INTRODUCTION

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Hepatitis delta virus (HDV) is a small RNA virus that is transmitted from human to human only in conjunction with hepatitis B virus (HBV) infection (23). HDV is a clinically important pathogen, since dual infection with HBV and HDV is often associated with liver injury that is more severe than that due to HBV alone (13, 24, 25). The HDV genome is a 1.7-kb covalently closed, single-stranded, circular RNA that displays 70% self-complementarity, allowing it to base pair into a highly compact, rod-like structure (reviewed in references 16 and 18). These genomic features are found in no other animal virus but recall those of plant viroids. But unlike viroid RNAs, which are noncoding, HDV RNA harbors a single open reading frame that encodes a key RNA-binding protein known as hepatitis delta antigen (HDAg). HDAg specifically binds HDV RNA (7) and these HDAg-RNA complexes form the core of the HDV virion. As a result of an RNA editing event that occurs during replication (4, 19), HDAg occurs in two isoforms differing at their C termini. The small (S) isoform is absolutely required for RNA replication (14), while the larger (L) isoform inhibits RNA replication but promotes the envelopment of HDV RNPs by the envelope proteins of HBV (5, 8). This envelopment is the basis of the requirement for HBV coinfection, since only HDV genomes enveloped by HBV glycoproteins can exit the cell and propagate the infection.

No HBV functions are required for HDV RNA replication, which proceeds via RNA-based rolling circular intermediates (9). Unlike all other RNA viruses, HDV does not appear to encode its own RNA-dependent RNA polymerase. HDAg, its sole gene product, has no homology to known polymerases and no detectable polymerase activity. The responsible polymerase is presumed to be of cellular origin, but its identity is unknown and little is known of host factors that affect viral replication.

Several years ago, we discovered that HDV RNA replication, which occurs efficiently in most cultured mammalian cells, proceeds poorly or not at all in certain avian cell lines (cited in reference 6), findings which have since been confirmed by others (6). Here we have further explored this observation, with the aim of assessing the generality of the block to replication in avian cells and whether it is due to the presence of inhibitory factors or to the absence of permissive ones. Our results strongly favor the latter hypothesis and provide a viable strategy for the identification of such factors.

	MATERIALS AND METHODS

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Cell culture. QT6 (20), QT35 (a gift from M. Linial, Fred Hutchinson Cancer Center) (20), LMH, and chicken embryonic fibroblast cells were maintained in Dulbecco's modified Eagle's medium with 10% tryptose phosphate broth, 5% fetal bovine serum, and 1% chick serum. QM7 was obtained from the American Type Culture Collection (ATCC) (1). QCL-3 and 8C93 cells were kindly provided by B. Cullen (Howard Hughes Medical Institute and Duke University) (11) and H. Yin (University of Texas Southwestern Medical Center), respectively. All of the adherent mammalian cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, except for CHO-K1 cells, for which the medium also contained 40 µg of proline per ml. These cells were transfected with either Superfect (Qiagen) or Fugene-6 (Roche). DT40 and BJAB cells were maintained and transfected as described previously (10, 15). Interspecific cell fusion between avian and mammalian cells was induced by PEG-1500 (Roche) for 1 min.

Plasmids. The expression plasmids for 1.1-mer HDV genomic (pSVL-D1.1 wild type [WT] and mutant) or antigenomic [pSVL-D1.1 (AG)] cDNA and SHDAg (pcDNA3-SHDAg) have been described (2, 3, 17, 26).

Immunofluorescence and in situ hybridization. To detect HDV replication at the single-cell level, short oligonucleotide DNA probes were used for in situ hybridization (12). A genome-specific 45-mer oligoprobe (2) 5' labeled with fluorescein isothiocyanate was synthesized by Life Technology. Cells were first fixed and permeabilized with methanol at 20°C for 15 min. Immunolocalization of -galactosidase was performed with a mouse monoclonal antibody (Promega) and tetramethyl rhodamine isothiocyanate (TRITC)-labeled goat anti-rabbit secondary antibody. Subsequently, cells were postfixed with in situ fixation buffer (Ambion), stored in 70% alcohol overnight at 4°C, and rehydrated with 50% formamide in 4× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 5 min at room temperature. Cells were denatured at 65°C with Zip-Hyb buffer (Ambion) for 10 min, hybridized with probes overnight at 37°C, washed twice with 50% formamide in 2× SSC and in 0.1× SSC at 37°C, and finally mounted with Vectashield containing 4',6'-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Northern blotting and flow cytometry. These analyses were performed as previously described (3, 26).

	RESULTS

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Block to HDV replication in avian cells. To assay for HDV RNA replication, cells were transfected with 1.1-mer clones of HDV cDNA, the expression of whose genomic RNA strand was driven by a simian virus 40 (SV40) late promoter (pSVL-D1.1) or cytomegalovirus (CMV) immediate-early promoter (pCMV-D1.1). Expression of this RNA by conventional pol II transcription results in production of overlength genomic RNAs which undergo self-cleavage to unit-length linears by the intrinsic ribozyme activity of the RNA. Ligation of these RNAs yields monomer circles of genomic polarity; in the presence of S-HDAg, these structures are replicated to antigenomic RNA. Thus, the presence of antigenomic RNA in Northern blots of RNA from such cells provides proof of authentic HDV RNA replication. Table 1 shows a summary of the lines in which we have detected HDV replication following transfection of HDV cDNA. Not only can cells of many mammalian species support replication, but virtually all cell types tested appear competent for this activity. In fact, no mammalian cell line we have ever tested has failed to support HDV RNA synthesis (3, 26, 27; Y.-T. Liu, R. Brazas, and D. Ganem, unpublished observation).

View larger version (86K): [in this window] [in a new window]   FIG. 1.   Northern and Western analysis for HDV replication in mammalian and avian cells. Monkey CV1 and quail QT6 cells were transfected with pSVL-D1.1 (lanes 1 and 6), mutant pSVL-D1.1 (lanes 2 and 7), pSVL-D1.1 + pcDNA3-SHDAg (lanes 3 and 8), mutant pSVL-D1.1 + pcDNA3-SHDAg (lanes 4 and 9) and pCMV-D1.1 (lanes 5 and 10) and were analyzed by Northern blotting for HDV antigenomic RNA synthesis (top panel). The total RNA of each sample is shown in the middle panel. Expression of S-HDAg was detected by Western blotting (bottom panel).

The defect in avian cells is recessive. The above data (and those of reference 6) indicate that species-specific factors affect the replication of HDV RNA. Two general models can be considered for how such factors may operate: avian cells may lack permissive factor(s) that mammalian cells possess, or avian cells may harbor an inhibitor(s) of HDV replication missing from mammalian hosts. To distinguish between these models, we employed a somatic cell genetic approach involving interspecific cell fusion. First, pSVL-D1.1 was transfected into QM7 quail cells. One day later, CHO-K1 (hamster) cells were added to these cultures. Once the hamster cells were stably attached, half of the cocultures were fused with 50% polyethylene glycol (PEG), while the remainder were simply cocultured in the absence of fusogen. All cultures were incubated in 10 µM 1--D-arabinofuranosylcytosine to prevent overgrowth of unfused cells, and 3 days later RNA from the cocultures was examined by Northern blotting for antigenomic RNA. As shown in Fig. 2A, products of HDV replication were readily detected when the cocultures were fused with PEG, but no HDV replication was observed in the absence of fusion. Similar results are observed in QT6 cells transfected with pCMV-D1.1 (Fig. 2C), although as expected, the basal level of HDV replication driven by this construct is higher (Fig. 1, lane 10). These results indicate that avian cells do not contain dominant inhibitors of replication. Rather, they suggest that avian cells lack permissive factors. Also, the fact that fusion is required for complementation to be observed indicates that the missing permissive factors are cell autonomousthey cannot be supplied in a paracrine fashion from nearby cells but must be directly introduced into the nonpermissive cell.

View larger version (49K): [in this window] [in a new window]   FIG. 2.   HDV Replication in heterokaryons of mammalian and avian cells. (A) Quail cells (QM7) were transfected with HDV cDNA of genomic polarity in pSVL vector and cocultured with mammalian cells (CHO-K1). Cell fusion was induced by PEG. HDV antigenomic RNA synthesis was analyzed by Northern blotting for cocultures with (left lane) or without (right lane) fusogen. (B) Quail cells (QT6) were transfected with HDV cDNA of antigenomic polarity and cocultured with a C2C12 derived mouse cell line, which had been stably transfected with -Gal plasmid. The -Gal protein was labeled with TRITC (red, upper panel) by indirect immunofluorescence. HDV genomic RNA was detected by fluorescein isothiocyanate-labeled (green, upper panel) oligonucleotide probes. The nuclei of cells were shown by DAPI staining (blue, lower panel). (C) QT6 cells were transfected with pCMV-D1.1. Transfected QT6 cells were cultured either alone (lane 3) or with CHO-K1 cells (lanes 1 and 2); cells of lanes 1 and 3 were induced to fuse with PEG, and then RNA was extracted and analyzed as described in the legend to Fig. 2A.

Impaired HDV replication in avian cells is not due to cell toxicity or cell death. Recently, Chang et al. (6) have suggested that the inability of chicken LMH cells to support HDV replication is due to toxicity resulting from HDAg expression within these cells. This was based upon the observation that when HDAg expression vectors were cotransfected with green fluorescent protein (GFP) reporter plasmids, GFP expression was impaired in a dose-dependent fashion. These authors also claimed that the replication defect of HDV in LMH cells could be partially suppressed by the broad-spectrum caspase inhibitor ZVAD-fmk, and they suggested on this basis that induced host cell apoptosis was an important component of the nonpermissive phenotype. Here we show that neither of these assertions is generally true of avian cells. QT6 cells were cotransfected with an enhanced GFP (EGFP) expression vector and a fivefold excess of either pcDNA3, pcDNA3-SHDAg, pcDNA3-lacZ, or pSVL-D1.1; 3 days later, cells were examined by flow cytometry for GFP expression. As shown in Fig. 3, the percentages of transfected cells expressing GFP and the mean levels of GFP expression per cell were not significantly different in any of the groups. In particular, expression of HDAg (panel A) had no adverse impact on GFP expression.

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Flow cytometry analysis of the effect of S-HDAg on GFP coexpression. pEGFP was cotransfected with pcDNA3-SHDAg (A), pSVL-D1.1 (B), pcDNA3 vector (C), and pcDNA3-LacZ (D) into QT6 cells. A negative control without pEGFP transfection is shown (E). The GFP expression was analyzed 3 days after transfection by flow cytometry using a Becton Dickinson FACSCalibur.

View larger version (63K): [in this window] [in a new window]   FIG. 4.   Annexin V staining to analyze the effect of S-HDAg on cell death. QT6 cells were transfected with pEGFP either alone (A and G) or together with pcDNA3-SHDAg (B), pSVL-D1.1 (C), mutant pSVL-D1.1 (D), pcDNA3 vector (E), and pcDNA3-LacZ (F). Cells were stained with annexin V before fixation at day 3 after transfection. Some cells were treated with 0.5 µg of staurosporine per ml (A) to induce apoptosis as a positive control for annexin V-Cy3 staining (middle panel). GFP expression is shown in the left panel and DAPI staining is shown in the right panel.

	DISCUSSION

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These experiments show that there is a profound (though not absolute) block to HDV RNA replication in cells of avian origin and define this block as being due to the absence of one or more permissive factors that are present in mammalian cells of many species and cell types. This block does not appear to be the trivial consequence of cytotoxicity of HDV products in avian cells, nor is it due to enhanced susceptibility to apoptosis. Although it is possible that some avian cell lines may be more sensitive to putative toxicities of this type, our results clearly show that such idiosyncratic toxicities, if they exist at all, cannot explain the general nonpermissiveness of avian cells. This interpretation is also consistent with the fact that, in recent experiments conducted by others (see Fig. 5 in reference 6), ZVAD-fmk treatment of HDV-transfected LMH cells did not appreciably restore viral antigenomic RNA levels, despite improved expression of cotransfected GFP reporters. Thus, even in that cell line, apoptosis was not the reason for impaired HDV replication.

We do not yet know the nature of the permissive factor(s) absent from avian cells. The fact that expression of HDAg in trans does not reverse the block suggests that the defect is likely to be downstream of HDAg expression. For example, if HDAg must interact with one or more host components to implement its replicative function, such components would be candidates for the missing factor(s), as would subunit(s) of the host-encoded polymerase required for HDV RNA synthesis. Other possibilities include (but are not limited to) host factors that might be involved in facilitating ribozyme-mediated RNA cleavage, the ligation of the RNA cleavage products, or the stabilization of newly replicated genomes.

Our results have two important implications for the study of HDV replication and pathogenesis. First, they suffice to explain earlier failures to transmit HDV to ducks bearing the duck hepatitis B virus (DHBV). Although HDV was readily transmitted by experimental inoculation of sera from HBV- and HDV-infected subjects into woodchucks bearing the woodchuck hepatitis virus (21), similar attempts at transmission to DHBV-infected ducks have generally been unsuccessful, despite early reports to the contrary (22). Second, the results of the complementation tests illustrated in Fig. 2 suggest that it may be possible to design gene transfer strategies to identify and clone the responsible missing factor(s). The identification of such factors should provide important clues to the nature of the host machinery required for HDV infection.