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The Hepatitis Delta Virus accumulation requires paraspeckle components and affects NEAT1 level and PSP1 localization

The Hepatitis Delta Virus (HDV) relies mainly on host proteins for its replication. We previously identified that PSF and p54nrb associate with the HDV RNA genome during viral replication. Together with PSP1, these proteins are part of paraspeckles, which are subnuclear bodies nucleated by the long non-coding RNA NEAT1. In this work, we established the requirement for PSF, p54nrb and PSP1 in HDV replication using RNAi-mediated knockdown in HEK-293 cells replicating the HDV RNA genome. We determined that HDV replication induces the delocalization of PSP1 to cytoplasmic foci containing PABP and increases NEAT1 level causing an enlargement of NEAT1 foci. Overall, our data support a role for the main paraspeckles proteins in HDV life cycle and indicate that HDV replication causes a cellular stress and induces both a delocalization of the PSP1 to the cytoplasm and a disruption of paraspeckles.


PSP1 associates with the HDV RNA genome in HEK-293 cells. Previously, we demonstrated that
PSF and p54nrb associate with the HDV RNA in cells replicating the HDV genome 23,24 . These two proteins and PSP1 are three of the main components of nuclear paraspeckles 26,28,29,46 . To determine whether PSP1 also interacts with the HDV RNA genome, we performed RNA-protein immunoprecipitation (RIP) assays for this protein in cells replicating HDV. For this, we used a cellular system where HEK-293 cells were stably transfected with a plasmid encoding HDAg-S under the control of a promoter activated by tetracycline and with an HDV RNA genome unable to produce HDAg (i.e. 293-HDV) 47 . Twenty-four hours after induction of HDAg-S expression with 1 μg/ml of tetracycline, and thus induction of HDV replication, the cells were treated with formaldehyde to cross-link the RNA-protein complexes. Following disruption of the cells, the ribonucleoprotein complexes were immunoprecipitated with an α-PSP1 antibody, or with either α-PSF or α-p54nrb antibodies as positive controls. As a negative control, α-β-actin was used as an unrelated antibody.
Following the co-immunoprecipitations, the cross-links were reversed by heating the samples and RT-PCR was carried out to amplify a fragment of the HDV RNA genome corresponding to the terminal domain containing the ribozyme sequences. When the co-immunoprecipitations were performed with either α-PSP1, α-PSF or α-p54nrb antibodies, we observed the presence of a band of the expected length for the HDV amplicon in the cells treated with tetracycline ( Fig. 1). The band observed was not due to a non-specific binding of the RNA to the protein G agarose beads or to antibody constant regions, because this amplicon was not observed in the negative controls. Taken together, our results indicate that PSP1 associates, directly or indirectly through protein:protein or protein:RNA:protein interactions, with the HDV RNA genome in this cellular system, and corroborate our previous observations of the association of both PSF and p54nrb with HDV RNA 23,24 . Knockdown of PSF, p54nrb and PSP1 leads to an inhibition of HDV replication. To clarify the involvement of PSF, p54nrb and PSP1 in HDV replication, we investigated whether knockdown of these proteins would affect HDV accumulation following induction of HDAg-S production. 293-HDV cells were transfected with commercial pools of siRNAs targeting mRNAs coding for PSF, p54nrb or PSP1. As negative controls, we also transfected cells with scrambled siRNAs or only water. Twenty-four hours post-transfection with the siR-NAs, the expression of HDAg-S, and thus HDV replication, was induced by addition of 1 μg/ml tetracycline. Twenty-four hours post-induction, total cellular lysates were prepared and separated in two samples: one was used for protein-extraction and the other was used for RNA extraction. Under our conditions, PSF, p54nrb and PSP1 were significantly knocked down when compared to the cells transfected with scramble siRNAs (Fig. 2a-c).
The effects of the knockdown of these three proteins on the accumulation of HDV RNA was assessed by quantitative RT-PCR (RT-qPCR) using the level of β-2-microglobulin mRNA to normalize the results. As expected when HDV replication was induced with the addition of tetracycline, we observed about 200-fold increase of HDV RNA compared to the untreated cells (Fig. 2d). Upon PSF, p54nrb or PSP1 knockdown, we observed a decrease of more than 90% of HDV RNAs accumulation compared to the cells only treated with tetracycline (Fig. 2d). In order to rule out the possibility that this effect could result from a reduced HDAg-S levels, which ScIentIFIc RePORTS | (2018) 8:6031 | DOI:10.1038/s41598-018-24500-1 is required for HDV replication, we performed a Western Blot on HDAg-S. There was no substantial difference in HDAg-S levels in cells transfected with the siRNAs targeting mRNAs coding for PSF, p54nrb or PSP1 compared to cells transfected with the pool of scramble siRNAs in cells replicating HDV (Fig. 2e). In summary, the knockdown of PSF, p54nrb and PSP1 leads to a decrease of the HDV genome accumulation, indicating that these proteins are required directly or indirectly for the accumulation of HDV genomes in these cells.
HDV replication induces the formation of PSP1 foci outside of the nucleus. Since the HDV RNA genome interacts with at least three proteins found in paraspeckles (PSF, p54nrb and PSP1), and that these proteins are required for the accumulation of HDV RNA, we hypothesized that paraspeckles might be disrupted upon HDV replication. Since PSP1 has often been used as marker for paraspeckles 28 , we investigated the localization of this protein upon induction of HDV replication. Briefly, HEK-293 cells (293), HEK-293 cells stably transfected with a plasmid encoding the HDAg-S under the control of a promoter inducible by tetracycline (293-Ag), and 293-HDV cells were treated or not with 1 μg/ml tetracycline. Forty-eight hours after this treatment, we performed an immuno-staining using an antibody directed against the C-terminal domain of PSP1. Upon induction of HDV replication, we observed that PSP1 delocalizes outside of the nucleus as large bright foci (Fig. 3a). This pattern was not observed in 293 cells nor in 293-Ag cells treated with tetracycline.
To confirm these results using a complementary approach, we performed sub-cellular fractionations of the cells replicating HDV, followed by a Western Blot for PSP1. The nuclear and cytoplasmic fractions were properly separated and validated using α-tubulin and α-H3 antibodies as cytoplasm and nucleus markers, respectively. In the samples where HDV replication was induced, we observed lower level of PSP1 in both cellular sub-fractions (Fig. 3b). Because H3, α-tubulin and β-actin levels were not significantly reduced by the cellular fractionation, and that PSP1 levels were not greatly affected by the induction of HDV replication (Fig. 3b, see Total lysate), our results suggest the presence of PSP1 into insoluble aggregates. In order to determine if the localization of the other two paraspeckles proteins, PSF and p45nrb, are similarly affected by HDV replication, we repeated the sub-cellular fractionation experiments described above for PSF and p54nrb. In contrast to PSP1, we observed no substantial changes in the localization of p54nrb when HDV replication was induced (Fig. 3b). Interestingly, we observed that induction of HDV replication promotes PSF cytoplasmic localization (Fig. 3b), which was previously shown to be linked to cell cycle arrest 48 .
We and others observed morphological changes in the 293-HDV cells when HDV replication was induced: the cells were round shaped compared to the uninduced cells and detached very easily from the culture dish surface after more than two days post induction (Fig. 4a) 47 . It was also reported that HDV induction causes a significant cell cycle arrest in G1/G0 47 . Given the phenotype, the cellular cycle arrest reported, the PSF cytoplasmic localization, and our results suggesting that PSP1 is present in insoluble aggregates, one hypothesis is that HDV replication could cause a cellular stress and that such a stress might induce the delocalization of PSP1 to the cytoplasmic stress granules. To investigate this hypothesis, we immuno-stained PSP1 and the stress granule marker PABP with or without induction of HDV replication 49 . Upon induction, we observed a strong co-localization of the immunofluorescence signal corresponding to both PABP and PSP1 (Pearson coefficient of 0.94 and Manders coefficient of 0.97; Fig. 4b and c). In contrast, we did not observe this effect in non-induced cells. This result suggests that HDV replication cause a delocalization of PSP1 to PABP-containing stress granules in this cellular system. HDV replication decreases PSP1 co-localization with NEAT1. It is proposed that the long non-coding RNA NEAT1 serves as a scaffold for the formation of paraspeckles 36 . Since it is required for the formation of paraspeckles, we performed an RNA-FISH on NEAT1 along with an immuno-staining of PSP1 to investigate whether NEAT1 remained in the nucleus. Additionally, we investigated if the relative co-localization of NEAT1 and PSP1 changes in cells replicating HDV. Under our condition, NEAT1 foci remain in the nucleus Figure 1. Association of HDV RNA with PSF, p54nrb and PSP1 in HEK-293 cells. HDV replication in 293-HDV cells was induced (+) or not (−) by tetracycline, then the cells were treated with formaldehyde to crosslink the RNA-protein complexes and lyzed. The lysates were used for RIP using α-PSF, α-p54nrb and α-PSP1 antibodies. The α-β-actin antibody was used for RIP as a negative control. Following co-immunoprecipitation, the cross-links were reversed by heating the samples and RT-PCR was carried out to amplify a 300 bp fragment of the HDV RNA genome located on the terminal domain containing the ribozyme sequences. The resulting PCR product was resolved on an agarose gel. The 100 bp DNA Ladder (NEB) was used as marker.
ScIentIFIc RePORTS | (2018) 8:6031 | DOI:10.1038/s41598-018-24500-1 during HDV replication (Fig. 5a). Without induction of HDV replication, about 40% of the PSP1 foci co-localized with NEAT1, consistent with previous studies (Fig. 5a-c) [28][29][30] . When the cells were treated with tetracycline to induce HDV replication, we detected a two-fold decrease in PSP1/NEAT1 co-localization ( Fig. 5a and c). This decrease also supports our observation that PSP1 delocalizes from the nucleus to cytoplasmic PABP-containing granules. Additionally, because PSP1 is still present in the nucleus and co-localizes with NEAT1 at a lower frequency, this result is consistent with our observation of their association upon induction of HDV replication.
To confirm the decrease of PSP1 interaction with NEAT1 upon HDV replication, we performed RIP assays, as described above. 24 hours after induction of HDV replication with tetracycline, the cells were treated with formaldehyde to cross-link the RNA-protein complexes. Following disruption of the cells, the ribonucleoprotein complexes were immunoprecipitated with an α-PSP1 antibody, α-PSF and α-p54nrb antibodies as positive controls, or an α-β-actin antibody as an unrelated antibody. The cross-links were reversed by heating the samples, and the resulting RNA used for RT-PCR with primers designed to amplify a fragment of 200 base pairs derived from NEAT1_2. For the cells where HDV replication was not induced, the immunoprecipitation assay performed using α-PSP1, α-PSF or α-p54nrb antibodies showed bands corresponding to the expected size for the NEAT1_2 amplicon (Fig. 5d). This result is in agreement with the reported interaction of NEAT1_2 with these three proteins in paraspeckles. We did not observe this band with the negative control antibody (i.e. using the α-β-actin antibody), indicating that the band observed is not due to a non-specific binding of NEAT1_2 with the protein G agarose beads or the antibody used for the RIP (Fig. 5d). For the cells where HDV replication was induced, no band corresponding to the NEAT1_2 amplicon was observed when the RIP was performed with the α-PSP1 antibody (Fig. 5d). Estimated values for the relative binding of NEAT1_2 by PSP1 using non-normalized differences of the Ct values after qPCR indicated that NEAT1_2 interaction with PSP1 is decreased by about 5-fold, which is in agreement with our results showing that PSP1 delocalizes from the nuclear paraspeckles upon HDV replication. In contrast, bright bands corresponding the NEAT1_2 amplicon was detected when either α-PSF or α-p54nrb antibodies were used (Fig. 5d). Estimated values for the relative binding of NEAT1_2 by PSF suggest that there is a 7-fold increase of amount of NEAT1_2 interacting with PSF. This could be due to an increased ability of NEAT1_2 to interact with PSF or to higher levels of NEAT1_2 transcripts.

Change in expression levels of NEAT1 upon HDV replication in HEK-293 cells.
To assess whether NEAT1 foci are affected by HDV replication, RNA-FISH on NEAT1 was performed and the foci quantified (Fig. 6a). Our analysis indicates that the number of NEAT1 foci does not vary significantly upon induction of HDV replication (Fig. 6b). However, we observed that, in cells replicating HDV, the NEAT1 foci were larger (Fig. 6c) and their intensity were ~2-fold increased compared to the cells without HDV replication (Fig. 6d). To independently confirm these results, we performed RT-qPCR on NEAT1_2 RNA in 293-HDV, with or without induction. Upon induction of HDV replication, we observed an increase of approximately 3-fold of NEAT1_2 level (Fig. 6e), which is consistent with the quantifications obtained by RNA-FISH. It was previously demonstrated that an increase of NEAT1_2 level is often accompanied by an increase in the expression of several antiviral proteins, such as IL8 42 . To assess if a similar phenomenon could be observed in cells replicating HDV, RT-qPCR was performed on both IL8 and IFNβ mRNA, under the same condition as above. Our results indicate that IL8 mRNA level is increased by approximately 2-folds and that IFNβ mRNA level is not significantly affected by HDV when compared to the uninduced cells ( Fig. 6f and g). These results are consistent with previous results showing that the expression of IL8, but not IFN-β, is regulated by NEAT1 42 . Taken together, these results indicate that HDV replication causes an up-regulation of NEAT1_2 and enlargement of NEAT1 foci in this cellular system.

Discussion
Several cellular proteins associating with HDV RNA have been previously identified 23,24,50,51 . However, only a few interactions have been confirmed in a cellular system replicating the HDV RNA genome, and a role for most of those proteins in HDV biology is still unknown. To identify proteins involved in HDV replication, we previously identified PSF and p54nrb as host proteins interacting with segments of the HDV RNA genome possessing promoter activity, both in vitro and in cells replicating HDV 23,24 . Because both of these proteins are part of paraspeckles, we hypothesized that components of these nuclear foci might have a role in HDV replication, and consequently that paraspeckles might be disrupted by HDV. In the current investigation, we established that three paraspeckles proteins, PSF, p54nrb and PSP1, associate with the HDV RNA genome and are required for HDV replication in HEK-293 cells. Furthermore, we determined that HDV replication induces the delocalization of PSP1 to cytoplasmic foci containing PABP, increases NEAT1_2 level and enlarges NEAT1 foci.
Knockdown of PSF, p54nrb or PSP1 inhibits HDV replication. PSF and p54nrb are involved in multiple RNA processes 26 , while the cellular role of PSP1 remains largely unknown and its involvement in RNA pathways remains to be characterized. These proteins also affect the expression of other proteins involved in multiple processes such as cell cycle arrest, regulation of RNA transport and stabilization, changes in transcripts and protein expression, and regulation of the cellular immunity cytokines 27 . For instance, PSF acts as transcription repressor for several genes, and its titration results in an overexpression of these genes 52 . Therefore, we cannot exclude the possibility that the reported decrease of HDV levels upon knockdown of PSF, p54nrb or PSP1 could be indirect and caused by the deregulation of other genes, RNA and proteins affected by PSF, p54nrb and PSP1. However, the need for these three paraspeckle proteins in HDV replication is consistent with previous the observation that HDAg and HDV RNA foci localize to sites usually associated with paraspeckles, in the periphery of nuclear SC35-containing speckles 53,54 .
Overall our results are consistent with a cellular stress caused by HDV replication. PSP1 is usually present in the nucleoplasm and concentrated in paraspeckles 28 . Upon HDV replication in HEK-293 cells, we observed that PSP1 delocalizes outside of the nucleus as large foci co-localizing with PABP. The cytoplasmic PABP foci we observed are similar to previously described stress granules, which are cytoplasmic structures composed of RNAs and proteins forming upon stress stimuli 49 . PABP usually associates with the polyA tail of mRNA in the cytoplasm and, upon stress, a subset of PABP cytoplasmic pool is recruited to the stress granules 55,56 . In the cellular system used, induction of HDV replication leads to cellular morphological changes, to the blockage of the majority of cells in the G1/G0 phase, and to a loss of cell adherence after two days 47 . This effect is caused by HDV replication, as it is not observed in 293 or 293-Ag cells upon addition of tetracycline 47 , and is consistent with a cellular stress caused by HDV replication which might be responsible for the cell cycle arrest 47,57 . Interestingly, other studies have shown that under transcriptional arrest and under stress conditions, PSP1 re-localizes instead to the perinucleolar region 28,58 . The reason for this difference in PSP1 re-localization is unknown, but since PSP1 interacts with HDV RNA, it is possible that shuttling of PSP1 might be affected by the viral RNA or viral ribonucleoprotein complexes. We also observed that the localization of both PSF and p54nrb is not considerably affected by HDV replication. These results are consistent with previous observations that changes in the PSP1 trafficking do not affect PSF foci localization and that knockout of PSP1 does not lead to paraspeckles disruption, indicating that PSP1 is not required for the formation of paraspeckles 30,59 .
HDV replication leads to an enlargement of NEAT1 foci and to an up-regulation of NEAT1 transcripts. This observation is consistent with previous reports suggesting that levels of NEAT1 RNA and/or formation of paraspeckles are part of a cellular response to stress events, such as those occurring during viral infections 37,39,41,[60][61][62] . Interestingly, both Influenza A virus and HSV-1 infections also upregulate NEAT1 expression causing larger paraspeckles, which correlates with the induction of a set of antiviral genes 42 . The authors proposed that increased sequestering of the transcriptional repressor PSF from host genes by NEAT1 activates the antiviral gene IL8, and thus innate immune response 42 . In agreement with this model, our results indicate that upon induction of HDV replication, IL8 mRNA levels is increased by approximately 2-folds when compared to the untreated cells, suggesting that a similar mechanism in which induction of NEAT1 by HDV might causes PSF sequestering and the activation of the innate immune response. High levels of NEAT1 were also reported to be associated with the expression of several oncogenes and shown to correlate with the occurrence of hepatocellular carcinoma [63][64][65][66][67] . Since PSF is a transcriptional repressor, it is tempting to speculate that higher levels of NEAT1 in HDV infected cells also perturbs the interaction of PSF with cellular promoters associated with the regulation of oncogenes. Further experiments are clearly needed to shed light on the impact of PSF sequestering by NEAT1 on host genes regulation and on the occurrence of fulminous hepatocellular carcinomas following HDV infection 68 .
In summary, we provide evidence of a role for three paraspeckle proteins in HDV replication, (PSF, p54nrb and PSP1), and demonstrate that HDV replication delocalizes PSP1 to cytoplasmic foci containing PABP and induces an increase of NEAT1 expression in HEK-293 cells. Although the cellular system used represent a valuable tool to study HDV replication and pathogenesis, further examination of a role for the paraspeckles components in HDV replication is needed. Using hepatocytes or infected samples might not only confirm our observation, but also provide important insights into how these foci are involved in the life cycle of HDV, its host response and the occurrence of hepatocellular carcinomas.
Protein knockdown. Knockdown of PSF, p54nrb and PSP1 was performed using commercially available pools of specific siRNAs directed against PSF mRNA (#sc-37007; Santa Cruz), p54nrb mRNA (#sc-38163; Santa Cruz) and PSP1 mRNA (#sc-76279; Santa Cruz). As negative controls, transfection with a "scrambled" siRNA (#sc-37007; Santa Cruz), no treatment and a mock control (H 2 O) were used. Transfection was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations with a ratio of 80 picomoles siRNA: 5 μL lipofectamine: 100 μL DMEM. Twenty-four hours post transfection, HDAg-S expression was induced by addition of 1 μg/mL tetracycline. Twenty-four hours post-induction, the total cellular lysates were centrifuged and separated in two samples. For one sample, the total RNA was extracted using TRIzol (Invitrogen). The RNA was used for RT-PCR and RT-qPCR with primers to amplify NEAT1and HDV. For the other samples, the total proteins were suspended in Laemmli 4x buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris-HCl pH 6.8), and the knockdown of the proteins was confirmed by Western Blotting.
Ribonucleoprotein immunoprecipitation assay (RIPA). The RIP analyses were performed as reported previously 23 . Briefly, 293-HDV cells were seeded and after twenty-four hours, HDV replication was induced by addition of 1 μg/mL tetracycline. Twenty-four hours later, 1% of formaldehyde was added to cross-link the ribonucleoprotein complexes and the reaction was quenched with 0.25 M glycine, pH 7. The cells were centrifuged at 3000 rpm for 4 min and washed twice with cold PBS, and then suspended in RIPA buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, 150 mM NaCl) supplemented with 1% protease inhibitor cocktail for mammalian extracts (Sigma-Aldrich) and 1 μg RNAse inhibitor (Biobasic). The cells were mechanically lysed by pipetting on ice, followed by agitation for 30 min at 4 °C. The cellular lysate was cleared by centrifugation at 20 000 rpm for 20 min at 4 °C. Then, the supernatant was sonicated and the lysates stored at −80 °C. Co-immunoprecipitation was performed with the Protein G immunoprecipitation kit (Sigma-Aldrich). 5 μg of antibody was added to the immunoprecipitation spin columns containing 50 μL of prewashed dynabeads. The antibodies used were targeted against PSF (#B92, Abcam), PSP1 (#SAB4200067, Sigma-Aldrich), p54nrb (#05-950 Upstate) and Beta Actin (mouse monoclonal, #6276, Abcam). The antibody-dynabeads complex was incubated on a rotator overnight at 4 °C and then washed in PBST buffer (PBS with 0.02% Tween). 500 μL of cellular lysate was added to the dynabeads-antibody complex and incubated for 1 h at 4 °C, followed by 4 washes in RIPA buffer. The dynabeads-antibody-antigen complex was eluted in 200 μL of a protein storage buffer (50 mM Tris-Cl, 0.5 mM EDTA, 10 mM DTT, 1% SDS, pH7.5). In order to reverse the crosslinking reaction, the samples were heated at 70 °C for 45 min. The RNA was extracted with TRIzol (Invitrogen), ethanol-precipitated and suspended in H 2 O. The HDV and NEAT1 RNA levels were assessed by RT-PCR.
Sub-cellular protein fractionation. Sub-cellular protein fractionation was performed according to Suzuki et al. 70 , with some modifications. Briefly, the cells were wash with PSB, suspended in 1 mL of Buffer A (10 mM HEPES, 10 mM KCL, 1.5 mM MgCl 2 ). 200 μl of the cell lysate was taken as "whole cell lysate" and mixed with Laemmli 4 × buffer. The rest of the lysate was incubated 10 minutes on ice, centrifuged 1 minute at 2000 × g and the supernatant was removed. The pellet was suspended in Buffer A containing 0.5 mM DTT, protease inhibitor cocktail (Roche, # RC4693116001), and 0.2% NP-40 (Igepal), incubated 2 minutes on ice and centrifuged 1 min at 2000 × g at 4 °C. The supernatant containing the "cytosolic fraction" was mixed with Laemmli 4× buffer. The pellet was suspended in NP-40 buffer (50 mM of Tris-HCL pH8.0, 0.4 M of NaCl, 5 mM EDTA pH 8.0, 1% NP-40, 0.2% SDS), incubated on ice with mixing for one hour. The lysate was then centrifuged at 4 °C, the supernatant labeled as "nuclear fraction" and mixed with Laemmli 4x sbuffer. Samples were heated 5 min at 95 °C and the samples were loaded on an sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with the blueye prestain protein ladder (GeneDireX, #PM007-0500).
Western-Blot. Bradford  Tween) and washed three times with TBST. The membrane was incubated with the following antibody: α-PSF (mouse monoclonal, #B92, Abcam), α-p54nrb (rabbit monoclonal, #05-950 Upstate), α-PSP1 (rabbit polyclonal, #SAB4200067, Sigma-Aldrich), α-Beta-actin (mouse monoclonal, #6276, Abcam). After three washes with TBST, the membranes were incubated with the appropriate secondary antibody in TBST with 3% BSA. The secondary antibodies used were the rabbit anti-mouse IgG HRP (polyclonal, # ab6728, Abcam), goat anti-rabbit IgG HRP (polyclonal, #ab6721, Abcam). The blots were visualized using ECL reagent according to the manufacturer's recommendations (Thermo Scientific #32106). The membrane was then exposed to a photosensitive film and the film was scanned. The densitometric measurement was performed for some digitized blots using Image-J software. Each experiment was performed in three independent assays. Fluorescence Immunostaining. Cells were plated in cell culture dishes with glass bottom (Ibidi).
Immunostaining was performed forty-eight hours after the induction of HDV replication with 1 μg/mL tetracycline. The cells were fixed with 4% paraformalhehyde in 1x PBS, washed three times in PBS containing 1% BSA and permeabilized with 0.5% Triton X-100. The cells were incubated with the following primary antibodies diluted in PBS containing 1% BSA: mouse monoclonal PSF (#B92, Abcam), rabbit monoclonal p54 (#05-950, Upstate), rabbit polyclonal antibody directed against the C-terminal domain PSP1 (#SAB4200067, Sigma-Aldrich), mouse monoclonal PABP (#SC32318, Cell Signaling). After three washes in PBS containing 1% BSA, the cells were washed with 0.1% Triton X-100 in PBS and incubated with the appropriate secondary antibodies in PBS containing 1% BSA. The secondary antibodies used were as following: polyclonal goat anti-mouse coupled to Alexa 488 (#A11001, Life Technologies), polyclonal anti rabbit coupled to Alexa 488 (# A11008, Life Technologies), polyclonal goat anti-mouse coupled to Alexa 594 (#A11005, Life Technologies) and polyclonal goat anti-rabbit coupled to Alexa 594 (#A11012, Life Technologies). After washing with 0.1% Triton X-100 in PBS, the cells were mounted in Vectashield containing DAPI (4′, 6-Diamidino-2-Phenylindole) staining. The cells were visualized using AxioImager inverted Z.1 microscope (Carl Zeiss Canada) with the objectives PLAN APOCHROMAT 40 × /0.95 and PLAN APOCHROMAT 63 × /1.4 OIL, the images were deconvolved using AxioVision Rel 4.8 and analyzed with either Axiocam or ImageJ. The immunofluorescence pictures shown are the combination of at least 25 Z stacks. Each experiment was performed in three independent assays.
In situ hybridization. RNA fluorescence in situ hybridization was performed using a probe against NEAT1 RNA (#SMF-2036-1Stellaris, Biosearch Technologies), using the protocol of the manufacturer for the simultaneous FISH and Immunofluorescence protocol was followed with minor modifications. Cells were plated in cell culture dishes with glass bottom (Ibidi). Forty-eight hours after the induction of HDV replication with 1 μg/mL tetracycline, the cells were washed twice in PBS and fixed with 4% paraformalhehyde in 1x PBS, washed three times in PBS containing 1% BSA and permeabilized with 0.1% Triton X-100. After washes in PBS, the cells were incubated with a polyclonal primary rabbit antibody directed against the C-terminal domain of PSP1 in PBS containing 1% BSA. After three washes in PBS with 0.1% Triton X-100 and the cells were incubated with the secondary antibody anti-rabbit coupled to Alexa 488 (Life Technologies). The cells were washed with PBS and incubated in the fixation buffer (3.7% formaldehyde in 1X PBS). After two washes in PBS, the cells were incubated in the wash buffer (10% formamide in 2x SSC) for 5 min at RT. Then, the cells were incubated overnight, at 37 °C, in the dark, in hybridization buffer (100 mg/mL dextran sulfate, 10% formamide in 2x SSC) containing 25 μM of NEAT1 probe. Then, the cells were washed in the hybridization buffer and they were mounted in Vectashield containing DAPI. The cells were visualized using AxioImager inverted Z.1 microscope (Carl Zeiss) with the objectives PLAN APOCHROMAT 40X /0.95 and PLAN APOCHROMAT 63X /1.4 OIL, the images were deconvolved using AxioVision Rel 4.8 and analyzed with either Axiocam or ImageJ. The immunofluorescence pictures shown are the combination of at least 25 Z stacks. Each experiment was performed in three independent assays.