It has long been assumed that the replication of HDV RNA, similar to that of plant viroids, is mediated by cellular polymerases. However, the evidence for this hypothesis is still very tenuous. For plant viroids, it has not been resolved whether the plant RdRP, pol II, or even pol I is involved in this process. For HDV replication, it was first suggested that pol II carries out HDV RNA replication based on a nuclear run-on experiment which showed that HDV RNA synthesis could be inhibited by a low concentration of α-amanitin (1 to 5 μg/ml) (39
). The most dramatic report came from Taylor's laboratory and showed that hepatocyte nuclear extracts were able to replicate the 1.7-kb genomic and antigenomic RNA faithfully in vitro without any extraneous factors (including HDAg) (18
). Furthermore, the reaction could be inhibited by an antibody against pol II and by α-amanitin. However, this claim is inconsistent with the rolling-circle mechanism, and this report has never been reproduced. Another report demonstrated that pol II was able to use a partial HDV antigenomic-sense RNA hairpin fragment as a template for an elongation reaction (elongation from the 3′ end of a cleavage fragment of the template) in vitro (17
). Significantly, the elongation reaction was stimulated by S-HDAg (76
). S-HDAg was also able to promote elongation in a DNA-dependent RNA transcription reaction. However, it is not clear whether such an elongation reaction (creating a genomic-antigenomic RNA chimeric molecule) is a biologically relevant process in HDV replication. Nevertheless, this was the most convincing evidence so far showing the capability of pol II in replicating HDV RNA in some fashion. Both S- and L-HDAg have also been shown to affect the cellular pol II-mediated, DNA-dependent RNA transcription either positively or negatively (36
). This effect further suggests the intimate association of HDAg with the cellular transcription machineries.
The evidence that HDV RNA replication in the cells is mediated by pol II came from a study of the effects of long-term α-amanitin treatment in a hepatocyte cell line after HDV RNA transfection (47
). This study showed that HDV mRNA transcription and RNA replication could be inhibited by α-amanitin at 1 to 5 μg/ml, consistent with the α-amanitin sensitivity of pol II (39
). Most significantly, when HDV RNA replication in a cell line expressing an α-amanitin-resistant pol II was carried out, HDV RNA replication became resistant to α-amanitin (47
). These data indicate strongly that pol II mediates HDV RNA replication. Unexpectedly, these studies also showed that the syntheses of the genomic and antigenomic strands have different sensitivities to α-amanitin. The genomic RNA synthesis (from the antigenomic RNA template), similar to mRNA transcription (from the genomic RNA template), is exquisitely sensitive to α-amanitin (1 to 5 μg/ml); in contrast, the antigenomic RNA strand synthesis (from the genomic RNA template) is surprisingly resistant (inhibited at >100 μg/ml) (40
). The latter finding suggests the involvement of other polymerases, such as pol I. Alternatively, the antigenomic RNA synthesis may occur in a compartment not permeable to α-amanitin. In either case, there appear to be separate transcription machineries for genomic and antigenomic RNA syntheses. This interpretation is further supported by the other differences in metabolic requirements between the syntheses of these two strands (Table ). It should be noted that there was another recent report claiming that there was no evidence for the involvement of polymerases other than pol II in HDV RNA replication (48
). However, that report actually examined only the genomic RNA synthesis. Thus, the evidence so far supports the novel idea that the genomic and antigenomic HDV RNA syntheses are carried out separately in different transcription machineries. For viroid RNA replication, there was also an earlier report suggesting that pol I and pol II mediate the synthesis of the separate RNA strands of potato spindle tuber viroid (62
The involvement of two separate transcription machineries in genomic and antigenomic RNA syntheses is consistent with the fates of these two RNA strands. The genomic RNA is exported to the cytoplasm immediately after synthesis, whereas the antigenomic RNA is retained in the nucleus (40
). The pol II transcription machinery, which makes HDV genomic RNA, is known to be coupled to the nuclear export machinery (14
). In contrast, HDV antigenomic RNA synthesis is postulated to occur in the nucleolus, where rRNA synthesis takes place; correspondingly, the antigenomic RNA, similar to rRNA, is not immediately transported to the cytoplasm. Furthermore, HDAg binds not only pol II transcription factors but also nucleolar proteins (24
). HDAg has been found in both the nucleoplasm and the nucleolus (74
). Most of the published studies examining HDV RNA replication in the cells are clouded by the fact that the genomic RNA synthesis is at least 10 times more robust than the antigenomic RNA synthesis; therefore, previous studies examining total HDV RNA synthesis in the cells may have overlooked the antigenomic RNA synthesis. More recent studies using BrUTP labeling have localized the newly synthesized genomic RNA in the nucleoplasm (near the PML body) and the antigenomic RNA near the nucleolus (Y.-J. Li and M. M. C. Lai, unpublished observation).
If HDV RNA synthesis is mediated by DNA-dependent RNA polymerases, how do these enzymes recognize RNA as a template? Conceivably, HDAg may convert the structure of HDV RNA (double-stranded form?) into the double-strand DNA conformation. HDAg binds to HDV RNA and serves as an RNA chaperone to alter the RNA conformation (25
). Interestingly, the double-stranded cDNA of HDV RNA contains a bidirectional promoter activity for pol II (38
); this promoter coincides very closely with the promoter determined for the natural HDV RNA (1
), further supporting the interchangeability of the DNA-RNA forms of the HDV genome. This promoter is localized to cDNA sequences corresponding to the end of the rodlike structure of HDV RNA. Another possibility is that HDAg may serve as a critical activator or coactivator in the HDV RNA transcription complex, conferring template specificity to either pol I or pol II for recognition of HDV RNA. This possibility is consistent with the finding that HDAg interacts with pol II (76
) and possibly other transcription factors.
Another interesting issue is how HDV RNA is differentially transported to the different subnuclear compartments to carry out different replication and transcription functions. The different modified forms of HDAg may carry out these diverse functions (34
). Significantly, the methylation-defective mutant of S-HDAg failed to aggregate in the speckle structure in the nucleus (34
); correspondingly, the genomic RNA synthesis, which is postulated to occur in the nucleoplasm, failed to take place. In contrast, the antigenomic RNA synthesis, which is postulated to take place in the nucleolus, can be mediated by this methylation-defective mutant (34
). Thus, there is a correlation between the HDAg localization and the replication of the separate HDV RNA strands. In addition, certain HDV RNA sequences may also determine the fate of the RNA, as HDV RNA can be shuttled in the absence of HDAg (66
). Finally, another intriguing question is whether HDV RNA replication involves a cytoplasmic phase. The genomic RNA, but not antigenomic RNA, is exported to the cytoplasm right after synthesis (40
). This fact is consistent with the eventual packaging of the genomic RNA into virions. However, the RNA export happens well before the virus assembly takes place. Also, HDV RNA can go back into the nucleus (66
). Whether certain steps of HDV replication cycle, e.g., RNA ligation, take place in the cytoplasm and whether shuttling of HDV RNA between the cytoplasm and the nucleus is necessary for successful RNA replication are important questions awaiting answers.