio of these two proteins in the virion is slightly variable, they are usually present in approximately equimolar amounts (1, 2, 26). Since L-HDAg is the protein that is required for HDV virion assembly (4), whereas S-HDAg is incorporated into the virion only when it is complexed with L-HDAg and viral RNA (34), the L-HDAg is invariably present in every virus particle. Furthermore, both L-HDAg and S-HDAg are complexed with HDV RNA (30), and both of them are transported into the nucleus along with HDV RNA (7). How, then, does HDV establish viral replication in the face of the shutoff of viral RNA synthesis by L-HDAg?

We recently developed a new HDV RNA transfection system for the study of HDV replication in cell culture, which avoids the use of artificial HDV cDNA intermediates (24). This system also allows the specific detection of newly synthesized 0.8-kb HDAg mRNA, which was difficult to detect in cDNA-based transfection systems. We previously used this approach to show that HDAg does not suppress the synthesis of HDAg mRNA, an observation that is in direct contrast to previous reports using cDNA-based experiments, in which a down-regulation of the HDAg mRNA polyadenylation was observed (10, 11). Thus, the cDNA-based transfection system used in the latter studies may have created an artifact by introducing artificial involvement of a DNA intermediate. In view of the conceptual difficulty in explaining how the incoming HDV can initiate RNA replication if L-HDAg can inhibit RNA replication so potently, we reexamined this issue using the cDNA-free transfection system.

We found that L-HDAg can potently inhibit the HDV genomic-sense RNA synthesis in a dominant-negative inhibitory manner. However, in contrast to the current understanding of the function of L-HDAg, the synthesis of the 1.7-kb antigenome and the 0.8-kb HDAg mRNA from the HDV genomic RNA template was not significantly inhibited by L-HDAg. As a result, the synthesis of the 1.7-kb antigenome, the 0.8-kb mRNA, and HDAg can occur even in the presence of equimolar amounts of L-HDAg and S-HDAg. This finding explains why HDV can establish RNA replication despite the presence of L-HDAg in the virion. Thus, the sensitivities of the genomic and antigenomic-sense RNA to L-HDAg are clearly different. These findings resolve a critical issue in the understanding of HDV replication and further suggest that the mechanisms of synthesis of the genomic and antigenomic HDV RNA are distinct.

MATERIALS AND METHODS

Cell culture and transfection.

Huh7 cells (25) were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU of penicillin per ml, 100 mg of streptomycin per ml, 2 mM l-glutamate, and 1% nonessential amino acids (complete DMEM). Tsδ3 cells, which were derived from a temperature-sensitive hamster cell line (33) and stably express the S-HDAg from an integrated cDNA copy of the HDAg-encoding mRNA under the cytomegalovirus promoter (12), were cultured at 33°C in DMEM supplemented with 10% fetal bovine serum, 100 IU of penicillin per ml, and 7.5 μg of gentamicin per ml. All transfections were performed using the DMRIE-C reagent (GibcoBRL) according to the protocol provided by the manufacturer, with some modifications. Briefly, 1 day prior to transfection, cells were seeded onto 60-mm-diameter dishes. On the following day, cells were transfected with an appropriate amount of RNA (typically 5 to 10 μg) in 2 ml of transfection mixture in serum-free media. After 1 to 2 h, 2 ml of culture medium containing 20% fetal bovine serum was added to the cells, giving a final concentration of 10% fetal bovine serum. Following incubation overnight, the culture medium was replaced with fresh medium and the cells were incubated for an additional 1 to 5 days.

Vectors and plasmid construction.

Plasmid PB1-3-I/II, which expresses an mRNA encoding the genotype I/II chimeric L-HDAg under the T7 promoter, was developed from the plasmid PB1-3, which expresses an mRNA encoding L-HDAg of the American isolate of genotype I (23). This plasmid was constructed by the same method used to construct plasmid PX9-I/II, which encodes the genotype I/II chimeric S-HDAg (24). PB1-3 contains the pT7-3 plasmid backbone (32) and HDV sequence from nucleotide (nt) 21 to 658 (reading through nt 0) inserted in the BamHI-PstI site. This plasmid differs from PX9 only in that PB1-3 contains the ORF for L-HDAg rather than S-HDAg. To construct plasmid PB1-3-I/II, the EcoRI (in the multiple cloning site)-StuI (at HDV nt 1334) fragment from the plasmid PB1-3 was replaced with the corresponding fragment from plasmid 63 of an HDV genotype II cDNA clone (19). Thus, genotype I nt 21 to 1334 (reading through nt 0) were replaced with the corresponding genotype II nt 1663 to 1334. Plasmid pKS/HDV1.9m expresses 1.9-kb genomic-sense HDV RNA, which contains a premature stop codon in the ORF for S-HDAg such that a truncated form of HDAg (m-HDAg) is translated. pKS/HDV1.9m was constructed by digesting pKS/HDV1.9 with AflII (site located at nt 1209), followed by a fill-in reaction with the Klenow fragment to blunt the ends. The blunt-ended product was ligated to produce the final plasmid, which contains an insertion of 5 nt. This insertion causes both a frameshift in the HDAg ORF and the introduction of a stop codon. Plasmid pBS/T7G-SP, used to detect antigenomic-sense HDV RNA in the noncoding region of the genome, was constructed by inserting the SacII (at nt 25)-PstI (at nt 658) fragment of the American HDV isolate (23) into the same two sites in the multiple cloning site of pBSII/KS+. The final construct expresses genomic-sense HDV RNA from nt 25 to 658 under the T7 promoter.

In vitro transcription.

Genomic HDV RNA (1.9 kb), which contains the entire HDV genome plus approximately 200 additional nucleotides of the HDV sequence encompassing the ribozyme domain (24), was transcribed from the EcoRV-digested plasmid pKS/HDV1.9, which contains both the T7 and SP6 promoters flanking the insert, using T7 MEGAscript (Ambion). Mutant genomic HDV RNA (1.9 kb) was transcribed by the same protocol, from plasmid pKS/HDV1.9m. Antigenomic HDV RNA (1.9 kb) was transcribed from the SnaBI-digested plasmid pKS/HDV1.9 using SP6 MEGAscript (Ambion) according to the manufacturer's directions. Capped mRNAs for in vitro translation of the wild-type S-HDAg and L-HDAg were transcribed from PX9-I/II and PB1-3-I/II, respectively, using T7 mMESSAGE mMACHINE (Ambion) after linearization of plasmids by HindIII digestion.

Northern blot analysis.

Total RNA was extracted from various cell lines using the guanidinium thiocyanate method (6). Polyadenylated RNA was isolated with an oligo(dT) cellulose column (Sigma) according to the standard method (31). The RNA was digested with RQ1 DNase (Promega), treated with formaldehyde, electrophoresed through formaldehyde-containing 1.2% agarose gels, blotted onto a nitrocellulose membrane (Hybond C extra; Amersham), and probed with 32P-UTP-labeled HDV strand-specific riboprobes. Riboprobes were transcribed with T7 RNA polymerase (Promega) from plasmids S18 (to detect genomic HDV RNA) (23) or pBS/T7G-SP (to detect antigenomic HDV RNA in the noncoding region of the genome) (24), following linearization of plasmids by EcoRV digestion. To detect newly synthesized HDAg mRNA in Huh7 cells transfected with HDV RNA (1.9 kb) of genotype I and the chimeric genotype I/II HDAg mRNA, blots were probed with 32P-end-labeled oligonucleotide 1565A (24) specific for the American isolate of genotype I HDV (23). The protocol for Northern blots using oligonucleotide probes was adapted from a published protocol (8). Northern blots probed with the full-length HDV riboprobes were hybridized and washed as described previously (16). RNA extracted from H1δ9 cells, which express and replicate HDV RNA from an integrated cDNA trimer (12), or RNA from Huh7 cells transfected with HDV RNA, was used for positive controls. After autoradiography, computer images were generated by using Canvas, version 5.0.

Western blot analysis.

Protein was extracted from transfected Huh7 or Tsδ3 cells according to the standard method (31). After denaturation by boiling in 2× sample buffer (100 mM Tris-HCl [pH 6.8], 200 mM dithiothreitol, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, and 20% glycerol), 40 μg of protein from each sample was loaded onto a sodium dodecyl sulfate–12.5% polyacrylamide gel electrophoresis minigel and electrophoresed for 60 to 90 min at 150 V. Proteins were then transferred to a nitrocellulose membrane (Hybond C extra; Amersham). S- and L-HDAg were detected by the ECL Western blot detection system (Amersham) using a combination of three monoclonal antibodies against both forms of HDAg (14) and were visualized by autoradiography.

RESULTS

Synthesis of the 1.7-kb antigenomic RNA occurs in the presence of equal amounts of L- and S-HDAg.

Previous reports demonstrated that L-HDAg is a potent inhibitor of HDV RNA replication in cells transiently transfected with HDV cDNA (3, 5, 9, 13, 36). However, our laboratory recently demonstrated that both S-HDAg and L-HDAg can inhibit cellular polymerase II-mediated transcription from a DNA template (21). Thus, it is possible that the observed inhibitory effects of L-HDAg on HDV RNA synthesis may have been due to inhibition of the DNA-templated HDV RNA synthesis. To examine such a possibility, we studied the effects of L-HDAg on HDV RNA synthesis in the cDNA-free RNA transfection system (24). This approach also enabled us to examine the effects of L-HDAg on the synthesis of the 0.8-kb mRNA, since an abundant amount of this mRNA can be detected in the HDV RNA-transfected cells (24).

We first studied synthesis of the 1.7-kb genomic RNA in the presence of varying ratios of S-HDAg and L-HDAg. Huh7 cells were transfected with the in vitro transcribed 1.9-kb antigenomic RNA and varying amounts of mRNAs encoding both L-HDAg and S-HDAg, and the genomic-sense RNA in the cells was examined at various time points after transfection (24). Similar to the previous results obtained in the HDV cDNA-transfected cells, we found that the presence of a small amount of the L-HDAg-encoding mRNA relative to the S-HDAg-encoding mRNA potently inhibited the synthesis of genomic-sense HDV RNA (Fig. 1). For example, when L-HDAg- and S-HDAg-encoding mRNAs were present in a ratio of 1:5 or higher, almost complete inhibition of the genomic RNA synthesis was observed (Fig. 1, lanes 3 to 5). As expected, transfection of 1.9-kb antigenomic HDV RNA with L-HDAg-encoding mRNA but without S-HDAg-encoding mRNA did not allow synthesis of genomic HDV RNA (Fig. 1, lane 6).

FIG. 1.

FIG. 1

Inhibition of HDV genomic RNA synthesis by L-HDAg. Shown are the results of Northern blot analysis of total RNA from Huh7 cells transfected with 1.9-kb antigenomic HDV RNA, mRNA encoding S-HDAg, and increasing amounts of mRNA encoding L-HDAg. (All the numbers represent micrograms of RNA per transfection.) RNA was harvested at day 4 posttransfection and was probed with 32P-labeled riboprobe detecting genomic-sense HDV RNA. Lane 1, positive control indicating the position of the 1.7-kb genomic monomer.

We then examined the synthesis of 1.7-kb antigenomic HDV RNA in the cells transfected with HDV genomic RNA and L-HDAg- and S-HDAg-encoding mRNAs at various ratios (Fig. 2). Surprisingly, there was only a very slight inhibition (25 to 50%) of antigenomic-sense HDV RNA synthesis even when equal amounts of L-HDAg- and S-HDAg-encoding mRNAs were transfected (Fig. 2A, lanes 2 and 3). Only when L-HDAg was present in fivefold excess over the amount of S-HDAg was significant inhibition of HDV antigenomic RNA synthesis observed (Fig. 2A, lane 4). This result is in stark contrast to the effect of L-HDAg on genomic-strand RNA synthesis, where L-HDAg mRNA present in a ratio of 1:5 to S-HDAg mRNA almost completely abolished genomic-strand RNA synthesis (Fig. 1, lane 3).

FIG. 2.

FIG. 2

Resistance of HDV antigenomic RNA synthesis to L-HDAg. (A) Northern blot analysis of antigenomic RNA from Huh7 cells transfected with 1.9-kb genomic HDV RNA, mRNA encoding S-HDAg, and increasing amounts of mRNA encoding L-HDAg. Cells were harvested at day 4 and probed for antigenomic-sense HDV RNA. (B) Western blot of protein from untransfected Huh7 cells (lane 1) and cells transfected with a fixed amount of mRNA encoding S-HDAg and increasing amounts of mRNA encoding L-HDAg (lanes 2 to 4). Cells were harvested at day 2 posttransfection. All the numbers represent micrograms of RNA per transfection.

To ensure that the transfected HDAg mRNAs synthesized the expected amounts of S- and L-HDAg, we examined HDAg levels in Huh7 cells transfected with S-HDAg mRNA and increasing amounts of L-HDAg mRNA (Fig. 2B). When equal amounts of mRNAs encoding S-HDAg and L-HDAg were transfected, equal amounts of S-HDAg and L-HDAg proteins were produced (Fig. 2B, lane 4). Since the first viral RNA species synthesized in the cells following natural HDV infection is antigenomic-strand RNAs, our results strongly suggest that HDV replication can be initiated even if the incoming virion contains equal amounts of L-HDAg and S-HDAg.

Synthesis of the 0.8-kb HDAg mRNA occurs in the presence of equal amounts of L- and S-HDAg.

Since the 0.8-kb mRNA and its translated product S-HDAg are required for the replication of the 1.7-kb RNA, the finding that the synthesis of the 1.7-kb antigenomic RNA is not inhibited by L-HDAg could be the result of two possible scenarios: (i) neither the 1.7-kb antigenome nor the 0.8-kb HDAg mRNA is inhibited by L-HDAg; or (ii) only the 0.8-kb mRNA, not the 1.7-kb antigenomic RNA, is resistant to inhibition by L-HDAg. However, once a sufficient amount of S-HDAg is translated from the newly synthesized 0.8-kb mRNA, the 1.7-kb antigenomic RNA synthesis can be rescued. To distinguish between these possibilities, we sought to examine the inhibitory effects of L-HDAg under conditions where the synthesis of the 0.8-kb mRNA and of the 1.7-kb RNA could be uncoupled.

First, we attempted to examine the inhibitory effects of L-HDAg on the 0.8-kb mRNA transcription independent of the 1.7-kb antigenomic RNA synthesis. Previously, an experimental system was developed in which the 0.8-kb mRNA synthesis occurs in the absence of 1.7-kb antigenomic RNA synthesis (24a). In this system, Huh7 cells were cotransfected with an in vitro transcribed mRNA encoding the wild-type S-HDAg and a 1.9-kb mutant genomic RNA that contains a premature stop codon in the ORF for HDAg. Under this condition, there is no replication of the 1.7-kb antigenomic RNA; only the 0.8-kb mRNA encoding a truncated mutant HDAg (m-HDAg) is transcribed (24a). The m-HDAg translated from this mRNA does not possess trans-activation activity and does not inhibit genomic RNA replication. Because the amount of mRNA transcribed is small and difficult to detect when the RNA template is not replicated, the accumulated protein products of the mRNAs were used as an indirect measure of mRNA synthesis. This system has several advantages for the analysis of 0.8-kb mRNA synthesis. First, since there is no genomic RNA replication, it is possible to measure the amount of 0.8-kb mRNA synthesized based on the same amount of RNA template, i.e., the transfected genomic RNA. Second, this system allows direct measurement of the S-HDAg and L-HDAg derived from the transfected mRNA and of the translated product (m-HDAg) derived from the newly transcribed mRNA. Finally, because the mRNA encoding m-HDAg is unstable and difficult to detect, the protein product (m-HDAg) provides an alternative way of measuring mRNA synthesis, inasmuch as HDAg can be translated only from the 0.8-kb mRNA (20).

Using this system, we found that when equal amounts of mRNAs encoding wild-type S-HDAg and L-HDAg were transfected together with the mutant HDV genomic RNA into Huh7 cells, the production of m-HDAg, which was translated from the newly synthesized mRNA, was only slightly reduced compared with cells transfected with the mutant genome and S-HDAg alone (Fig. 3). Western blot analysis confirmed that L-HDAg and S-HDAg, both of which were translated from the transfected mRNAs, were present in equal amounts (Fig. 3, lane 2). These results strongly suggested that the synthesis of the 0.8-kb mRNA is not significantly inhibited by L-HDAg.

FIG. 3.

FIG. 3

Resistance of the 0.8-kb HDV mRNA synthesis to L-HDAg as revealed by Western blot analysis of the protein products. Shown are the results of Western blot analysis of protein from Huh7 cells transfected with a 1.9-kb HDV mutant genomic RNA and S-HDAg-encoding mRNA alone (lane 1) or together with an equivalent amount of L-HDAg-encoding mRNA (lane 2). Cells were harvested at day 2 posttransfection. Western blotting was performed using a mixture of three monoclonal antibodies against S-HDAg (15). All the numbers represent micrograms of RNA per transfection.

Synthesis of the 1.7-kb antigenome occurs in the presence of L-HDAg, independently of mRNA synthesis.

Having established that de novo transcription of HDAg mRNA can occur in cells containing equal amounts of S- and L-HDAg, we next examined the direct effects of L-HDAg on the synthesis of the 1.7-kb antigenomic RNA. For this purpose, we used an experimental condition in which S-HDAg was provided from a source not inhibited by L-HDAg, and the newly synthesized 0.8-kb mRNA does not produce a functional HDAg to affect the 1.7-kb RNA synthesis. Tsδ3 cells, which stably express S-HDAg from an integrated cDNA containing the ORF for S-HDAg (12), were transfected with the 1.9-kb mutant genomic or antigenomic RNA and increasing amounts of mRNA encoding L-HDAg. In this system, the amount of S-HDAg remains constant, since the only source of the functional S-HDAg is the integrated cDNA; de novo transcription from the transfected genome will produce an mRNA which encodes a truncated m-HDAg and thus will not affect the 1.7-kb genomic or antigenomic RNA synthesis.

We first used this system to reexamine the effect of L-HDAg on HDV genomic RNA synthesis. We transfected Tsδ3 cells with the 1.9-kb antigenomic RNA and increasing amounts of L-HDAg-encoding mRNA, and genomic RNA synthesis in the cells was examined. The results showed that these cells indeed could support HDV RNA replication, even without a transfected mRNA encoding S-HDAg (Fig. 4A, lane 3). When 5 μg or more of mRNA encoding L-HDAg was cotransfected, the genomic RNA synthesis was completely inhibited (Fig. 4A, lanes 4 to 6). Western blot analysis showed that approximately equal amounts of S-HDAg and L-HDAg were detected in Tsδ3 cells transfected with 5 μg of L-HDAg-encoding mRNA (data not shown). This result confirmed the result seen in Fig. 1. The reverse experiment was then performed, in which the 1.9-kb mutant genomic RNA was transfected together with various amounts of the L-HDAg-encoding mRNA, and antigenomic RNA synthesis in the cells was examined. The results (Fig. 4B) showed a clear contrast to those of the genomic RNA synthesis (Fig. 4A). When 5 μg of mRNA encoding L-HDAg was transfected, there was no inhibition of 1.7-kb antigenomic RNA synthesis (Fig. 4B, lane 2). Even at 20 μg of L-HDAg-encoding mRNA, there was still a small amount of antigenomic 1.7-kb RNA (Fig. 4B, lane 4). The synthesis of the 0.8-kb mRNA appeared to be slightly more sensitive than the 1.7-kb antigenomic RNA to inhibition by L-HDAg. Nevertheless, at 5 μg of L-HDAg-encoding mRNA, there was still a substantial amount of 0.8-kb mRNA. Under the same condition, the transcription of the HDAg mRNA (1.1 kb) from the integrated cDNA was not affected by L-HDAg. Since the newly transcribed 0.8-kb mRNA did not produce a functional HDAg to affect the 1.7-kb antigenomic RNA synthesis, this result suggests that the antigenomic RNA is intrinsically resistant to inhibition by L-HDAg. These results combined suggest strongly that, even in the presence of existing S-HDAg, L-HDAg differentially affects HDV genomic and antigenomic RNA synthesis. We conclude that, like the 0.8-kb mRNA, the synthesis of 1.7-kb antigenome is not significantly inhibited by L-HDAg. Therefore, HDV genomic RNA can be replicated into antigenomic RNA even in the presence of L-HDAg. These results combined indicate that HDV genomic and antigenomic RNA synthesis are differentially sensitive to inhibition by L-HDAg, further suggesting that the mechanisms of synthesis of these two RNA species are different.

FIG. 4.

FIG. 4

Comparison of the sensitivity of 1.7-kb genomic and antigenomic RNA synthesis to inhibition by L-HDAg. (A) Northern blot of genomic RNA from Tsδ3 cells transfected with 1.9-kb antigenomic HDV RNA and increasing amounts of mRNA encoding L-HDAg (lanes 3 to 6) and probed with an antigenomic-sense HDV RNA. Lane 1, positive control from HDV RNA-transfected Huh7 cells marking the position of the 1.7-kb genome; lane 2, RNA from untransfected Tsδ3 cells. (B) Northern blot of antigenomic RNA from Tsδ3 cells transfected with a 1.9-kb mutant genomic HDV RNA and increasing amounts of mRNA encoding L-HDAg (lanes 1 to 4). Lane 5, total RNA from untransfected Tsδ3 cells; lane 6, positive control from RNA-transfected Huh7 cells marking the positions of the 1.7-kb antigenome and the 0.8-kb mRNA.

DISCUSSION

In this study we demonstrate that in the HDV RNA transfection system, as others have found in the HDV cDNA transfection systems, HDV genomic RNA synthesis is potently inhibited by the presence of a small amount of L-HDAg. However, unlike previous findings, the synthesis of HDV antigenomic RNA, including both the 1.7-kb antigenome and the 0.8-kb HDAg mRNA, is considerably more resistant to inhibition by L-HDAg. When equal amounts of S-HDAg and L-HDAg are present, such as those found in the incoming HDV virion, the antigenomic RNA synthesis is inhibited by no more than 50%, allowing a significant level of RNA synthesis to occur. In contrast, the genomic RNA synthesis is almost completely inhibited under the same conditions. This finding answers a long-standing question concerning the ability of HDV to carry out RNA synthesis immediately after infection. Previous data on the role of L-HDAg in the HDV replication cycle suggested that synthesis of both genomic and antigenomic HDV RNA had essentially zero tolerance for the presence of L-HDAg; for example, the presence of as low as 10% of L-HDAg in the pool of HDAg molecules in the cells inhibits HDV RNA synthesis almost entirely (5, 9). However, the infecting HDV virion contains almost equal amounts of L- and S-HDAg, both of which can be translocated with the viral RNA into the nucleus (7). Thus, our finding that L-HDAg does not inhibit HDV antigenomic RNA synthesis very efficiently will enable the synthesis of antigenomic RNA and HDAg mRNA from the incoming genome. In this scenario, the synthesis of genomic RNA will be delayed until the L-HDAg is degraded or a sufficient amount of the S-HDAg is synthesized from the newly transcribed mRNA. Significantly, there was some dose-dependent inhibition of antigenomic RNA synthesis by L-HDAg when the amount of L-HDAg exceeded the amount of S-HDAg. This will assure efficient inhibition of HDV RNA synthesis during the late stage of the viral replication cycle. This finding also suggests that the ratio of S-HDAg to L-HDAg packaged in the virion is critical for the infectivity of virus particles.

Our findings also provide strong evidence that the mechanisms of synthesis of HDV genomic and antigenomic RNAs are different. Previous mutagenesis studies of HDV RNA have shown that some mutations of HDV RNA affected RNA synthesis only from genomic RNA but not from antigenomic RNA, and vice versa, suggesting that the synthesis of these two RNAs have different cis-acting RNA sequence requirements (35). Our findings on the different effects of L-HDAg on genomic versus antigenomic RNA synthesis further suggest that the syntheses of these two RNAs require different trans-acting factors. Preliminary evidence from our laboratory also showed that the syntheses of these two RNAs have significantly different sensitivities to α-amanitin, suggesting that the replication machineries for these two RNAs are different (our unpublished data). Further insights into the role of HDAg in the synthesis of HDV genomic and antigenomic RNAs may come from a better understanding of the molecular basis for the trans-activation of HDV replication by S-HDAg and of the mechanism by which L-HDAg is thought to inhibit this process. It has been proposed that oligomerization of S-HDAg is essential for the assembly of the HDV transcription complex, since mutations of the coiled-coil domain in S-HDAg abolished HDV replication (36). Mutations of this same domain in L-HDAg destroyed its ability to inhibit replication (36). Furthermore, S-HDAg from different HDV genotypes may not support the RNA replication of other genotypes and may even serve as a trans-dominant inhibitor of genotype I HDV RNA replication (3). Studies of S-HDAg chimeras between different genotypes supported the hypothesis that the coiled-coil domain of S-HDAg was responsible for the inhibitory activity (3). These results suggest the importance of oligomerization between S-HDAg itself or between S-HDAg and L-HDAg for the trans-acting and inhibitory functions of these two forms of HDAg, respectively. It is possible that antigenomic RNA synthesis does not require oligomerization of S-HDAg, so that the presence of L-HDAg would not inhibit antigenomic RNA synthesis via interruption of such oliogmerization.

In summary, our findings reported here indicate that the syntheses of genomic and antigenomic RNAs are under different mechanisms of regulation. More importantly, these findings provide a possible answer to the question of why HDV can initiate replication despite the presence of L-HDAg in the virion. The precise mechanism of synthesis for these two RNAs will require further studies.

ACKNOWLEDGMENTS

We thank Thomas B. Macnaughton for his comments.

L.E.M. is supported by a scholarship from the Life and Health Insurance Medical Research Fund. M.M.C.L. is an Investigator of the Howard Hughes Medical Institute.

REFERENCES

  • 1.Bergmann K F, Gerin J L. Antigens of hepatitis delta virus in the liver and serum of humans and animals. J Infect Dis. 1986;154:702–706. doi: 10.1093/infdis/154.4.702. [DOI] [PubMed] [Google Scholar]
  • 2.Bonino F, Heermann K H, Rizzetto M, Gerlich W H. Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope. J Virol. 1986;58:945–950. doi: 10.1128/jvi.58.3.945-950.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Casey J L, Gerin J L. Genotype-specific complementation of hepatitis delta virus RNA replication by hepatitis delta antigen. J Virol. 1998;72:2806–2814. doi: 10.1128/jvi.72.4.2806-2814.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Chang F-L, Chen P-J, Tu S-J, Wang C-J, Chen D-S. The large form of hepatitis δ antigen is crucial for assembly of hepatitis δ virus. Proc Natl Acad Sci USA. 1991;88:8490–8494. doi: 10.1073/pnas.88.19.8490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Chao M, Hsieh S-Y, Taylor J. Role of two forms of hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication. J Virol. 1990;64:5066–5069. doi: 10.1128/jvi.64.10.5066-5069.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  • 7.Chou H-C, Hsieh T-Y, Sheu G-T, Lai M M C. Hepatitis delta antigen mediates the nuclear import of hepatitis delta virus RNA. J Virol. 1998;72:3684–3690. doi: 10.1128/jvi.72.5.3684-3690.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Geliebter J, Zeff R A, Schulze D H, Pease L R, Weiss E H, Mellor A L, Flavell R A, Nathenson S G. Interaction between kb and Q4 gene sequences generates the Kbm6 mutation. Mol Cell Biol. 1986;6:645–652. doi: 10.1128/mcb.6.2.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Glenn J S, White J M. trans-Dominant inhibition of human hepatitis delta virus genome replication. J Virol. 1991;65:2357–2361. doi: 10.1128/jvi.65.5.2357-2361.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Hsieh S-Y, Taylor J. Regulation of polyadenylation of hepatitis delta virus antigenomic RNA. J Virol. 1991;65:6438–6446. doi: 10.1128/jvi.65.12.6438-6446.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Hsieh S-Y, Yang P-Y, Ou J T, Chu C M, Liaw Y F. Polyadenylation of the mRNA of hepatitis delta virus is dependent upon the structure of the nascent RNA and regulated by the small or large delta antigen. Nucleic Acids Res. 1994;22:391–396. doi: 10.1093/nar/22.3.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Hwang S B, Jeng K S, Lai M M C. Studies of functional roles of hepatitis delta antigen in delta virus RNA replication. In: Dinter-Gottleib G, editor. The unique hepatitis delta virus. R. G. Austin, Tex: Landes Company; 1995. pp. 95–109. [Google Scholar]
  • 13.Hwang S B, Lai M M C. Isoprenylation masks a conformational epitope and enhances trans-dominant inhibitory function of the large hepatitis delta antigen. J Virol. 1994;68:2958–2964. doi: 10.1128/jvi.68.5.2958-2964.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Hwang S B, Lai M M C. Isoprenylation mediates direct protein-protein interactions between hepatitis large delta antigen and hepatitis B virus surface antigen. J Virol. 1993;67:7659–7662. doi: 10.1128/jvi.67.12.7659-7662.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Hwang S B, Lai M M C. A unique conformation at the carboxyl terminus of the small hepatitis delta antigen revealed by a specific monoclonal antibody. Virology. 1993;193:924–931. doi: 10.1006/viro.1993.1201. [DOI] [PubMed] [Google Scholar]
  • 16.Jeng K S, Daniel A, Lai M M C. A pseudoknot ribozyme structure is active in vivo and required for hepatitis delta virus RNA replication. J Virol. 1996;70:2403–2410. doi: 10.1128/jvi.70.4.2403-2410.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kuo M Y-P, Chao M, Taylor J. Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen. J Virol. 1989;63:1945–1950. doi: 10.1128/jvi.63.5.1945-1950.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lai M M C. The molecular biology of hepatitis delta virus. Annu Rev Biochem. 1995;64:259–286. doi: 10.1146/annurev.bi.64.070195.001355. [DOI] [PubMed] [Google Scholar]
  • 19.Lee C-M, Changchien C S, Chung J C, Liaw Y F. Characterization of a new genotype II hepatitis delta virus from Taiwan. J Med Virol. 1996;49:145–154. doi: 10.1002/(SICI)1096-9071(199606)49:2<145::AID-JMV12>3.0.CO;2-D. [DOI] [PubMed] [Google Scholar]
  • 20.Lo K, Hwang S B, Duncan R, Trousdale M, Lai M M C. Characterization of mRNA for hepatitis delta antigen: exclusion of the full-length antigenomic RNA as an mRNA. Virology. 1998;250:94–105. doi: 10.1006/viro.1998.9364. [DOI] [PubMed] [Google Scholar]
  • 21.Lo K, Sheu G-W, Lai M M C. Inhibition of cellular RNA polymerase II transcription by delta antigen of hepatitis delta virus. Virology. 1998;247:178–188. doi: 10.1006/viro.1998.9253. [DOI] [PubMed] [Google Scholar]
  • 22.Luo G, Chao M, Hsieh S Y, Sureau C, Nishikura K, Taylor J. A specific base transition occurs on replicating hepatitis delta virus RNA. J Virol. 1990;64:1021–1027. doi: 10.1128/jvi.64.3.1021-1027.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Makino S, Chang M F, Shieh C K, Kamahora T, Vannier D M, Govindarajan S, Lai M M C. Molecular cloning and sequencing of a human hepatitis delta virus RNA. Nature (London) 1987;329:343–346. doi: 10.1038/329343a0. [DOI] [PubMed] [Google Scholar]
  • 24.Modahl L E, Lai M M C. Transcription of hepatitis delta antigen mRNA continues throughout hepatitis delta virus (HDV) replication: a new model of HDV RNA transcription and replication. J Virol. 1998;72:5449–5456. doi: 10.1128/jvi.72.7.5449-5456.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24a.Modahl, L. E., T. B. Macnaughton, N. Zhu, D. L. Johnson, and M. M. C. Lai. RNA-dependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerases. Mol. Cell. Biol., in press. [DOI] [PMC free article] [PubMed]
  • 25.Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth of human hepatoma cell lines with differentiated function in chemically defined medium. Cancer Res. 1982;42:3858–3863. [PubMed] [Google Scholar]
  • 26.Pohl C, Baroudy B M, Bergmann K F, Cote P J, Purcell R H, Hoofnagle J, Gerin J L. A human monoclonal antibody that recognizes viral polypeptides and in vitro translation products of the genome of the hepatitis D virus. J Infect Dis. 1987;156:622–629. doi: 10.1093/infdis/156.4.622. [DOI] [PubMed] [Google Scholar]
  • 27.Polson A G, Bass B L, Casey J L. RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase. Nature (London) 1996;380:454–456. doi: 10.1038/380454a0. [DOI] [PubMed] [Google Scholar]
  • 28.Polson A G, Ley H L, Bass B L, Casey J L. Hepatitis delta virus RNA editing is highly specific for the amber/W site and is suppressed by hepatitis delta antigen. Mol Cell Biol. 1998;18:1919–1926. doi: 10.1128/mcb.18.4.1919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ryu W-S, Bayer M, Taylor J. Assembly of hepatitis delta virus particles. J Virol. 1992;66:2310–2315. doi: 10.1128/jvi.66.4.2310-2315.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Ryu W-S, Netter H J, Bayer M, Taylor J. Ribonucleoprotein complexes of hepatitis delta virus. J Virol. 1993;67:3281–3287. doi: 10.1128/jvi.67.6.3281-3287.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]
  • 32.Tabor S, Richardson C C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA. 1985;82:1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Wang E H, Tjian R. Promoter-selective transcriptional defect in cell cycle mutant ts 13 rescued by hTAFII250. Science. 1994;263:811–814. doi: 10.1126/science.8303298. [DOI] [PubMed] [Google Scholar]
  • 34.Wang H-W, Chen P-J, Lee C-Z, Wu H-L, Chen D-S. Packaging of hepatitis delta virus RNA via the RNA-binding domain of hepatitis delta antigens: different roles for the small and large delta antigens. J Virol. 1994;68:6363–6371. doi: 10.1128/jvi.68.10.6363-6371.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Wang H-W, Wu H-L, Chen D-S, Chen P-J. Identification of the functional regions required for hepatitis D virus replication and transcription by linker-scanning mutagenesis of viral genome. Virology. 1997;239:119–131. doi: 10.1006/viro.1997.8818. [DOI] [PubMed] [Google Scholar]
  • 36.Xia Y-P, Lai M M C. Oligomerization of hepatitis delta antigen is required for both the trans-activating and trans-dominant inhibitory activities of the delta antigen. J Virol. 1992;66:6641–6648. doi: 10.1128/jvi.66.11.6641-6648.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]