Phosphorylation of Ebola Virus VP30 Influences the Composition of the Viral Nucleocapsid Complex

Background: Ebola virus VP30 is an essential transcription factor dispensable for viral replication whose activity is regulated via phosphorylation. Results: Phosphorylation of VP30 impacts viral transcription and replication by modulating interaction with the nucleocapsid proteins VP35 and NP. Conclusion: VP30 phosphorylation influences the composition of the viral polymerase complex via phosphorylation-dependent interaction with VP35. Significance: VP30 phosphorylation status modulates both viral transcription and replication. Ebola virus is a non-segmented negative-sense RNA virus causing severe hemorrhagic fever with high fatality rates in humans and nonhuman primates. For transcription of the viral genome four viral proteins are essential: the nucleoprotein NP, the polymerase L, the polymerase cofactor VP35, and VP30. VP30 represents an essential Ebola virus-specific transcription factor whose activity is regulated via its phosphorylation state. In contrast to viral transcription, VP30 is not required for viral replication. Using a minigenome assay, we show that phosphorylation of VP30 inhibits viral transcription while viral replication is increased. Concurrently, phosphorylation of VP30 reciprocally regulates a newly described interaction of VP30 with VP35, and strengthens the interaction with NP. Our results indicate a critical role of VP30 phosphorylation for viral transcription and replication, suggesting a mechanism by which VP30 phosphorylation modulates the composition of the viral polymerase complex presumably forming a transcriptase in the presence of non-phosphorylated VP30 or a replicase in the presence of phosphorylated VP30.

Ebola virus (EBOV) 4 together with Marburg virus constitutes the family Filoviridae within the order Mononegavirales. Filoviruses are characterized by the filamentous shape of their particles and contain a non-segmented negative-sense RNA genome (1). EBOV is classified as a BSL4 pathogen, and human infection results in a severe hemorrhagic fever with case fatality rates of up to 90% (2,3). Currently, neither an approved vaccine nor antiviral therapy is available for humans (4,5). The enveloped EBOV particle is composed of seven structural proteins, five of which form the helical nucleocapsid that represents the template for viral transcription and replication. The viral genome is encapsidated by the major nucleocapsid protein NP, and VP35, VP30, and VP24 interact with NP to form the mature nucleocapsid (6 -8). The enzymatically active part of the viral polymerase L is bound to the nucleocapsid by interaction with the polymerase cofactor VP35 (9). The hexameric zinc-finger protein VP30 is an EBOV-specific viral transcription factor (10 -12).
After entry into the host cell, the EBOV envelope fuses with host cell membranes to release the nucleocapsid into the cytoplasm where transcription and replication take place (9,13). Initial transcription of the newly entered encapsidated RNA genome is entirely accomplished by the nucleocapsid proteins that are associated with the intruding virus (primary transcription). Transcription is regulated by conserved transcription start and stop signals at the viral gene borders (7). The gene start signals are part of RNA secondary structures, and it has been proposed that VP30 binds to the RNA at the first gene start signal to initiate transcription (14,15). In addition, VP30 was shown to be important for transcription reinitiation of subsequent genes (16). After transcription, EBOV mRNA is then translated by the cellular translation machinery, and newly formed viral proteins accumulate in inclusion bodies. Subsequently, a positive-sense full-length genome is replicated, which is concomitantly encapsidated by newly synthesized NP molecules. The produced antigenomic intermediate serves as a template for the replication of new viral genomes that, in turn, are available for transcription. In contrast to its essential role in viral transcription, VP30 is dispensable for viral replication, which is accomplished by L and VP35 alone (9). How the viral polymerase complex switches between its transcription and replication activity is still elusive.
Transcriptional support activity of VP30 is modulated via phosphorylation at two N-terminal serine clusters, each con-sisting of three serine residues (S29-S30-S31, and S42-S44-S46) ( Fig. 1A) (17). Mutation of serine residues to alanine mimicking nonphosphorylated serine or to aspartate mimicking phosphorylated serines indicated that non-or weakly phosphorylated VP30 supports initiation of viral transcription, while fully phosphorylated VP30 does not. The VP30-specific cellular kinase is unknown. VP30 can be dephosphorylated by the cellular phosphatases PP1 and PP2A, and inhibition of these enzymes by okadaic acid blocked viral transcription comparable to a VP30 mutant, which mimics the fully phosphorylated protein (17).
Recently, a positive influence of VP30 phosphorylation mutants on replication of EBOV was described by Martinez et al. (18). This result was of great interest because, so far, EBOV replication was thought to be independent of VP30 (9). Most of these experiments were performed by overexpressing VP30 phosphorylation mutants in EBOV-infected cells, where naturally wild type VP30 (VP30_wt) is present. Therefore, the question arose whether in the absence of VP30_wt the same results were to be expected. In addition, in the infected cell, it is difficult to differentiate effects on transcription and on replication since viral replication enhances the number of templates for transcription. Therefore, it remained unclear whether the observed influence of VP30 phosphorylation was the result of a direct effect on replication, or an indirect effect of down-regulation of viral transcription. As a consequence, the mechanism of how VP30 exerts its effect on viral transcription is still unknown.
In this study, we used previously characterized VP30 phosphorylation mutants mimicking either completely phosphorylated or completely non-phosphorylated VP30 to study their impact on viral replication and viral transcription (16 -18). To this end we employed two different minigenome systems allowing investigation of viral genome replication and transcription in absence of infectious virus and, more importantly, differentiation of these two processes (9,19,20). Positive influence of VP30 phosphorylation on viral replication and a negative influence on transcription were confirmed, suggesting VP30 phosphorylation to balance viral transcription and replication. To understand the mechanism by which phosphorylation conveys VP30 influence on transcription and replication, we investigated the role of VP30 phosphorylation for interaction with other viral nucleocapsid proteins, which might influence the composition of the polymerase complex. We showed for the first time an interaction of VP30 with VP35, which was reciprocally regulated by VP30 phosphorylation. We suggest that phosphorylation leads to dissociation of VP30 from the VP35-L complex, thereby modulating transcription and replication activities of the polymerase. In addition, we could demonstrate that the interaction of VP30 and NP was positively influenced by phosphorylation of VP30. The VP30-NP complex was not required for transcription and replication, but was essential for the incorporation of VP30 into new virus particles and the prerequisite for primary viral transcription in target cells.

EXPERIMENTAL PROCEDURES
Cell Culture-HEK293 (human embryonic kidney) and HUH7 (human hepatoma) cells were cultivated with Dulbecco's modified Eagle's medium with penicillin and streptomycin, 5 mM glutamine, and 10% fetal calf serum at 37°C and 5% CO 2 .
EBOV-specific Transcription and Replication Competent Virus-like Particle (trVLP) Assay-The assay was performed as described in Refs. 19,22. Transfection of plasmids was performed using TransIT (Mirus). Where indicated, the regular minigenome was replaced by a replication-deficient minigenome to analyze virus-specific transcription without the influence of replication (see Ref. 20). Additional transfection of pGL4.70 (Promega) encoding a firefly luciferase was performed for normalization of transfection efficiency. Reporter activity in producer cells was measured 72 h post-transfection (p.t.) using the Dual Luciferase assay (Promega). Released trVLPs were purified from the supernatant via ultracentrifugation over a 20% sucrose cushion and used for infection of target cells, which were either naïve or pretransfected with 500 ng of pCAGGS VP30_wt FLAG or VP30_E197A FLAG , respectively. An aliquot of trVLPs was analyzed regarding the incorporation of VP30 mutants using proteinase K digestion assay as described in Ref. 23. 60 h postinfection target cells were lysed and a Renilla reporter assay (Promega) was performed. Results obtained with VP30_wt were set to 100%.
Strand-specific Two-step Reverse Transcription and Quantitative Real-time PCR-Replication of the minigenome was analyzed using a strand-specific two-step RT quantitative real-time PCR (PeqLab). Briefly, producer cells (see EBOV-specific trVLP assay) were harvested 72 h p.t. and RNA was isolated using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol, including on-column digestion with the RNase-Free DNase Set (Qiagen) to avoid plasmid contamination. RNA samples were normalized to firefly luciferase levels, which correspond to transfection efficiency. First, RNA was reverse transcribed using a specific primer for replicated vRNA (Luc(ϩ): 5Ј-GGC CTC TTC TTA TTT ATG GCG A-3Ј, see Ref. 20). Next, cDNA was subjected to quantitative real-time PCR running on an ABI StepOne TM PCR cycler using Primer Luc(ϩ) and Luc(Ϫ) (5Ј-AGA ACC ATT ACC AGA TTT GCC TGA-3Ј). A probe with sequence specificity for the Renilla luciferase reporter (5Ј-CCA CAT ATT GAG CCA GTA GCG CGG-3Ј) containing 5Ј FAM (6-FAM-Phosphoramidit) and 3Ј DDQ-1 (Deep Dark Quencher-1, Biomers) was used for detection of amplificates. Serial 10-fold dilutions (10 6 to 10 9 ) of the minigenome plasmid were used as standards to determine the amount of minigenome copies in the samples. PCR conditions: Initial denaturation of cDNA was obtained at 95°C for 5 min, followed by 40 cycles with denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and elongation at 95°C for 15 s.
Coimmunoprecipitation Analysis-HEK293 cells were transfected using TransIT (Mirus) with the corresponding plasmids (1 g each). DNA amounts were adjusted, if necessary, with empty vector. Cells were lysed at 24 or 48 h p.t. for 20 min at room temperature in ice-cold coimmunoprecipitation buffer ((20 mM Tris-HCl, pH 7.5/100 mM NaCl/1% Nonidet P-40/17.5 mM EDTA) with 1ϫ Complete protease inhibitor mixture (Roche) and 0.1% Triton X-100). Cell debris was removed by centrifugation (10 min at 8.000 ϫ g, 4°C). An aliquot was taken for expression control (input). The clarified supernatant was added to 35 l of equilibrated mouse anti-FLAG M2 affinity gel agarose or mouse anti-HA affinity gel agarose (both Sigma-Aldrich). Precipitation was performed for 2 h at 4°C. Precipitated protein complexes were washed three times with coimmunoprecipitation buffer without Triton X-100 via centrifugation at 1.500 ϫ g. The pellet was resuspended in 45 l of loading buffer (100 mM Tris-HCl, pH 6.8/0.2% bromphenol blue/20% glycerol/10% 2-mercaptoethanol/4% SDS), and subjected to SDS-PAGE and Western blot analysis.

Influence of VP30 Phosphorylation on Viral Replication and
Transcription-Recent data revealed that VP30 is not only essential for viral transcription but influenced viral replication as well (18). EBOV replication was boosted upon simultaneous expression of a phosphomimetic VP30 mutant in trans (VP30_DD, Ref. 18)). This was astonishing since the presence of VP30 is not required at all for replication (9,20). With respect to these findings, we investigated the role of VP30 phosphorylation for viral replication. We used two different VP30 mutants mimicking either a completely non-phosphorylated VP30 (VP30_AA FLAG ), or a completely phosphorylated VP30 (VP30_DD FLAG ), which were described earlier (16,17) (Fig.  1A). Using a transcription and replication competent virus-like particle (trVLP) assay that monitors viral transcription and/or replication under BSL2 conditions, the effects of the two mutants on replication were analyzed. In contrast to the experimental set up used by Martinez et al. (18), this assay is independent of the presence of VP30_wt, allowing to investigate effects of completely phosphorylated or non-phosphorylated VP30 without interfering VP30_wt. The trVLP assay is based on the intracellular expression of an EBOV-specific minigenome containing a reporter gene (Renilla luciferase), which is flanked by sequences encoding the minimal required cis-acting transcription/replication and encapsidation signals of the viral genome (Fig. 1B). The minigenome is under the control of the T7 promoter allowing the transiently expressed T7 polymerase to synthesize the negative-sensed RNA minigenome, which is then encapsidated by NP and serves as a template for the EBOV polymerase complex. Simultaneous plasmid-based expression of VP24, VP40, and GP leads to assembly and release of trVLPs thus imitating an authentic infection cycle. (Fig. 1C, Ref. 19).
Influence of the different VP30 phosphomimetic mutants on the activity of the T7 promoter, was excluded by verifying that expression of the different VP30 mutants did not differentially impact the activity of a luciferase reporter gene under the control of the T7 promoter (data not shown).
First, we investigated the impact of VP30 phosphorylation on viral replication by using a negative-strand-specific two-step quantitative real-time PCR targeting specifically the replicated luciferase reporter gene (vRNA, see Ref. 20). Similar to the results gained by Martinez et al., expression of phosphomimetic VP30_DD FLAG stalled transcription and favored viral replication (180% compared with VP30_wt FLAG ) (18). In contrast, expression of non-phosphorylated VP30_AA FLAG reduced the amount of replicated minigenomes to 73% (Fig. 1C).
Increase of viral replication positively impacts viral transcription because replicated minigenomes are again available as templates for viral transcription (20). With respect to the newly discovered influence of VP30 phosphorylation on viral replication, we reconsidered previous data concerning the transcriptional support activity of VP30 phosphorylation mutants (16,18). To exclude the influence of viral replication for the activity of VP30 as transcription factor, we used a modified trVLP assay where the classical minigenome (template for transcription and replication) was substituted by a replication-deficient minigenome (Ref. 20), Fig. 1, B and D). Similar to previous results obtained with the classical minigenome (16,17), VP30_AA FLAG supported transcription of the replication-deficient minigenome comparable to VP30_wt FLAG , while VP30_DD FLAG was transcriptionally inactive (Fig. 1D). These results indicate that phosphorylation of VP30 influences transcription even in the absence of simultaneous replication.
Taken together, employing VP30 mutants mimicking either the completely phosphorylated or non-phosphorylated protein in a trVLP assay revealed a balancing influence of VP30 phosphorylation on viral transcription and replication. In presence of a non-phosphorylated VP30 (VP30_AA FLAG ), viral transcription was enhanced and viral replication decreased, while in the presence of a phosphorylated VP30 (VP30_DD FLAG ) viral transcription was stalled and viral replication increased. These results suggest that either VP30 directly influenced replication, or the phosphorylation state of VP30 regulates only the process of transcription, and effects on replication are the result of transcription regulation. This could be the case if the transcription complex comprising L, VP35, NP, and VP30 is transformed into a replication complex by phosphorylation of VP30.
Interaction of VP30 Phosphorylation Mutants with VP30 and NP-We therefore examined whether the phosphorylation of VP30 has an impact on the interaction with nucleocapsid proteins, which could regulate the composition of the viral polymerase complex. First, we focused on self-assembly of VP30, which is a hexameric protein composed of three VP30 dimers (10,11). Only VP30 hexamers support viral transcription. While hexamerization of VP30 is readily detected by coimmunoprecipitation, dimerization can only be detected after chemical crosslinking (10,11). We tested whether a permanent negative charge at the phosphorylation site influenced the oligomerization of VP30, which would then explain why VP30_DD also is unable to support viral transcription (11). We used FLAG-tagged VP30 phosphorylation mutants (Fig. 1A) and a Myc-tagged VP30_wt to perform coimmunoprecipitation analyses. VP30_AA FLAG , VP30_DD FLAG , or VP30_wt FLAG were able to coprecipitate the FIGURE 1. Influence of VP30 phosphorylation for viral transcription and replication. A, schematic presentation of VP30 phosphorylation mutants. VP30 serine residues S29 -31, S42, S44, S46 were mutated to either uncharged alanine mimicking a non-phosphorylated VP30 (VP30_AA FLAG ) or to aspartate mimicking a fully phosphorylated VP30 (VP30_DD FLAG ). B, schematic presentation of the Ebola virus-specific minigenome. All open reading frames coding for the Ebola virus proteins are substituted by a reporter gene (here: Renilla luciferase), which is flanked by the 3Ј leader (ldr) and 5Ј trailer (trl) regions of the genome containing essential transcription, replication, and encapsidation signals. Deletion of the 5Ј trailer region (nt 1-155) results in a replication-deficient minigenome. C, trVLP assay monitoring viral transcription and replication in producer cells. Transfection of plasmids encoding all viral proteins as well as T7-driven expression of a minigenome containing a Renilla luciferase reporter gene leads to EBOV-specific minigenome transcription and replication by the nucleocapsid proteins. Additionally, a firefly luciferase is transfected as control for transfection efficiency. Reporter gene activity can be measured 72 h p.t. using the Dual luciferase assay (Promega) reflecting viral transcription and replication. The measurement of replicated minigenomes is obtained by a two-step strand-specific reverse transcription and quantitative RealTime PCR targeting replicated minigenome copies. For that, RNA was isolated 72 h p.i., normalized to firefly luciferase levels and subjected to qRT-PCR as described in Ref. 20. Quantities of replicated minigenome copies obtained with VP30_wt FLAG were set to 100%, Ø: without VP30 D, tVLP assay monitoring viral transcription. The assay was performed as described under B. Instead of the regular minigenome, a replication-deficient minigenome was used to monitor viral transcription without the amplifying influence of replication. Luciferase activity obtained by VP30_wt FLAG was set to 100%. VP30 was replaced by the respective VP30 phosphorylation mutant as indicated. *: p Յ 0.05, **: p Յ 0.01, ***: p Յ 0.001. same amount of VP30_wt myc indicating that oligomerization with VP30_wt is not affected by the phosphorylation state of the protein ( Fig. 2A, lanes 3-5). Coimmunoprecipitation experiments with Myc-and FLAG-tagged VP30_AA or Myc-and FLAG-tagged VP30_DD revealed that both, VP30_AA as well as VP30_DD, were able to homooligomerize (Fig. 2B, lanes 3  and 6). Further, VP30_AA and VP30_DD were able to interact with each other supporting the conclusion that the charge at the VP30 phosphorylation sites does not influence the oligomerization of the protein (Fig. 2B, lane 7).
Next, we investigated the interaction between VP30 and NP. Previous work showed that in the presence of NP the nonphosphorylated VP30 was diffusely distributed in the cytoplasm, while phosphorylation leads to accumulation of VP30 in NP-induced inclusion bodies (17). Nevertheless, incorporation of VP30 into trVLPs was shown to be independent of the phos-FIGURE 2. Influence of VP30 phosphorylation on the interaction with VP30 and NP. A, coimmunoprecipitation of FLAG-tagged VP30 phosphorylation mutants with Myc-tagged VP30_wt. HEK293 cells transiently expressing VP30_wt myc and VP30_AA FLAG or VP30_DD FLAG were lysed 48 h p.t., and protein complexes were precipitated for 2 h, 4°C using mouse anti-FLAGா M2 agarose. An aliquot for expression control (input) was collected from the cell lysate before precipitation. Western blot analysis was performed using a biotinylated mouse anti-FLAGா BioM2 antibody and Alexa Fluorா 680-conjugated streptavidin (shown in red) and a rabbit anti-c-Myc and an IRDyeா 800-conjugated goat anti-rabbit antibody (shown in green). Detection of proteins was obtained with LiCor Odysseyா Imaging Systems. Precipitation of Myc-tagged VP30_wt by VP30_wt FLAG was set to 100%. Quantification of at least six independent experiments is shown to the left. B, oligomerization of VP30 phosphorylation mutants. Myc-and FLAG tagged plasmids of VP30_AA or VP30_DD were coexpressed and subjected to coimmunoprecipitation analysis. The experiment was performed as described under A. Note: due to technical reasons lanes 2, 5, and 8 are narrower than the other lanes. C, coimmunoprecipitation of VP30 phosphorylation mutants with NP. The experiment was performed as described under A. VP30 FLAG was stained by a biotinylated mouse anti-FLAGா BioM2 antibody and Alexa Fluorா 680-conjugated streptavidin, whereas NP staining was performed using a chicken anti-NP and an Alexa Fluorா 680-conjugated goat anti-chicken antibody. Precipitation of NP by FLAG-tagged VP30_wt was set to 100%. Quantification of at least four independent experiments is shown to the left. *: p Յ 0.05. phorylation state (16). In this study, we wanted to quantify the interaction of VP30 with NP by coimmunoprecipitation analysis (Fig. 2C). We observed that VP30_DD FLAG , mimicking the fully phosphorylated VP30, precipitated NP in significantly higher amounts compared with VP30_AA FLAG or VP30_wt FLAG (Fig.  2C, lanes 3-5) suggesting that the interaction of VP30_DD and NP is stronger. These data underlined the previous results indicating that phosphorylation of VP30 modulates the interaction with NP (17).
Interaction of VP30 and NP Is Essential for the Viral Life Cycle but Dispensable for Viral Transcription and Replication-To investigate whether the strengthened interaction between VP30 and NP contributed to the switch from viral transcription to replication, we wanted to analyze the significance of the NP-VP30 interaction for the viral life cycle. Based on the available crystal structure of the VP30 C terminus we therefore created a VP30 mutant without NP binding activity, which would then be employed in the minigenome assay to test its ability to support viral transcription (11). We generated a VP30 construct carrying a point mutation of glutamate 197 to alanine and analyzed the interaction of VP30_E197A FLAG with NP. Phenotypical characterization using immunofluorescence analysis revealed a diffuse intracellular distribution of VP30_E197A FLAG comparable to VP30_wt FLAG upon single expression (Fig. 4B). Upon coexpression with a Myc-tagged NP, VP30_E197A FLAG was not concentrated into NP-induced inclusion bodies in contrast to VP30_wt, indicating that the interaction between the proteins is impaired (Fig. 3A). The obtained results were confirmed by coimmunoprecipitation analysis. We observed that interaction between NP and VP30 was significantly inhibited upon mutation of glutamate 197 to alanine, indicating that E197 is necessary for the interaction of VP30 with NP (Fig. 3B). Next, we analyzed the significance of NP-VP30 interaction using the trVLP assay by replacing VP30_wt with VP30_E197A FLAG . Interestingly, VP30_E197A FLAG was able to support transcription and replication similar to VP30_wt FLAG indicating that interaction of VP30 with NP is not required for these processes (Fig. 3C). To elucidate the role of the NP-VP30 interaction for other steps of the viral life cycle, we used purified trVLPs from the supernatant of producer cells to infect target cells (Fig. 3D, Ref. 19). Infection with trVLPs produced by cells expressing VP30_E197A FLAG instead of VP30_wt did not result in reporter gene activity in target cells, similar to trVLPs containing no VP30 (AVP30). To confirm that this result was not due to the absence or inactivity of the minigenome, we provided the target cells with VP30_wt FLAG in trans, which were then infected with the generated trVLPs. Under these conditions, all trVLPs used for infection-induced reporter gene activity (Fig. 3D, ϩ VP30_wt FLAG ). In target cells complemented in trans with VP30_wt FLAG , trVLPs from cells expressing VP30_E197A FLAG induced approximately the same level of reporter gene activity as trVLPs containing no VP30 or VP30_wt FLAG . We therefore concluded that the amount of minigenomes in the different trVLPs was comparable, further showing that replication of minigenomes in producer cells was not affected by the missing interaction of VP30_E197A FLAG and NP. Similarly, when target cells were provided with VP30_E197A FLAG in trans, all tested trVLPs were able to induce reporter activity (Fig. 3D, ϩ VP30_E197A FLAG ). To understand why trVLPs purified from cells expressing VP30_E197A FLAG were unable to support primary transcription in target cells, we examined their protein composition. Western blot analysis revealed that only traces of VP30_E197A FLAG were recruited into trVLPs (Fig. 3E, lane 5). These data indicate that interaction of VP30 with NP is dispensable for activation of viral transcription and replication, but essential for incorporation of VP30 into trVLPs. Further, interaction of VP30 with NP does not seem to be necessary for the template function of the minigenome, since functional minigenomes were delivered to target cells in the absence of NP-VP30 interaction.
VP30 Does Interact with VP35 in a Phosphorylation-dependent Manner-Although phosphorylation of VP30 influenced its binding to NP, this interaction did neither explain the phosphorylation-dependent effects of VP30 on transcription nor on replication. Nevertheless, it is conceivable that VP30 interacts with the polymerase complex for its direct impact on viral transcription. We, therefore, investigated whether VP30 interacted with the polymerase cofactor VP35. Coimmunoprecipitation analysis of cell lysates expressing VP30_wt FLAG and VP35 HA revealed that, indeed, these proteins do interact with each other (Fig. 4A, lane 6). When trying to confirm this result by immunofluorescence analysis we were faced with the problem that both VP30 and VP35 are diffusely distributed in the cytoplasm preventing colocalization analyses (Fig. 4, BϩC). We therefore took advantage of the ability of NP to recruit VP35 as well as VP30_wt into inclusion bodies (Fig. 4, EϩF, see also Fig. 3A and Ref. 25). We coexpressed NP and VP35 together with VP30_ E197A FLAG which, due to its missing interaction with NP, was not concentrated in inclusions formed by NP alone (Fig. 4G, see also 3A). If VP30_E197A FLAG was recruited into NP-induced inclusions in the presence of VP35, this should be mediated by interaction with VP35. Immunofluorescence analyses revealed colocalization of VP30_E197A FLAG and VP35 HA in the NP mycinduced inclusions, confirming the interaction between VP30 and VP35 (Fig. 4G).
After having identified a complex between VP30 and VP35, it was interesting to test whether phosphorylation of VP30 might interfere with this interaction. Coimmunoprecipitation analyses of VP35 HA with VP30_AA FLAG and VP30_DD FLAG showed that VP35 HA readily coprecipitated VP30_wt FLAG and VP30_ AA FLAG , but only weakly VP30_DD FLAG (Fig. 5A, lanes 3-5). Quantification revealed that interaction between VP35 HA and VP30_AA FLAG was strengthened (155% compared with VP30_ wt), while interaction with VP30_DD FLAG was decreased (40% compared with VP30_wt).
Taken together, these results demonstrate that VP30 interacts with VP35, an essential component of the viral polymerase complex. The interaction between the two proteins is positively influenced when the phosphorylation sites of VP30 contain no charged amino acids, while a negatively charged phosphorylation site mimicking a fully phosphorylated VP30 inhibited interaction with VP35.
In summary, phosphorylation of VP30 modulates viral transcription and replication as well as interaction of VP30 with the polymerase complex protein VP35 and NP. It is suggested that effects of VP30 phosphorylation on viral RNA synthesis are triggered by modulation of the VP30-VP35 interaction, which changes the composition of the polymerase complex.

DISCUSSION
Phosphorylation of cellular proteins is a versatile and widely used tool to regulate their function and it is therefore not surprising that viruses use phosphorylation to modify the function of their proteins, as well. For example, the P proteins within the order Mononegavirales are phosphorylated and play an essential role in viral transcription and replication by acting as polymerase cofactors, analogous to EBOV VP35. Phosphorylation of Rinderpestvirus P protein, e.g. serves as a molecular switch balancing viral transcription and replication (26). Filoviruses represent an exception within the order Mononegavirales by having a non-or very weakly phosphorylated P-analog, VP35, and an additional fourth nucleocapsid protein involved in RNA synthesis, VP30, which is phosphorylated (12,17). The only protein among viruses in the order Mononegavirales with a similar function as VP30 is M2-1 of respiratory syncytial virus (RSV). M2-1 also acts as an essential phosphorylation-dependent viral transcription factor. However, in contrast to VP30 which is active in supporting transcription in its non-phosphorylated state, M2-1 supports transcription elongation and antitermination in its phosphorylated state (27,28). For RSV, the FIGURE 3. Interaction of VP30 and NP is dispensable for viral transcription and replication. A, coimmunofluorescence analysis of VP30_wt FLAG and VP30_E197A FLAG with NP myc . HUH7 cells on glass cover slides transiently expressing FLAG-tagged mutants of VP30 and Myc-tagged NP were fixed at 16 h p.t. VP30 staining was performed using a mouse anti-FLAGா M2 antibody and Rhodamin-conjugated goat anti-mouse. NP myc was stained using a rabbit anti-c-Myc and FITC-conjugated goat anti-rabbit antibody. B, coimmunopreciptiation analysis of VP30_wt FLAG and VP30_E197A FLAG with NP myc . HEK293 cells transiently expressing FLAG-tagged VP30_wt FLAG or VP30 E197A FLAG were lysed at 24 h p.t, and coimmunopreciptation was performed for 4 h using mouse anti-FLAGா M2 agarose. Samples were subjected to SDS-PAGE and Western blotting. VP30 staining was performed using a biotinylated mouse anti-FLAGா BioM2 antibody and Alexa Fluorா 680-conjugated streptavidin. NP myc was stained using a rabbit anti-c-Myc and Alexa Fluorா 680-conjugated goat anti-rabbit antibody. Detection of proteins was obtained with LiCor Odysseyா Imaging Systems. Quantification of three independent experiments is stated on the left. ***: p Յ 0,001. C, reporter gene activity in producer cells. Cells have been transfected as described under Fig. 1B. Reporter gene activity was measured 72 h p.t. using the Dual Luciferase assay (Promega). Results obtained with VP30_wt FLAG were set to 100%. D, reporter gene activity in target cells. trVLPs generated from producer cells (described under C) were purified from supernatants and used for infection of (i) naive target cells, (ii) cells that express VP30_wt FLAG (ii), or VP30_E197A FLAG (iii). Reporter gene activity obtained upon infection with trVLPs containing VP30_wt FLAG was set for each setup to 100%, Ø: without VP30. E, expression and incorporation of VP30_E197A into trVLPs. trVLPs were treated with proteinase K to analyze specific incorporation of VP30 into trVLPs. Western blotting of cell lysate and purified trVLPs was performed using a mouse anti-FLAGா M2 antibody and Alexa Fluorா 680 goat anti-mouse antibody for VP30 staining, GP and NP were detected using a goat anti-GP/NP antibody and Alexa Fluorா 680-conjugated donkey anti-goat antibody. Detection was obtained using LiCor Odysseyா Imaging Systems. APRIL 19, 2013 • VOLUME 288 • NUMBER 16 activity of the transcription complex consisting of M2-1, P, and L has been proposed to be influenced by phosphorylation of the P protein which weakens its interaction with M2-1 resulting in a release of M2-1 from the polymerase complex to facilitate viral replication (29).

Phosphorylation of Ebola Virus VP30
For EBOV, the mechanism that regulates the switch from transcription to replication is currently not understood. Recent experiments demonstrated an increase in EBOV replication upon expression of phosphomimetic VP30_DD in trans (18).
The same mutant has been shown earlier to be unable to support viral transcription (17). These results led us to presume that EBOV transcription and replication might be interconnected. When transcription was switched on in presence of VP30_wt or VP30_AA, mimicking the nonphosphorylated VP30, replication was simultaneously reduced. Vice versa, viral transcription was not activated in presence of VP30_DD, while replication activity was enhanced ( Fig. 1; Ref. 18). The mechanism of how the phosphorylation state of VP30 is conveyed to FIGURE 4. VP30 interacts with VP35. A, coimmunoprecipitation of FLAG-tagged VP30_wt with HA-tagged VP35. HEK293 cells expressing VP30_wt FLAG and VP35 HA were lysed 48 h p.t., and protein complexes were precipitated for 2 h, 4°C using mouse anti-HA agarose. An aliquot for expression control (input) was collected from cell lysate before precipitation. Western blot analysis was performed using a biotinylated mouse anti-HA-Bio antibody and Alexa Fluor 680ாconjugated streptavidin (shown in red). FLAG-tagged VP30 constructs were stained by rabbit anti-FLAGா and IRDyeா 800-conjugated goat anti-rabbit (shown in green). Detection of proteins was obtained with LiCor Odysseyா Imaging Systems. B-E, immunofluorescence analysis. HUH7 cells on glass cover slides expressing VP30_E197A FLAG , NP myc and VP35 HA were fixed at 16 h p.t. Staining of VP30_E197A FLAG was performed using guinea pig anti-VP30 and an Alexaா 488-conjugated goat anti-guinea pig antibody. VP35 HA was stained using mouse anti-HA and Alexaா 594-conjugated goat anti-mouse. NP myc was stained using rabbit anti-c-Myc and AMCA-conjugated horse anti-rabbit. B, single expression of VP30_wt FLAG , VP30_E197A FLAG , VP35 HA , and NP myc . C, coexpression of VP30_wt FLAG and VP35 HA . D, coexpression of VP30_E197A FLAG and NP myc . E, coexpression of VP35 HA and NP myc . F, coexpression of VP30_wt FLAG , VP35 HA , and NP myc . G, coexpression of VP30_E197A FLAG , VP35 HA , and NP myc .
its transcriptional support activity and its effect on viral replication remained unclear. Since phosphorylation of VP30 has an opposing effect on viral transcription and replication, we suggest that this post-translational modification balances viral transcription and replication. One possibility to explain this result is that phosphorylation of VP30 induced changes in the activity of the polymerase complex, triggering a transition between transcription and replication. We, therefore, analyzed whether phosphorylation of VP30 influenced the interaction between protein components of the EBOV RNA synthesis machinery. For the first time, an interaction of VP30 and VP35 was demonstrated, which was reciprocally affected by the phosphorylation state of VP30. Since VP35 is a cofactor of the viral polymerase, and VP30 was shown to be essential for transcription, the phosphorylation-dependent interaction VP35 and VP30 is presumed to push the polymerase complex into a transcription state. We propose that trimeric complexes of the VP30, VP35, and L are formed that represent the viral transcriptase activity, whereas VP30-less complexes of VP35 and L represent replication complexes. Our hypothesis is in line with observations made in the RSV system where the interaction between M2-1 and P seems to determine the balance between transcription and replication (see above) (29). Similarly, for vesicular stomatitis virus, a member of the Rhabdoviridae, two polymerase complexes have been suggested to promote either viral transcription or replication. While transcription is achieved by a complex consisting of phosphorylated P proteins, the polymerase L and also cellular proteins like translation elongation factor-1␣ and heat-shock proteins, viral replication is accomplished by a tripartite complex composed of L and N-P oligomers (30 -32). For EBOV, so far host cell proteins that might be involved in putative transcription or replication complexes have not yet been identified.
Phosphorylation of VP30 might also have effects on its binding to viral RNA. It has been published that VP30 is able to bind specifically EBOV RNA, which is assumed to be essential for its function in transcription activation (15). Since the phosphorylation sites of VP30 overlap with the putative RNA binding site, it is conceivable that phosphorylation of VP30 impairs its interaction with RNA due to electrostatic repulsion. Thus, phosphorylation of VP30 and subsequent dissociation of VP30 from the RNA template might additionally contribute to a release of VP30 from the transcription complex. This assumption needs further investigation.
Our experiments revealed that phosphorylated VP30 was better precipitated by NP than non-phosphorylated VP30 or VP30_wt suggesting the interaction of VP30 with NP is also influenced by VP30 phosphorylation. This result complements earlier findings that showed VP30_DD to be concentrated in the NP-induced inclusions harboring freshly formed nucleocapsids. In contrast, VP30_AA was homogenously distributed throughout the cell (17,33). This leads to the assumption that the phosphorylated VP30 is predominantly associated with nucleocapsids while the non-phosphorylated VP30 interacts with soluble NP.
The study demonstrated that interaction of NP and VP30 is not necessary for viral transcription, but essential for the viral life cycle. This is because of the recruitment of VP30 into trVLPs is necessary to have VP30 at hand supporting primary transcription in the target cell.
Based on data obtained within this study, we propose the following model for VP30 phosphorylation status-dependent transition from transcription to replication (Fig. 6). Non-phosphorylated VP30 helps to initiate viral transcription, and the polymerase complex elongates the nascent RNA to produce capped and polyadenylated mRNAs (17,34). Upon phosphorylation by a yet unidentified cellular kinase VP30 dissociates from the polymerase complex, due to the impaired interaction with VP35 and possibly the viral RNA. Released phosphor- FIGURE 5. Interaction of VP30 and VP35 is dependent on VP30 phosphorylation. A, coimmunoprecipitation of VP30 phosphorylation mutants with VP35 HA . HEK293 cells expressing FLAG-tagged VP30 constructs and HA-tagged VP35 were lysed 48 h p.t., and protein complexes were precipitated for 2 h, 4°C using mouse anti-HA agarose. An aliquot for expression control (input) was collected from the cell lysate before precipitation. Western blot analysis was performed using a biotinylated mouse anti-HA-Bio antibody and Alexa Fluorா 680-conjugated streptavidin (shown in red). FLAGtagged VP30 constructs were stained by a rabbit anti-FLAG antibody and IRDyeா 800-conjugated goat anti-rabbit (shown in green). Detection of proteins was obtained with LiCor Odysseyா Imaging Systems. Precipitation of VP30_wt FLAG by VP35 HA was set to 100%. Quantification of at least three independent experiments is shown to the left. Statistical significance: **, p Յ 0.01. FIGURE 6. Proposed model for VP30 phosphorylation modulating viral transcription and replication. Viral transcription of the NP-encapsidated genome is accomplished by the polymerase L and the polymerase cofactor VP35 together with VP30 (1). Non-phosphorylated VP30 is bound to the viral polymerase complex via VP35. In contrast, viral replication proceeds in the absence of VP30. Phosphorylation of VP30 leads to dissociation of VP30 from the polymerase complex because of the impaired interaction with VP35 and VP30 (2). VP30 remains in contact with the nucleocapsid by its interaction with NP (3). These VP30-less polymerase complexes represent the viral replicase.
ylated VP30 remains bound to the nucleocapsid via a strengthened interaction with NP (17). If VP30 becomes dephosphorylated by the cellular phosphatases PP1 or PP2A, the non-phosphorylated VP30 is again recruited to the polymerase complex to switch on the transcription mode.
In summary, our data suggest that VP30 phosphorylation regulates the transcription and replication activities of the polymerase complex by binding to the polymerase complex via interaction with the polymerase cofactor VP35.