Genotype-Specific Complementation of Hepatitis Delta Virus RNA Replication by Hepatitis Delta Antigen

Plasmids for HDV genome expression.

The genotype III expression plasmids pHDV·III(+) and pHDV·III(−) were created from cloned PCR amplification products from the isolate Peru-1 (7). Briefly, amplification products spanning nucleotides (nt) 307 to 1290 and 1078 to 424 of the circular 1,677-nt genome were cloned into the plasmid vector pT7Blue (Novagen) to yield clones pNV1-2 and pNV3-1, respectively. The 1-kb PstI fragment from pNV1-1 was cloned into the PstI site of pNV3-1 to yield the monomeric clone pNV3/1. The 1.7-kb KpnI fragment from pNV3/1 was cloned between the KpnI sites of clone pNV1-3, a sister clone to pNV1-2, to yield pPeru-1×1.5. From clone pNV1-3, the 466-bp SmaI fragment encompassing nt 522 to 988 of the Peru-1 genome was cloned into the SmaI site of pGEM-3Zf(+) (Promega) to yield clone p293. The 1,677-bp Esp3 I fragment from pPeru-1×1.5 (the Esp3 I site is at position 795 in the Peru-1 isolate) was cloned into the Esp3 I site of p293 to yield pPeru-1×1.2, which contained a duplication of both the genomic and antigenomic ribozyme autocleavage domains and encoded the short form of HDAg. The EcoRI-XbaI fragment from pPeru-1×1.2 was then inserted into the same sites of the plasmids pCMV2 and pCMV3 (8) to yield the expression plasmids pHDV·III(+) and pHDV·III(−), which direct the synthesis of antigenomic and genomic HDV RNA, respectively, in transfected cells.

Constructs for the expression of HDV RNA defective for HDAg production, HDAg(−), were created by site-directed mutagenesis with PCR primers containing the following mutations: for genotype I, an A was inserted between positions 1577 and 1578 (genotype I nucleotide numbering according to reference 32), thereby introducing a stop codon and frameshift at codon 7; for genotype III, an A was inserted between positions 1579 and 1580 (genotype III nucleotide numbering according to reference 7), thereby introducing a stop codon and frameshift at codon 6. PCR-amplified fragments containing these mutations were inserted between the SstII and BglII sites of pCMV2-DC1×1.2 and pCMV3-DC1×1.2 (8) for genotype I, and between the Bsu36 I and XhoI sites of pHDV·III(+) and pHDV·III(−) for genotype III. The sequences of the PCR-amplified mutated segments were verified after cloning. Plasmids pHDV·I(−)Ag(−) and pHDV·III(−)Ag(−) were constructed to produce genomic genotype I and genotype III RNA, respectively, in transfected cells, and pHDV·I(+)Ag(−) and pHDV·III(+)Ag(−) were constructed to produce antigenomic genotype I and genotype III RNA, respectively, in transfected cells.

Plasmid pHDV·III(+)Δ-NR was constructed to express nonreplicating genotype III antigenomic RNA in transfected cells. It is similar to the nonreplicating genotype I HDV antigenomic RNA expression construct pCMV3-DC-Δ1×1(A) (8), which will be referred to here as pHDV·I(+)Δ-NR. pHDV· III(+)Δ-NR was created by cloning the genome-length NarI (position 722) fragment of pPeru-1×1.2 into the AccI site of pGEM-3Zf(−) (Promega) to yield pGPeru(+)(Nar), in which the HDV genome was oriented such that the T7 polymerase promoter would produce antigenomic sense HDV RNA. Plasmid pGPeru(+)(Nar) was digested with XhoI and partially with BamHI, treated with Klenow DNA polymerase, and ligated to yield pGPeru(+)(Nar ΔBam-Xho), which, in the process of deleting 262 nt, places an XhoI site at position 90 of the Peru-1 sequence. The XhoI-BamHI fragment (Peru-1 positions 90 to 722; the BamHI site comes from plasmid vector sequences) from pGPeru(+)(Nar ΔBam-Xho) was substituted for the same fragment in pHDAg-S·III (see below) to yield pHDV·III(+)Δ-NR, in which genotype III positions 1505 to 1677 and 1 to 90 have been deleted.

Plasmids for HDAg expression.

The genotype I HDAg expression plasmid pCMV-AgS, which encodes the short form of HDAg, is described elsewhere (28). For clarity, we refer to this construct here as pHDAg-S·I. The expression construct pHDAg-L·I, which encodes the long form of genotype I HDAg, was created by substituting the PstI-SalI fragment of the plasmid pGDC-1M2 (4), which contains the nucleotide C at position 1012, for the same fragment in pHDAg-S·I. The genotype III expression plasmid pHDAg-S·III was obtained by substituting the 872-bp SstI-ApaI fragment from pPeru-1×1.2 for the same fragment in pHDAg-S·I. Plasmid pHDAg-S·III contains positions 779 to 1651 from the Peru-1 isolate and includes the coding sequence for HDAg and the polyadenylation signal. Expression plasmid pHDAg-L·III, which encodes the long form of genotype III HDAg, was obtained by substituting the 310-bp PstI-ApaI fragment from pNV1-3, which contains the nucleotide C at position 1014, for the same fragment in pHDAg-S·III.

Expression plasmids for HDAg-S from additional isolates were obtained by PCR amplification. Genotype I clones were obtained by ligating PCR-amplified fragments into the Ecl136 II site of plasmid pCMV3 (8). PCR amplification was performed with primers 5414 (26) and 6657 (5′-CAGCAGTCTCCTCTTTACAGA-3′; nt 1658 to 1638); conditions were as reported previously (6) except that the extension step was increased to 2 min and Pfu polymerase was used. Genotype I isolates were from Italian patients I27 and I43, who were chronically infected with HDV (26), and U02, an American patient with chronic HDV infection (7). For genotype III, HDAg-coding sequences were amplified with primers 5414 and 7676 (5′-GCTCCTTCCTCCTTAGGAGAGATAAG-3′; nt 1650 to 1625). Fragments digested with Bsu36I (position 1635) and SmaI (position 988) were substituted for the same fragment in pHDAg-L·III. Genotype III isolates were Peru 5 and 13, from Peruvian soldiers who experienced acute HDV infection while stationed at different base camps in the Amazon jungle (10), and P20 and P21, from two natives of the Amazon basin in Peru (provided by M. Sjögren and A. Colichon). Clones were analyzed by restriction digestion and sequencing to determine the correct orientation relative to the cytomegalovirus promoter and to determine which form of HDAg (short or long) was encoded.

Chimeric HDAg expression plasmids.

The chimeric constructs pHDAg-S·III·III·I and pHDAg-S·I·I·III were obtained by switching 0.7-kb NgoA IV fragments between pHDAg-S·I and pHDAg-S·III. The NgoA IV site, located at position 1161 in the genotype I isolate, is identical in both isolates. Clones were screened by restriction digestion for the correct orientation. Plasmid pHDAg·III·I·I contained genotype III sequences from the beginning of the HDAg sequence up to the ApaI site in genotype I (nt 1380); it was created by a two-step procedure. Genotype III sequences between nt 1380 and 215 were amplified with primers 5′-TTTCCTGCCTCGGGCCCTCTTCGCC-3′ and 5′-TTTGCTGCCGATGGGCCCTCGGACCGGGGTC-3′ and cloned between the ApaI sites of the genotype I plasmid pGDC-1 to yield pG{Peru/DC-1} (Apa); clones were checked by restriction digestion for proper orientation. The primers used were specific for genotype III sequences, but internal ApaI sites were added; the ApaI site in the primer for the 1380 region is located such that the coding sequence is uninterrupted through this site when the genotype I and genotype III sequences are combined. The 601-bp BbsI-Esp3 I fragment from pG{Peru/DC-1}(Apa) was inserted between the same sites in plasmid pHDAg-S·III to yield pHDAg-S·III·I·I. Plasmid pHDAg-S·III·I·III, containing genotype I HDAg-S sequences between the ApaI and NgoA IV sites and genotype III sequences elsewhere, was created by substituting the 708-bp NgoA IV segment from pHDAg-S·III for the same fragment in pHDAg-S·III·I·I. Plasmid pHDAg-S·I·III·I, containing genotype III HDAg-S sequences between the ApaI and NgoA IV sites and genotype I sequences elsewhere, was created by a three-step procedure. The region between nt 1380 and 883 was amplified with primers 5′-GCGAAGAGGGCCCGGCAGGAAACCATG-3′ and 5414 (26); the primer around 1380 contains an internal ApaI site which is located such that the coding sequence is uninterrupted through this site when genotype I and genotype III sequences are combined. The ApaI-PstI fragment of the PCR amplification product was substituted for the same fragment in plasmid pHDAg-S·III·I·I to give pHDAgP/C(Apa-Pst). The NgoA IV fragment of pHDAgP/C(Apa-Pst) was replaced by the same fragment of pHDAg-S·I to give pHDAgP/C(Apa-Ngo); the ApaI fragment of this plasmid was substituted for the same fragment in pHDAg-S·I to yield pHDAg-S·I·III·I. Plasmid pHDAg-S·I·III·III was created by substituting the NgoA IV fragment of pHDAg-S·III for that in pHDAg-S·I·III·I.

Transfections.

Huh-7 cells were plated in six-well (35-mm-diameter) dishes and transfected with 5 μg of DNA per well by the calcium phosphate method as described previously (6). Transfections included the plasmid pSEAP2Control (Clontech), which was used to correct for transfection variability by monitoring secreted alkaline phosphatase expression (14). Transfection efficiencies varied by 2.5-fold or less. All experiments included duplicate transfections, and all were repeated at least twice.

RNA analysis.

Cellular RNA was harvested as described previously (6) between days 5 and 7 posttransfection, as indicated, except that proteinase K (1 mg/ml) was included in the cell lysis buffer. For gel electrophoresis and blot hybridization analysis, 15% of the total RNA isolated was treated with DNase (Life Technologies) for 30 min prior to electrophoresis in 6.7% formaldehyde–1.5% agarose gels. Gels were stained with ethidium bromide and examined by UV illumination to verify equal loading and sample integrity. Samples were electroblotted onto Nytran (Boehringer Mannheim) membranes and fixed by UV illumination (Stratalinker; Stratagene, La Jolla, Calif.). Complete and even transfer of RNA samples to the membrane was verified by inspection of ethidium bromide-stained RNAs under UV illumination. Hybridization was for 16 to 36 h at 60°C in buffer containing 50% formamide, 1 M NaCl, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), 0.5 mg of sheared salmon sperm DNA per ml, and hybridization probe (10⁶ cpm/ml; 5 × 10⁸ cpm/μg). Blots were washed with 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 65°C and then exposed to film or quantified by radioanalytic imaging (InstantImager; Packard Instruments, Meriden, Conn.). Hybridization probes were monomeric genotype I or genotype III RNAs, as indicated, except where noted otherwise. Genotype I probes were used to detect genotype I RNAs, and genotype III probes were used to detect genotype III RNAs. Under the stringent hybridization and washing conditions used, homologous hybridization is favored over heterologous hybridization because the sequences of genotypes I and III are only 60% identical overall, and the longest contiguous segment of sequence identity is 35 nt. However, it is possible to detect genotype III RNA with a genotype I RNA probe, and vice versa. For purposes of quantitation, hybridization signal intensities were corrected for transfection efficiency by cotransfection of a reporter expression construct for secreted alkaline phosphatase (14).

Immunoblot analysis.

Cell lysates were obtained by treatment with 2% SDS–0.2 M Tris-HCl (pH 7.5)–1 mM EDTA and analyzed for hepatitis delta antigen by SDS-polyacrylamide gel electrophoresis in 12% acrylamide gels and immunoblotting with human monoclonal anti-HD T1/39 as described elsewhere (27).

A replicating HDV genotype III cDNA clone.

Analyses of HDV replication in cultured cells have thus far been limited to cDNA clones of HDV genotype I isolates. Two of the clones used (6, 15, 22) were derived from animals infected in related passage series and differed by about 1% (16, 32), a third (11, 23) was obtained from a genotype I isolate (24) that is about 10% different in nucleic acid sequence over the entire genome. Sequence variations among the genotypes, on the other hand, are as high as 40% (7), and inspection of sequence differences within some functional elements (such as the RNA editing site and the C terminus of the long form of HDAg) suggests that there could be important functional differences among the genotypes (3, 9). To compare the replication of genotypes I and III, we obtained a full-length cDNA clone from the prototype genotype III isolate Peru-1 (7). Expression constructs in which the cytomegalovirus promoter directs the synthesis of HDV RNA containing duplications of the ribozyme elements and encoding the short form of HDAg were created (Fig. 1). These constructs are similar to those used for analysis of HDV genotype I replication (6).

Following transfection of Huh-7 cells, the genotype III expression construct pHDV·III(+) yielded replicating HDV genotype III RNA, as indicated by the accumulation of both genomic (Fig. 2A) and antigenomic RNA (not shown) between 6 and 12 days posttransfection, even though only antigenomic RNA synthesis is directed by the cytomegalovirus promoter. The results shown are representative of numerous similar transfections in which either genomic or antigenomic RNA synthesis was directed by the transfected plasmids. In these experiments, the level of HDV RNA was determined by radioanalytic imaging to be about two- to threefold lower for the transfected genotype III expression construct than for a similar genotype I expression construct. In addition to HDV RNA expression, HDAg was also detected in the transfected cells. Although only the short form of HDAg is encoded in the transfected constructs, both forms of HDAg are produced (Fig. 2B) because of an RNA editing event that occurs during viral RNA replication (6, 21). Comparison of the relative levels of genotype I and III HDAg expression is difficult because of antigenic and structural variations. Curiously, genotype III HDAg-S migrated faster in the gel than did genotype I HDAg (Fig. 2C). Genotype III HDAg-L also migrated faster than its genotype I counterpart, but the difference in the migration rates was not as great. The different migration pattern was observed in numerous experiments in which either replicating HDV expression constructs or HDAg expression constructs were transfected (not shown).

Genotype-specific support of RNA replication by HDAg.

In cultured cells, HDV RNA replication requires expression of the short form of HDAg (16). Thus, transfection of HDV cDNA constructs deficient in HDAg expression yields replicating HDV RNA only if an expression construct for HDAg is also cotransfected and if the defect which abrogates HDAg expression leaves intact structural elements required for RNA replication (1, 15, 17, 35). To examine the ability of HDAg to support replication of genotype I and genotype III HDV RNAs, constructs for intracellular expression of RNAs defective for HDAg synthesis were created (Fig. 1B). The defect was created by inserting a thymidine nucleotide in the cDNA at the beginning of the seventh and sixth codons, respectively, of the wild-type genotype I and genotype III expression constructs. This insertion created a stop codon as well as a frameshift. A single-base insertion was used in order to minimize potential effects on the RNA secondary structure that could also affect the ability of the RNA to replicate (35).

Transfection of Huh-7 cells with HDAg(−) RNA expression constructs but without cotransfected expression plasmids for HDAg yielded no evidence of HDV RNA replication, as determined by the inability to detect opposite-sense transcripts 6 days posttransfection (Fig. 3). As previously shown for similar genotype I HDAg(−) expression constructs, RNA replication was observed upon cotransfection of an expression construct for genotype I HDAg (Fig. 3). Antigenomic RNA was readily detectable 6 days after cotransfection of the defective genomic RNA expression construct pHDV·I(−)Ag(−) with the HDAg expression construct pHDAg-S·I, and genomic HDV was observed after cotransfection of the defective antigenomic RNA expression construct pHDV·I(+)Ag(−) with the same genotype I HDAg expression construct. The same result was obtained for genotype III: cotransfection of genotype III HDAg(−) constructs with a genotype III HDAg expression construct yielded high levels of replication of genotype III HDAg(−) RNAs (Fig. 3). However, genotype III HDAg did not support appreciable replication of genotype I HDAg(−) RNAs, and genotype I HDAg supported replication of genotype III HDAg(−) RNAs at only about 5% of the level observed with genotype III HDAg (Fig. 3). Thus, the ability of HDAg to support replication of HDAg(−) RNA was genotype specific.

HDAg clones from different HDV isolates.

The results presented in Fig. 2 and 3 were obtained with clones derived from the prototype isolates of HDV genotypes I and III. To determine whether the observed effects might be due to genotype-specific differences rather than a peculiarity of the two isolates used, additional HDAg expression clones were obtained by cloning PCR-amplified cDNAs. Three clones were obtained from additional unrelated genotype I isolates and four from additional unrelated genotype III isolates. Within a 357-bp region (nt 908 to 1265) corresponding to the C-terminal half of the HDAg-coding region, extents of sequence divergence among these genotype I and genotype III isolates were from 6 to 9% and from 4 to 8%, respectively (4, 10, 26, 31). Upon cotransfection of Huh-7 cells, all four genotype I HDAg clones supported replication of HDAg(−) genotype I RNA, and all five genotype III HDAg clones supported replication of HDAg(−) genotype III RNA (Fig. 4). However, none of the HDAg clones was able to support replication of HDAg(−) RNA of the other genotype at more than 5% of the level supported by the prototype HDAg clones of the same genotype (Fig. 4). These results were verified in three separate transfection experiments. Variations in transfection efficiency, as determined by analysis of a cotransfected reporter gene (14), were less than twofold and could not account for the different levels of RNA replication.

Interestingly, the level of RNA replication supported varied considerably among the HDAg clones, particularly for those from genotype I. Isolates I27 and I43 were 5-fold less active and 10-fold more active, respectively, than the HDAg clone from the prototype (Fig. 4). Variations in the level of HDAg expression were less than 25%, as determined by immunoblot analysis (not shown), and do not likely explain the differences in the levels of genotype I RNA replication supported by the genotype I HDAg clones. The low activity of the I27 clone was not likely due to deleterious mutations introduced by misincorporation during PCR amplification prior to cloning, because a second clone from a separate PCR amplification produced the same result. Isolates U02 and I27 are more closely related to the prototype (6.5 and 7.3% divergence, respectively) than is isolate I43 (9% divergence), suggesting that genetic distance alone does not account for the different levels of genotype I RNA replication supported. For the genotype I HDAg clones, the relative levels of activity in supporting RNA replication were similar for genotype I and genotype III RNA, although the levels of genotype III RNA replication supported were much lower than those supported by any of the genotype III HDAg clones. There was much less variation among the level of RNA replication supported by genotype III HDAg clones from different isolates.

Stabilization of HDV RNA by HDAg.

HDAg has been shown to bind specifically to HDV RNA, and this binding is required for HDV RNA replication (19). In transfected cells, the interaction between HDAg and HDV RNA has also been shown to stabilize HDV RNAs that contain large deletions which preserve the ability of the RNA to form the characteristic unbranched rod structure but abolish the replication activity of the RNA (17). To assess whether inefficient interactions between RNAs and HDAg of different genotypes could be responsible for the diminished ability to support replication, Huh-7 cells were cotransfected with pHDAg-S·I or pHDAg-S·III and either pHDV·I(+)ΔApa or pHDV·III(+)ΔBX. The latter two constructs direct the synthesis of antigenomic RNAs containing internal deletions that preserve the ability of the RNA to form the predicted unbranched rod structure but prevent RNA replication even in the presence of HDAg. Six days posttransfection, RNAs were harvested and analyzed by Northern blotting for levels of antigenomic transcripts transcribed from pHDV·I(+)ΔApa or pHDV·III(+)ΔBX. The hybridization probes did not contain HDAg coding sequences and did not detect appreciable RNA in cells transfected with either pHDAg-S·I or pHDAg-S·III alone (Fig. 5).

Both the genotype I and genotype III RNAs accumulated to higher levels in cells cotransfected with either pHDAg-S·I or pHDAg-S·III than in cells cotransfected with a control plasmid that did not encode HDAg (Fig. 5). This accumulation was likely due to stabilization of the RNA, as previously suggested (17), and not due to RNA replication, because genomic RNAs were not detectable (not shown). Genotype III RNA was apparently stabilized equally by both genotype I and III HDAg. However, genotype I RNA was stabilized three- to fourfold more effectively by genotype I HDAg than by genotype III HDAg (Fig. 5). This difference was not as great as that between the abilities of these two HDAg species to support RNA replication of genotype I RNA (Fig. 2). These data suggest that the low level of replication of genotype I RNA supported by genotype III antigen might be due, in part, to weak interactions between this RNA and this antigen, but that additional factors are also likely to be important. Conversely, because genotype I HDAg stabilized genotype III RNA about as well as did genotype III HDAg, it seems unlikely that weak RNA-HDAg interactions can explain the low level of replication of genotype III RNA supported by genotype I HDAg.

Chimeric HDAg constructs.

Comparison of the predicted amino acid sequences of genotype I and III HDAg indicates both conserved and variable regions (Fig. 6A). Studies of genotype I HDAg have identified several functional elements, including a dimerization domain (30, 36), nuclear localization signals (36), and a bipartite RNA-binding domain containing two arginine-rich motifs (19). To attempt to identify regions and possible functional elements responsible for the genotype-specific support of HDV RNA replication, chimeric HDAg constructs containing mixtures of type I and type III sequences were created and analyzed for the ability to support replication of type I and type III RNAs. The HDAg coding region was divided into three approximately equal-size regions, which were exchanged between the genotype I and III clones to create a series of six chimeric HDAg expression constructs (Fig. 6B). The amino-terminal third included the dimerization domain, the middle third included the RNA-binding region, and the C-terminal third included a glycine-proline-rich region of unknown function. Amino acid sequence identities were 60, 70, and 78% for the N-terminal, middle, and C-terminal segments, respectively. Huh-7 cells were cotransfected with these chimeric HDAg expression constructs and the HDAg(−) RNA expression construct pHDV·I(+)Ag(−) or pHDV·III(+)Ag(−), each of which directs expression of HDAg(−) antigenomic RNA. Seven days posttransfection, RNA was harvested and analyzed for the presence of HDV genomic RNA by Northern blot hybridization. RNA levels were quantified by radioanalytic imaging and are presented relative to that observed for cotransfection with the prototype HDAg constructs (Fig. 6).

Analysis of replication supported by the chimeric constructs indicated that multiple regions of type I and type III antigens play important roles in supporting replication. The most dramatic effect was attributed to the N-terminal segment of genotype I HDAg, which was required for replication of genotype I RNA. None of the HDAg constructs containing the genotype III N-terminal segment supported high levels of replication of genotype I RNA; these constructs did, however, support replication of genotype III RNA. Interestingly, the N-terminal region of genotype III HDAg was not as critical for replication of genotype III RNA. Substitution of the genotype III N-terminal region (i.e., I·III·III) reduced replication of genotype III RNA to 56% of the prototype level, compared with no detectable replication of genotype I RNA with the complementary construct III·I·I (Fig. 6). As suggested by the results of the RNA stabilization experiment, substitution of the genotype I RNA-binding region with that from type III led to a substantial decrease in replication of type I RNA (32% of the prototype level for construct I·III·I [Fig. 6]) but did not eliminate activity altogether. The complementary substitution within genotype III HDAg reduced replication of genotype III RNA, but to a lesser extent (67% of the prototype level for construct III·I·III). Overall, replication of genotype I RNA was more sensitive to substitutions within genotype I HDAg compared to the sensitivity of genotype III RNA replication to substitutions within genotype III HDAg. Curiously, the C-terminal regions of both HDAg species not only exhibited no genotype specificity but increased replication upon grafting onto the other HDAg. Compared with the prototypes, construct I·I·III supported fourfold-higher replication of genotype I RNA and construct III·III·I produced twofold-higher levels of genotype III RNA.

Effects of HDAg on replication of wild-type HDV.

The inability of HDAg from genotypes I and III to fully support replication of HDAg(−) RNA from the other genotype raised the question of what effects might occur on replication of wild-type HDV. Huh-7 cells were cotransfected with HDV expression constructs for genotype I and genotype III and expression constructs for the short form of HDAg from both genotypes; for comparison, expression constructs for the long form of HDAg were also cotransfected independently. For wild-type genotype I, cotransfection of an expression construct for HDAg-S(I) had little effect on RNA replication, and as expected (15), coexpression of the long form of HDAg suppressed replication (Fig. 7). Surprisingly, coexpression of the short form of genotype III HDAg strongly inhibited genotype I RNA replication; remarkably, this inhibition was even greater than that due to the long form of genotype I HDAg (Fig. 7). This inhibition was not limited to the prototype genotype I isolate; RNA replication of another genotype I isolate (clone L [5]; GenBank accession no. L22066) was also strongly inhibited by coexpression of genotype III HDAg (4). The strongest suppression of genotype I RNA replication was observed with the long form of genotype III HDAg. Genotype III RNA replication was less affected by the presence of genotype I HDAg. Cotransfection of pHDAg-S·I with pHDV·III(+) resulted in a two- to threefold inhibition of replication, while pHDAg-S·III increased replication of the wild-type genotype III RNA. However, genotype I HDAg-L strongly inhibited replication of genotype III RNA, as did genotype III HDAg-L.

• 1.Beard M R, MacNaughton T B, Gowans E J. Identification and characterization of a hepatitis delta virus RNA transcriptional promoter. J Virol. 1996;70:4986–4995. doi: 10.1128/jvi.70.8.4986-4995.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 2.Bonino F, Hoyer B, Ford E, Shih J W, Purcell R H, Gerin J L. The delta agent: HBsAg particles with delta antigen and RNA in the serum of an HBV carrier. Hepatology. 1981;1:127–131. doi: 10.1002/hep.1840010207. [DOI] [PubMed] [Google Scholar]
• 3.Casey J L. Hepatitis delta virus: genetics and pathogenesis. Clin Lab Med. 1996;16:451–464. [PubMed] [Google Scholar]
• 4.Casey, J. L. 1997. Unpublished results.
• 5.Casey J L, Bergmann K F, Brown T L, Gerin J L. Determinants of RNA editing in hepatitis delta virus. Prog Clin Biol Res. 1993;382:5–11. [PubMed] [Google Scholar]
• 6.Casey J L, Bergmann K F, Brown T L, Gerin J L. Structural requirements for RNA editing in hepatitis delta virus: evidence for a uridine-to-cytidine editing mechanism. Proc Natl Acad Sci USA. 1992;89:7149–7153. doi: 10.1073/pnas.89.15.7149. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 7.Casey J L, Brown T L, Colan E J, Wignall F S, Gerin J L. A genotype of hepatitis D virus that occurs in northern South America. Proc Natl Acad Sci USA. 1993;90:9016–9020. doi: 10.1073/pnas.90.19.9016. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 8.Casey J L, Gerin J L. Hepatitis D virus RNA editing: specific modification of adenosine in the antigenomic RNA. J Virol. 1995;69:7593–7600. doi: 10.1128/jvi.69.12.7593-7600.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 9.Casey J L, Gerin J L. Hepatitis delta virus: RNA editing and genotype variations. In: Dinter-Gottlieb G, editor. The unique hepatitis delta virus. R. G. Austin, Tex: Landes Company; 1995. pp. 111–124. [Google Scholar]
• 10.Casey J L, Niro G A, Engle R E, Vega A, Gomez H, McCarthy M, Watts D M, Hyams K C, Gerin J L. Hepatitis B virus (HBV)/hepatitis D virus (HDV) coinfection in outbreaks of acute hepatitis in the Peruvian Amazon Basin: the roles of HDV genotype III and HBV genotype F. J Infect Dis. 1996;174:920–926. doi: 10.1093/infdis/174.5.920. [DOI] [PubMed] [Google Scholar]
• 11.Chang M F, Sun C Y, Chen C J, Chang S C. Functional motifs of delta antigen essential for RNA binding and replication of hepatitis delta virus. J Virol. 1993;67:2529–2536. doi: 10.1128/jvi.67.5.2529-2536.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 12.Chao M, Hsieh S Y, Taylor J. The antigen of hepatitis delta virus: examination of in vitro RNA-binding specificity. J Virol. 1991;65:4057–4062. doi: 10.1128/jvi.65.8.4057-4062.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 13.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]
• 14.Cullen B R, Malim M H. Secreted placental alkaline phosphatase as a eukaryotic reporter gene. Methods Enzymol. 1992;216:362–368. doi: 10.1016/0076-6879(92)16033-g. [DOI] [PubMed] [Google Scholar]
• 15.Kuo M Y, 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]
• 16.Kuo M Y, Sharmeen L, Dinter G G, Taylor J. Characterization of self-cleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J Virol. 1988;62:4439–4444. doi: 10.1128/jvi.62.12.4439-4444.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 17.Lazinski D W, Taylor J M. Expression of hepatitis delta virus RNA deletions: cis and trans requirements for self-cleavage, ligation, and RNA packaging. J Virol. 1994;68:2879–2888. doi: 10.1128/jvi.68.5.2879-2888.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 18.Lazinski D W, Taylor J M. Relating structure to function in the hepatitis delta virus antigen. J Virol. 1993;67:2672–2680. doi: 10.1128/jvi.67.5.2672-2680.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 19.Lee C Z, Lin J H, Chao M, McKnight K, Lai M M. RNA-binding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication. J Virol. 1993;67:2221–2227. doi: 10.1128/jvi.67.4.2221-2227.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 20.Lin J H, Chang M F, Baker S C, Govindarajan S, Lai M M. Characterization of hepatitis delta antigen: specific binding to hepatitis delta virus RNA. J Virol. 1990;64:4051–4058. doi: 10.1128/jvi.64.9.4051-4058.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 21.Luo G X, 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]
• 22.MacNaughton T B, Gowans E J, Jilbert A R, Burrell C J. Hepatitis delta virus RNA, protein synthesis and associated cytotoxicity in a stably transfected cell line. Virology. 1990;177:692–698. doi: 10.1016/0042-6822(90)90535-y. [DOI] [PubMed] [Google Scholar]
• 23.MacNaughton T B, Wang Y J, Lai M M. Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites. J Virol. 1993;67:2228–2234. doi: 10.1128/jvi.67.4.2228-2234.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 24.Makino S, Chang M F, Shieh C K, Kamahora T, Vannier D M, Govindarajan S, Lai M M. Molecular cloning and sequencing of a human hepatitis delta (delta) virus RNA. Nature. 1987;329:343–346. doi: 10.1038/329343a0. [DOI] [PubMed] [Google Scholar]
• 25.Maynard J E, Hadler S C, Fields H A. Delta hepatitis in the Americas: an overview. Prog Clin Biol Res. 1987;234:493–505. [PubMed] [Google Scholar]
• 26.Niro G A, Smedile A, Andriulli A, Rizzetto M, Gerin J L, Casey J L. Predominance of hepatitis delta virus genotype I among chronically infected Italian patients. Hepatology. 1997;25:728–734. doi: 10.1002/hep.510250339. [DOI] [PubMed] [Google Scholar]
• 27.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]
• 28.Polson, A. G., H. L. Ley III, B. L. Bass, and J. L. Casey. Hepatitis delta virus RNA editing is highly specific for the amber/W site, and is suppressed by hepatitis delta antigen. Mol. Cell. Biol., in press. [DOI] [PMC free article] [PubMed]
• 29.Rizzetto M. The delta agent. Hepatology. 1983;3:729–737. doi: 10.1002/hep.1840030518. [DOI] [PubMed] [Google Scholar]
• 30.Rozzelle J J, Wang J G, Wagner D S, Erickson B W, Lemon S M. Self-association of a synthetic peptide from the N terminus of the hepatitis delta virus protein into an immunoreactive alpha-helical multimer. Proc Natl Acad Sci USA. 1995;92:382–386. doi: 10.1073/pnas.92.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 31.Shakil A O, Hadziyannis S, Hoofnagle J H, DiBisceglie A M, Gerin J L, Casey J L. Geographic distribution and genetic variability of hepatitis delta virus genotype I. Virology. 1997;234:160–167. doi: 10.1006/viro.1997.8644. [DOI] [PubMed] [Google Scholar]
• 32.Wang K S, Choo Q L, Weiner A J, Ou J H, Najarian R C, Thayer R M, Mullenbach G T, Denniston K J, Gerin J L, Houghton M. Structure, sequence and expression of the hepatitis delta viral genome. Nature. 1986;323:508–514. doi: 10.1038/323508a0. [DOI] [PubMed] [Google Scholar]
• 33.Wu J C, Chen C M, Sheen I J, Lee S D, Tzeng H M, Choo K B. Evidence of transmission of hepatitis D virus to spouses from sequence analysis of the viral genome. Hepatology. 1995;22:1656–1660. [PubMed] [Google Scholar]
• 34.Wu J C, Choo K B, Chen C M, Chen T Z, Huo T I, Lee S D. Genotyping of hepatitis D virus by restriction-fragment length polymorphism and relation to outcome of hepatitis D. Lancet. 1995;346:939–941. doi: 10.1016/s0140-6736(95)91558-3. [DOI] [PubMed] [Google Scholar]
• 35.Wu T T, Netter H J, Lazinski D W, Taylor J M. Effects of nucleotide changes on the ability of hepatitis delta virus to transcribe, process, and accumulate unit-length, circular RNA. J Virol. 1997;71:5408–5414. doi: 10.1128/jvi.71.7.5408-5414.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 36.Xia Y P, Yeh C T, Ou J H, Lai M M. Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex. J Virol. 1992;66:914–921. doi: 10.1128/jvi.66.2.914-921.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 37.Zierman R, Ganem D. Homologous and heterologous complementation of HBV and WHV capsid and polymerase functions in RNA encapsidation. Virology. 1996;219:350–356. doi: 10.1006/viro.1996.0260. [DOI] [PubMed] [Google Scholar]