We have found that a C-terminally truncated form of HDAg, HDAg-160, expressed in E. coli
and purified under native conditions, exhibits specific binding to unbranched rod segments of HDV RNA, as indicated by the formation of a discrete complex in an electrophoretic mobility shift assay. The use of bacterially expressed HDAg-160 in binding assays is a significant improvement over bacterially expressed full-length protein, which produced heterogeneously migrating complexes, bound to non-HDV RNAs, and formed aggregates (Fig. ). Similar to our results with HDAg-195, Chao et al. observed that nondenatured full-length HDAg, expressed as a fusion protein in bacteria, exhibited no clear specificity for HDV RNA (7
). Previous studies that have shown specific binding of full-length HDAg to the HDV unbranched rod structure used HDAg expressed in bacteria as a fusion protein that was denatured and renatured prior to RNA binding (7
). These previous analyses were limited by potential effects of large fusion protein partners on HDAg structure, the sensitivity of binding to renaturation conditions, and the inability to evaluate the extent or quality of the renatured protein. The nonspecific RNA binding activity of native, full-length, bacterially expressed HDAg is most likely an effect of the bacterial expression. Secondary structure prediction analyses performed on the segment of the protein removed to create HDAg-160 indicated no defined structures. Possibly, in bacterially expressed HDAg-195, this proline/glycine-rich 35-aa region contributes to structural variability and/or aggregate formation that contributes to nonspecific RNA binding activity. Removal of this region is unlikely to directly affect RNA binding; it is not among those regions of the protein implicated in RNA binding by other studies that have used either site-directed mutagenesis or deletional analysis (18
). An HDAg protein created by a 50 aa C-terminal truncation (HDAg-145) yielded binding results similar to those of HDAg-160, except that the discrete complex formed migrated slightly faster (not shown).
Binding of HDAg-160 was specific for the HDV RNA unbranched rod structure; fully double-stranded RNAs and single-stranded RNAs incapable of forming this structure, even if derived from HDV, were not bound (data not shown). Our deletional analysis of the RNA requirements for binding indicated that binding is not determined by just one or two unique local structural features in the RNA; rather, complex formation in vitro required that the unbranched rod be greater than 298 nt (Fig. ). We also observed a size-dependent relationship in cells via analysis of RNA stabilization by HDAg that verified the biological relevance of the complexes formed by HDAg-160 (Fig. ). Previous studies that were able to demonstrate specific binding of HDAg to HDV RNA in vitro indicated that binding required the HDV unbranched rod structure (7
), but the RNAs analyzed in those studies were larger than those analyzed here and the dependence of binding on the size of the RNA was not investigated. The length requirement that we have observed for HDV RNA binding to HDAg could be significant for the virus in that it may provide a means by which HDAg discriminates between binding to its cognate RNA rather than shorter similarly structured RNAs (i.e., extended hairpins) in infected cells.
Cells infected by HDV contain both genomic and antigenomic RNAs, as well as two isoforms of HDAg that differ by 19 or 20 aa at the C terminus. Our deletional analysis was conducted using a segment of the antigenomic RNA, but it seems probable that similar size-dependent binding will be obtained for the genome. Chang et al. observed that segments of genomic HDV RNA 311 nt or longer were efficiently packaged into virus-like particles from cells expressing the long isoform of HDAg, while a 258-nt segment was not (5
). The nature of the difference in packaging was not further explored at the time, but could be explained by our in vitro binding results: the failure of the shorter 258-nt RNA to be efficiently packaged is likely due to its inability to be bound by HDAg. It is not yet clear whether RNPs with varying HDAg isoform compositions differ structurally. HDAg-160 contains all of the known RNA-binding regions within both isoforms of HDAg.
The results presented in Fig. indicate that HDAg-160 bound several different unbranched RNA segments derived from the HDV antigenome and genome. Previous binding studies, which used denatured bacterially expressed HDAg, were qualitative, and could not distinguish binding characteristics of different RNAs. With natively expressed HDAg-160, we found that binding to different unbranched RNAs was not identical, in terms of either affinity or the maximum levels of binding (Fig. ). Variations in affinity could be due to subtle structural differences in the RNAs that modulate binding activity. It is not clear why some RNAs failed to be completely bound during the assay. Perhaps, structural heterogeneity in the RNA that affects binding, without affecting gel mobility, is responsible. Another possibility is that binding requires conformational changes—in the RNA, the protein, or both—and the time required for these changes varies among the different RNA-protein interactions. Consistent with these models, we have observed that binding is strongly dependent on both the temperature and time of incubation (D. A. Defenbaugh and J. L. Casey, unpublished data). Regardless of the mechanistic explanation, the variable binding to different segments of the RNA suggests that binding in the context of the full-length unbranched rod RNA might occur preferentially at certain sites. In cells, such preferential binding could lead to an ordered assembly of HDAg on the RNA that is likely to play a role in RNA transcription of both the genome and antigenome as well as packaging of the genome.
HDAg is known to form dimers, and several reports have indicated that the protein assembles into higher-order structures, possibly octamers (9
). The results of our electrophoretic mobility shift assays are most consistent with the formation of an HDV RNA-HDAg complex containing a large HDAg multimer, rather than an RNP consisting of either an HDAg monomer or dimer. The mobility of the complex formed by HDAg-160 and 395L, a 395-nt RNA derived from the left end of the antigenomic unbranched rod, was approximately half that of unbound 395L RNA (Fig. ). The molecular mass of an HDAg-160 monomer is about 19 kDa; complexes formed with 395L and either a single monomer or dimer of HDAg-160 would have molecular masses 15% (149 kDa) to 30% (168 kDa) greater than that of the RNA alone, respectively. On the other hand, a complex involving 395L RNA and an HDAg-160 octamer (152 kDa), for example, would produce an RNA-protein complex with a mass more than twice the size of the RNA alone (282 kDa versus 130 kDa), consistent with the large shift in mobility observed. Of course, in addition to effects of the mass of the protein on RNP mobility, conformational changes in the RNA that might occur upon protein binding could also alter the mobility in a native gel. However, two additional findings support the conclusion that a larger mass of protein is bound. Comparison of the mobility of RNA-protein complexes formed by HDAg-195 and HDAg-160 (Fig. ) and HDAg-145 (not shown) on a given RNA demonstrates readily detectable changes in mobility, consistent with a large contribution of the protein mass to the mobility of the complex. Furthermore, the resistance of RNA-protein complexes to nuclease digestion (Fig. ) is more consistent with binding of a large amount of protein to the RNA. Because no RNPs of intermediate mobility were observed as the level of HDAg-160 increased (Fig. ), the protein either assembles on the RNA with a high degree of cooperativity or binds as a preassembled protein complex. While others have suggested that HDAg exists as an octameric structure in cells and in vitro, even in the absence of HDV RNA (25
), our results presented here cannot distinguish between these two possibilities, nor can we determine the number of HDAg subunits involved.
The complex formed between HDAg-160 and 395L RNA was resistant to digestion with micrococcal nuclease. Interestingly, high concentrations of nuclease reduced the size of the complex formed by 395L RNA, which is greater than the minimum-length RNA required for binding, to the same as that formed by 311L RNA, which is the smallest RNA bound by HDAg-160 (Fig. ). These results indicate that (i) most of the bound RNA is tightly associated with HDAg-160, and (ii) the 395-nt RNA does not bind more protein than the 311-nt RNA. This result, together with the minimum length requirement for binding and our conclusion that HDAg binds as a large multimer, suggests that binding of the multimeric unit occurs in discrete amounts. Thus, given that the minimum length for binding of HDAg-160 to HDV unbranched rod RNA is ~300 nt, we expect five HDAg-160 multimers are able to bind per full-length HDV RNA. If the multimeric unit is an octamer (as suggested by Zuccola et al. ), then we would expect the RNA/protein ratio to be 1:40. This number is near the low end of the range of RNA/protein ratios determined by previous analyses of HDV RNA-protein complexes in cells and in virions (12
). In future studies, determination of the exact size of the HDAg multimer involved in binding, as well as the number of such complexes bound to progressively larger RNAs, will permit a more precise evaluation of the composition of the HDV RNA-protein complex.