INTRODUCTION

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Human hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV) and requires HBV envelope proteins for packaging, secretion and infection (reviewed in reference 24). HDV particles contain a ribonucleoprotein core composed of the circular 1.7-kb RNA genome and multiple copies of the only HDV-encoded protein, delta antigen (Ag) (23). There are two forms of the Ag. The first is a 195-amino-acid (aa) species, known as the small delta protein (Ag-S), which is essential for replication of the RNA genome (11). The second is 19 aa longer at its C terminus (Ag-L) and arises as a consequence of a posttranscriptional RNA editing event (17). This Ag-L is a dominant-negative inhibitor of genome replication (4), but it is essential for particle assembly (2).

These two Ag species share 195 aa of primary sequence and thus have some common features. These include (i) a coiled-coil domain located at aa 12 to 60, which facilitates protein-protein interactions (21); (ii) a bipartite nuclear localization signal, between aa 68 and 88 (28); and (iii) a bipartite RNA-binding domain, consisting of aa 97 to 107 and 136 to 146 (3).

The coiled-coil domain has been shown to be required for a number of the functions of both small and large delta antigens. (i) Mutations that destroy or alter this dimerization domain reduce or eliminate the ability of Ag-S to function as a trans activator of HDV replication (15). (ii) These same mutations when presented on Ag-L prevent the antigen from inhibiting HDV RNA replication and also block the ability to coassemble Ag-S into viral particles (15).

Biophysical studies by Rozzelle et al. showed that the synthetic peptide that corresponds to aa 12 to 60 of Ag was  helical in structure and was sufficient for dimerization and even multimerization (21). Recently, Zuccola et al. solved the crystal structure for this peptide and confirmed that it contains a long N-terminal and a short C-terminal -helical segment separated at aa 49 by proline (29). To form a dimer, the long helices of each of two monomers wrap around each other, forming an antiparallel coiled-coil (29). In addition, each dimer associates with three other dimers, forming what has been called a "doughnut-like octamer" (29). In support of this model, they used recombinant Ag-S, and chemical cross-linking followed by mass spectrometry, to show that octamers could form in vitro. Finally, based on the alignment of several different Ag sequences of this region, they noted that certain amino acids were both conserved and predicted to be important for dimerization and/or multimerization. Based on their study, we have selected nine such critical amino acids and evaluated their importance in the context of both full-length Ag-S and Ag-L. Each of these single amino acids was changed to alanine in order to avoid altering the secondary structure of the protein while disrupting the intermolecular associations. This series of Ag mutants was then evaluated by in vivo assays to determine whether they still (i) supported HDV replication, (ii) acted as dominant-negative inhibitors, (iii) had the ability to coassemble into particles, (iv) made complexes with an affinity-tagged form of Ag-S, and (v) were able to increase the accumulation of processed HDV RNA species. We consider that the results of (i), (ii), and to some extent (iv) are supportive of the predictions based upon the crystal structure, while those of (iii) reveal the diversity and complexity of Ag interactions in vivo and those of (v) indicate separate functions that are independent of such interactions.

	MATERIALS AND METHODS

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Plasmids. Most constructs were based on the vector pSVL (Pharmacia). pTW148 expresses 1.2× unit-length HDV cDNA and has a frameshift in the Ag-S open reading frame; thus, HDV replication can be achieved only by supplying Ag-S in trans (26). pDL444 and pDL445 were used to express the wild-type Ag-S and Ag-L, respectively (15). Plasmids pDL448 and pDL449 express Ag-S(19-31) and Ag-L(19-31), respectively, that have a deletion of 13 aa from the coiled-coil region (15). pDL480 expresses antigenomic HDV RNA with a deletion spanning the region between nucleotides 215 and 1380 (14). The following constructs were based on the vector pcDNA3 (Invitrogen). Constructs pPB102 and pPB105 express Ag-S(19-31) and Ag-S(FLAG), respectively. The latter species is a form of Ag-S with the FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) at the N terminus. Plasmids pTW198 and pTW199 express wild-type Ag-S and Ag-L, respectively.

Construction of plasmids expressing Ag-S and Ag-L with amino acid substitutions in the coiled-coil region. To construct mutated plasmids, primers containing the sequences corresponding to the amino acid substitution were used to amplify that part of the Ag corresponding to aa 9 to 58, numbered according to the sequence of Kuo et al. (11). The PCR fragments were then inserted between the EcoRI and SacII sites of pDL444 or pDL445 to get the mutated forms of Ag-S and Ag-L, respectively. All mutants were confirmed by nucleotide sequencing.

DNA transfections. For all experiments, transfection of the human hepatoma cell line Huh7 (20) was performed using FuGENE 6 (Roche) following the manufacturer's instructions.

RNA analysis. Total cell RNA was isolated with Tri Reagent (Molecular Research Center), glyoxalated, and analyzed electrophoretically on gels of 1.7% agarose as previously described (15). RNA was transferred to a nylon membrane (Zeta-probe GT; Bio-Rad) and immobilized by UV cross-linking. Hybridization was performed with ³²P-labeled RNA probe specific for antigenomic HDV RNA. Levels of antigenomic RNA were detected and quantitated using a Bio-Imaging system (Fuji).

Protein analysis. Samples were resuspended in Laemmli buffer (13) and analyzed by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrotransferred to a nitrocellulose membrane, and Ag was detected by using a rabbit polyclonal antiserum and by incubation with ¹²⁵I-labeled protein A (Du Pont). Quantitation was via a Bio-Imaging system.

Coassembly of Ag into virus-like particles. Huh7 cells were transfected with plasmid pSV45H (from Don Ganem) for the expression of all three HBV envelope proteins, along with the appropriate plasmids expressing wild-type or mutant forms of Ag-S and Ag-L. Tissue culture medium was collected at 4, 6, and 8 days after transfection and clarified by low-speed centrifugation for 10 min. Then virus-like particles were collected through a 20% sucrose cushion containing 100 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA by centrifugation for 18 h at 23,000 rpm in a Beckman SW28 rotor at 4°C. The pellet was analyzed for delta proteins as described above.

Immunoaffinity purification of Ag complexes. Huh7 cells were cotransfected with (i) pPB105, which expresses Ag-S(FLAG), and (ii) pPB102, which expresses Ag-S(19-31), with a deletion in the coil-coiled domain (15), in combination with (iii) wild-type or mutant forms of Ag-S or Ag-L, as indicated in Fig. 6. At 5 days after transfection, we used a modification of the method described by Chiang et al. (5) to purify complexes containing the FLAG epitope. About 10⁶ cells were resuspended in 1 ml of 100 mM KCl in BC buffer (20 mM Tris-HCl [pH 7.9], 20% glycerol, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM 2-mercaptoethanol) plus 0.1% NP-40 and then lysed by sonication (five times for 30 s each, at microtip setting) on ice with a 550 Sonic Dismembrator (Fisher Scientific). After clarification for 5 min at 1,500 rpm, one-third of the supernatant was incubated with 50 µl of anti-FLAG M2-agarose (Sigma), which contains the anti-FLAG M2 monoclonal antibody conjugated onto the agarose, at 4°C for 16 h with rotation. After being washed four times in BC300 (BC buffer including 300 mM KCl) plus 0.1% NP-40, once in BC100 (BC buffer containing 100 mM KCl) plus 0.1% NP-40 and once in BC buffer, the bound protein was eluted directly with Laemmli buffer. Aliquots of both total protein and bound protein were subjected by SDS-PAGE and immunoblot analysis as described above.

Accumulation of processed genomic RNA circles in the presence of mutant and wild-type forms of Ag-S. Huh7 cells were cotransfected with plasmid pDL480, to express multimeric HDV RNA, along with a construct expressing one of the Ag-S mutants. Cells were harvested at days 2 and 4 after transfection, and total RNA was assayed by SDS-PAGE and Northern analysis as described above.

	RESULTS

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Negative controls. For the following in vivo assays of Ag interactions, we used as negative controls specific deleted forms of Ag-S and Ag-L that were unable to dimerize. These mutants, which have a deletion of aa acids 19 to 31, were characterized in the earlier study of Lazinski and Taylor (15). These previous studies showed that (i) both truncated proteins could be stably expressed in Huh7 cells, (ii) Ag-S(19-31) did not support replication, (iii) Ag-L(19-31) could not coassemble wild-type Ag-S, and (iv) Ag-L(19-31) was not a dominant-negative inhibitor. Further studies show that Ag-S(19-31) cannot be coassembled by wild-type Ag-L (data not shown).

Design of Ag mutants. The design was based on a two-step process, essentially as used by Zuccola et al. (29). First, they considered sequence conservation. The sequences of 10 independent HDV isolates were aligned through the region of the delta antigen, and many conserved amino acids were detected. Second, they used the determined three-dimensional structure to predict which of these conserved amino acids might be needed to maintain dimerization and multimerization. Figure 1A shows two HDV sequences through this region: first, the sequence used for the crystal structure (18); and second, the sequence used for our subsequent in vivo studies (12). Also indicated are the conserved amino acids and the nine individual ones chosen for mutagenesis to alanine. The intent of these substitutions was to maintain the primary and secondary structures while testing the predictions regarding tertiary structure. As summarized in Fig. 1B, we assigned a hierarchy of effects for each of these nine mutations, in terms of their potential role in dimerization and multimerization of Ag.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Predictions and observed consequences of mutating single amino acids of Ag that are both conserved and possibly critical for protein dimerization and/or multimerization. (A) Sequence at aa 12 to 60 of the coiled-coil domain of Ag of Makino et al. (18). Underlined are the amino acids predicted, using the data of Zuccola et al. (29), to be critical for dimerization and/or multimerization. Also shown is the corresponding sequence of Ag from Kuo et al. (12), which is subsequently used as the backbone for the construction of mutant proteins. (B) Individual amino acids that were changed to alanine to test the prediction that they were involved in either dimerization or multimerization. Also shown is a predicted ranking of their importance for multimerization. (C) Mutant proteins organized according to three subsequently observed in vivo distinguishing characteristics (Fig. 3, 4B, and 6) and summarized in Tables 2 and 4.

Expression and stability of Ag mutants. With this strategy in mind, the next question was to determine whether the mutant proteins were stably expressed. Vectors expressing the nine mutated forms of Ag-S and the corresponding nine forms of Ag-L were cotransfected into Huh7 cells, and the levels of accumulation of the individual proteins assayed at 4 days by immunoblotting to detect delta protein. As an internal control, each cotransfection included a plasmid that expressed Ag-S(19-31). Typical immunoblot results are shown in Fig. 2. From quantitation as presented in Table 1, it can be seen that for most cases somewhat less of the mutant protein accumulated. Furthermore, in most cases the levels of the large mutant were less than the corresponding mutant in small. In order to avoid artifacts due to such differences, in all subsequent cotransfection experiments we increased the input of plasmid DNA so that more equal amounts of protein might be accumulated.

View larger version (68K): [in this window] [in a new window]   FIG. 2.   Intracellular expression and accumulation of mutated forms of Ag-S and Ag-L. Huh7 cells were cotransfected with combinations of two plasmids that expressed different forms of Ag. Four days later, the proteins were assayed by electrophoresis and immunoblotting to detect Ag species. In each cotransfection, as an internal standard, we used a third plasmid which expressed Ag-S(19-31). Relative to this standard, the cells received threefold-greater amounts of the two plasmids that express forms of Ag-S and Ag-L. As indicated for lanes 1 to 10, these two forms were either wild type (wt) or one of the nine mutants.

View this table: [in this window] [in a new window]   TABLE 1.   Intracellular accumulation of mutated forms of Ag-S and Ag-L

trans support of genome replication by Ag-S mutants. The most demanding test for a mutant Ag-S is whether it can support the replication of a mutated HDV genome that is unable to express wild-type Ag-S. This test was applied to the nine mutants of Ag-S. As shown in Fig. 3 and summarized in Table 2, only three of the nine mutants supported the synthesis and accumulation of HDV genomes. Two of these, L17 and V51, were equivalent to wild type. For reasons we do not understand, the third mutant, V21, reproducibly supported 10 to 20% accumulation of antigenomic RNA relative to wild type.

View larger version (72K): [in this window] [in a new window]   FIG. 3.   Northern analysis to detect HDV replication supported in trans by Ag-S mutants. Huh7 cells were cotransfected with pTW148, which expresses 1.2× unit-length cDNA and has a frameshift in the Ag-S open reading frame, along with constructs expressing different forms of Ag-S. At 4 days after transfection, total cellular RNA was isolated and analyzed as described in Materials and Methods to detect antigenomic HDV RNA. Lanes 2 to 13 represent cells transfected with pTW148 alone (lane 2) or with constructs expressing wild-type Ag-S (lane 3), the indicated nine mutant constructs (lanes 4 to 12), or a construct expressing Ag-S(19-31) (lane 13). Lane M shows 5'-labeled single-stranded DNA size markers. Lane 1 is 2 ng of 1.7-kb HDV cDNA.

View this table: [in this window] [in a new window]   TABLE 2.   Distinguishing properties of mutated forms of Ag-S and Ag-L

Dominant-negative inhibition of replication by Ag-S and Ag-L mutants. Previous studies have shown that like Ag-L, most altered forms of Ag-S lose the ability to support replication. Furthermore, relative to Ag-S, even low amounts can act as potent dominant-negative inhibitors (4, 15). When tested in the presence of wild-type Ag-S, none of the nine mutants were able to act as dominant-negative inhibitors, even when present in amounts approximately comparable to that of wild-type Ag-S (Fig. 4A).

View larger version (117K): [in this window] [in a new window]   FIG. 4.   Northern analysis to detect dominant-negative inhibition of HDV replication by Ag mutants. (A) Huh7 cells were cotransfected with plasmid pTW148, plasmid pDL444 (which expresses Ag-S), and a construct expressing one of the Ag-S mutants. At 4 days after transfection, total RNA was analyzed and detected as for Fig. 3. Lane M, 5'-labeled single-stranded DNA size markers; lane 1, 2 ng of 1.7-kb HDV cDNA; lane 2, cells transfected with pTW148 alone; lane 3, cells cotransfected with pTW148 and the construct expressing wild-type Ag-S; lanes 4 to 6, triple cotransfections with pTW148, the construct expressing wild-type Ag-S, and a construct expressing Ag-L (lane 4), Ag-S(19-31) (lane 5), or Ag-L(19-31) (lane 6); lanes 7 to 15, alanine mutants as indicated. (B) Experiments essentially as for panel A except that the mutations were expressed on Ag-L rather than Ag-S. Lanes 1 to 4, as in panel A; lanes 5 to 13, alanine mutants as indicated.

Coassembly of Ag-S and Ag-L mutants. Our next test was whether mutants expressed on Ag-S would, in the presence of the envelope proteins of HBV, be coassembled by wild-type Ag-L and released as virus-like particles. We observed that each of the nine mutants expressed on Ag-S could be coassembled (Fig. 5). Quantitation of these data showed that the efficiencies were within a factor of 3 of that for wild-type Ag-S (Table 3). We do not consider these as significant differences. Furthermore, the molar ratio of mutant Ag-S to wild-type Ag-L in the released particles was less than 1 (Table 3). This was also true when the amount of mutant Ag-S expressed within the cell was increased fourfold (data not shown; see also reference 22). One interpretation of these data would be that coassembly efficiency reflects not multimerization ability but only dimerization, and that this dimerization was not affected by the nine mutations.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Immunoblot analysis to detect coassembly of Ag-S mutants into virus-like particles. Cotransfection of Huh7 cells was carried out using pSV45H (to express the envelope proteins of HBV) along with pDL445 (to express wild-type Ag-L) and constructs expressing mutants of Ag-S. As described in Materials and Methods, virus-like particles released by the transfected cells were subsequently collected and assayed by electrophoresis and immunoblotting to detect Ag species. Lane 1, particles from cells expressing wild-type Ag-S in the absence of Ag-L; lane 2, both Ag-S and Ag-L; lane 3, Ag-S(19-31) and Ag-L; lane 4, Ag-S and Ag-L(19-31); lanes 5 to 12, wild-type Ag-L and the indicated mutants in Ag-S. The mobilities of Ag-L and Ag-S are indicated at the right.

Immunoaffinity purification of complexes formed between Ag-S(FLAG) and Ag-S or Ag-L mutants. Another way to assay the interactions between Ag species is via immunoaffinity. We expressed within transfected cells both Ag-S(FLAG) and each of the mutants of either Ag-S or Ag-L. As shown in Fig. 6, we were able to demonstrate the formation of these complexes. As an indicator of the specificity of these complexes, we also expressed in these cells the protein Ag-S(19-31), which is known to be unable to make dimers (15). As shown in Fig. 6, this protein was present in the total sample but virtually absent from the fraction of bound protein.

View larger version (93K): [in this window] [in a new window]   FIG. 6.   Immunoblot analysis to detect complexes formed between Ag-S(FLAG) and mutant forms of Ag-S and Ag-L. (A) Huh7 cells were cotransfected with plasmids pPB102 [expressing Ag-S(19-31)] and pPB105 [expressing Ag-S(FLAG)], along with pTW198 (expressing wild-type Ag-S; lane 1) or plasmids expressing the indicated mutants of Ag-S (lanes 2 to 10). At 5 days after transfection, we isolated that protein which bound via the FLAG epitope to an immunoaffinity column. Samples of total and bound protein were assayed by gel electrophoresis and immunoblotting to detect all forms of Ag. The three electrophoretic forms of Ag-S are indicated at the right. Note that Ag-S(19-31) acts as a negative control which fails to bind to Ag-S(FLAG). (B) Experiments essentially as for panel A except that the mutations were expressed on Ag-L rather than Ag-S. That is, we used pTW199 (expressing wild-type Ag-L; lane 1) or plasmids expressing the indicated mutants of Ag-L (lanes 2 to 10).

View this table: [in this window] [in a new window]   TABLE 4.   Effect of mutations on ability to form complexes with Ag-S(FLAG)

Ability of Ag-S mutants to enhance the accumulation of processed HDV RNA circles. In conclusion, these assays were carried out as a negative control, that is, to determine whether the mutagenesis of Ag-S interfered with a biological property that does not depend on dimerization and multimerization.

View this table: [in this window] [in a new window]   TABLE 5.   Accumulation of processed HDV RNA in the presence of mutated forms of Ag-S

	DISCUSSION

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The aim of this study was to evaluate predictions for the dimerization and multimerization of Ag. These predictions were based on intermolecular interactions detected in the crystal structure of the domain of aa 12 to 60 on the Ag (29). We considered mutagenesis to alanine at nine sites that were (i) conserved between different isolates of HDV and (ii) predicted to be important for dimerization and/or multimerization (Fig. 1A). We tested these mutants in the context of both full-length Ag-S and Ag-L, via biological assays, using transfected cells.

The top six mutants of Ag-S, in terms of their predicted importance for dimerization/multimerization (Fig. 1B), were the ones that could no longer support replication (Fig. 3). To this extent these results followed the predictions. We also note that four of these mutations were of hydrophobic amino acids predicted to be important in the interactions that form the dimer (Fig. 1B).

Another consistent correlation was obtained when the nine mutants, as expressed on Ag-L, were tested as dominant-negative inhibitors of replication, as supported by wild-type Ag-S (Fig. 4B). Seven mutants were inhibitors. Three of these (L17, V21, and L51) were the same as those that when expressed on Ag-S were able to support replication. However, it was unexpected that the four additional mutants (R13, I54, R24, and I57) were dominant-negative inhibitors. One explanation might be that, as for mutants in the nucleocapsid of tomato spotted wilt virus (25), the heterotypic interaction between the mutant and the wild type (as in the dominant-negative inhibition assay) could be stronger than the homotypic interaction between two mutants (as in the support of replication assay).

Results of the dominant-negative inhibition assays also showed that mutations of W20 and W50 were the most potent of the nine amino acid mutations tested (Fig. 1C and 3B). This ranking also agrees with the predictions based on the crystal structure (Fig. 1B). It is important to note that it was originally suggested from examination of the structure that dimerization was stabilized by the hydrophobic interactions of I16, L17, and W20 from one monomer to W50, L51, and I54 on a second monomer, and vice versa (29). Therefore, the present data not only support this interpretation but also indicate that the two tryptophans, W20 and W50, are critical residues.

In contrast to the above good correlations between prediction and experiment, there were also less favorable situations. First, we found that none of the mutants, when expressed on Ag-S, was a dominant-negative inhibitor (Fig. 4A; Table 2). In contrast, when expressed on Ag-L, we did observe inhibition for L17, V21, and L51 (Fig. 4B; Table 2), and this was as expected, since these, as expressed on Ag-S, were able to support genome replication (Fig. 3; Table 2). We speculate that for these three mutations, as expressed on Ag-S, there was insufficient structural difference from wild-type Ag-S to produce any inhibition.

A second discrepancy arose when we tested the mutants in three different assays of protein coassembly into virus-like particles in the presence of the envelope proteins of HBV (Table 3). The first assay was for the coassembly by wild-type Ag-L of mutants expressed on Ag-S. Each mutant was coassembled as efficiently as wild-type Ag-S. This was unexpected since six of these mutants could neither support replication (Fig. 3) nor act as dominant-negative inhibitors (Fig. 4A). Since we consider that the coassembly assay detects dimerization but not multimerization, it may be unable to distinguish functional from nonfunctional dimers. Therefore, one interpretation is that the six mutants were able to make only nonfunctional dimers. In addition, it must be remembered that the coassembly assay involves more than just interactions between Ag species; there are also essential interactions with the envelope proteins of the helper virus in order to achieve assembly into the virus-like particles (9). Moreover, recent studies show that Ag-L is significantly more hydrophobic than Ag-S, and that some of the Ag-L species have to undergo isoprenylation as a prerequisite for interaction with the envelope proteins of the helper virus (7, 19). These factors might contribute to the diversity of results obtained for the coassembly by mutant Ag-L, of wild-type Ag-S, or of mutant Ag-S (Table 3).

Our interpretation of the subset of the present in vivo studies specifically concerned with the support of genome replication and the dominant-negative inhibition is that the essential interactions between the Ag species are not random but ordered and consistent with the predictions based on the crystal structure for the 12-60 peptide. Independent of these studies, from application of an immunoaffinity strategy, we obtained direct in vivo evidence for the ability of the mutated forms of Ag-S and Ag-L to make complexes with a form of Ag-S containing at its N terminus a FLAG epitope. These data show that all the mutated forms were able to bind to the tagged protein (Fig. 6; Table 5) and thus were at least able to form dimers. (Incidentally, this agrees with the results obtained by coassembly [Fig. 5].) Furthermore, analysis of the quantitation of these data (Table 4) reveals that the predicted hierarchy (Fig. 1B) is close to the directly observed values (Fig. 1C). This immunoaffinity strategy also allows us to begin to address the question of in vivo complexes larger than dimers. The wild-type forms of Ag-S and Ag-L made complexes with the tagged Ag-S that on average were at least greater than tetramers (Fig. 6, lane 1; Table 4, footnotes). Further studies using the same strategy, indicate complexes of at least decamers (data not shown). It will be necessary to determine to what extent these multimers are ordered. At this stage there is no in vivo evidence to suggest that Ag can behave like the core protein of the helper virus, HBV, and form ordered multimeric capsid structures (27). In contrast, we have already seen from electrophoretic analyses under nondenaturing conditions that within transfected cells Ag can become associated with high-molecular-weight complexes (6). Maybe Ag, like other proteins when expressed to high levels in bacterial or mammalian cells, can produce multimeric aggregates that are disordered (10). Furthermore, there may be additional components in the Ag complexes and some of the Ag interactions may be indirect. After all, we know that Ag species have to interact with HDV RNAs and, during assembly, interact with the envelope proteins of the helper virus. In addition, the Ag can also interact with host proteins; others have described candidate host protein interactors, namely, nucleolin (16) and delta-interacting protein A (1).