This report focuses on the use of immunostaining procedures to study the associations of δAg in the presence and absence of HDV RNAs. The advantage of this approach is that if the cells are fixed prior to staining, one can be confident that observed associations are not artifacts of rearrangement, such as can occur during cell fractionation procedures. This is a concern in our previous studies of δAg that made use of fractionation followed by rate zonal sedimentation, immunoprecipitation, and gel electrophoresis under nondenaturing conditions (8
). Even when fixation is followed by fractionation, there are concerns about the interpretability of the complexes detected (8
). However, even for studies using fixation followed by immunostaining, there is still the limitation that a colocalization of two components supports but is not sufficient to prove an interaction, whether direct or indirect interactions.
Most studies reported here made use of expression in 293 cells, but many aspects have been confirmed in Huh7 cells. Also, some of the results with the small δAg have been reproduced with large δAg. A difference was that when large δAg was relocalized to the nucleoplasm, it showed a greater tendency than small δAg to localize to SC35 speckles (data not shown).
There are three major questions addressed by this study. First, what are the nature and significance of δAg in the nucleolus? δAg, like nucleolin, is an RNA-binding protein, and we conclude that both proteins colocalize because they bind to rRNA precursors (15
); immunoprecipitation of δAg with nucleolin has been reported, but such studies did not exclude the possibility that the interaction was mediated by rRNA (24
). Since δAg is nucleolar only in the absence of HDV RNA transcription and/or accumulation, one might argue that nucleolar localization is irrelevant to replication. An alternative interpretation is that δAg goes to the nucleolus before it is utilized to facilitate the accumulation of processed HDV RNA transcripts. If such a nucleolar transit does occur, this raises the question of whether such a transit is somehow required for the viral life cycle. It can be said that the only forms of δAg that support replication are ones which, expressed in the absence of HDV RNAs, will accumulate in the nucleolus. At the same time, altered forms, such as large δAg that does not support replication, will also go to the nucleolus. As suggested in the introduction, we know that the RNA binding proteins of many viruses, both RNA and DNA viruses, can localize to the nucleolus (15
). Furthermore, some studies consider nucleolar transit to be of relevance. For example, in the case of orthomyxoviruses there are studies suggesting that prior nucleolar localization is required for the nucleocapsid protein early in replication, which is followed by a combination of nucleoplasmic and nucleolar localizations (12
Second, what are the nature and significance of δAg found in the nucleoplasm? Several situations have been found in which δAg localized to the nucleoplasm. Not just replicating HDV RNA (Fig. ) but also nonreplicating HDV RNA (Fig. ) can cause nucleoplasmic accumulation of δAg. Also, nucleoplasmic accumulation can occur in the absence of detectable HDV RNA species. For example, at 40 h after induction δAg is primarily localized to the nucleoplasm (Fig. ). Certain cell stress conditions lead to nucleoplasmic localization (Fig. ). Also, several altered forms of δAg localize to the nucleoplasm (Fig. ). In each of these situations we do not know what the δAg is binding to in the nucleoplasm. In some cases we can detect a component that colocalized with SC35 (Fig. ). This could reflect indirect rather than direct binding, but the localization apparently does not need δAg to possess either a dimerization or an RBD.
Third, does δAg bind, whether directly or indirectly, to nucleoplasmic Pol II? Some form of binding, direct or indirect, is plausible since it has been shown that Pol II is essential for at least some of the HDV RNA-directed transcription (8
). There is a report that δAg can interact in vitro with one or two subunits of host Pol II (41
) while a more recent study reconstituted Pol II transcription in the absence of δAg (1
). Be that as it may, our previous studies using immunoprecipitation indicate that for the majority of δAg expressed within cells in the presence or absence of HDV RNA, there is only minimal association with Pol II (8
). Furthermore, the immunostaining studies show that in the absence of HDV RNAs, the δAg is largely nucleolar while Pol II is nucleoplasmic (Fig. ); thus, δAg is not binding to Pol II and is somehow binding to nucleolar components, which we believe are probably rRNA precursors. Contrary interpretations are that δAg in the nucleolus is binding to nucleolin (24
) and B23 (17
), that it has fewer posttranslational modifications than nucleoplasmic δAg (38
), that δAg modified to contain a nucleolar localization signal supports the synthesis of antigenomic RNA but not genomic RNA (16
), and that Pol I in the nucleolus carries out the transcription of antigenomic RNA (25
In the presence of HDV RNAs the immunostaining results show that δAg and Pol II are in the same nucleoplasmic location, and it has been tempting to assert that they must be associated and participating in RNA-directed RNA synthesis (8
). In contrast, after the present studies we would be more cautious. First, at 40 h after induced replication, when accumulation of processed HDV RNAs has reached a maximum value and transcription may have ceased (7
), the same colocalization is observed (Fig. ). Furthermore, when HDV transcription is inhibited with amanitin, the colocalization remains (Fig. ). Therefore, we suggest that the majority of δAg that colocalizes with Pol II in the nucleoplasm is not currently involved with active RNA-directed transcription. In cells that have undergone HDV transcription, we have previously used immunoprecipitation and found at least 16% of the HDV RNAs in association with δAg (8
). Thus, we expect that the majority of δAg in the nucleoplasm is in association with HDV RNAs in a postreplicative state. However, we do not know why such ribonucleoprotein complexes remain largely in the nucleoplasm. In addition, we have to remember that δAg can have a similar nucleoplasmic localization in the presence of HDV RNAs that cannot replicate (Fig. ) or in the total absence of full-length HDV RNAs (Fig. and ), and so also will certain altered δAg forms that lack an RBD or a dimerization domain (Fig. ).
In summary this study used immunostaining of cells under defined conditions of δAg expression, without and with the accumulation of associated HDV RNA species, to examine the intracellular localizations of δAg and its colocalizations with host components. The findings reveal the complications of extrapolating from such localizations to interpretations of functional significance.