ABSTRACT
Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus transcription and replication currently is not well understood. An important factor regulating viral transcription is VP30, an Ebola virus-specific transcription factor associated with the viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts viral transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports viral transcription. Upon VP30 phosphorylation, viral transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the viral RNA. VP30 thereby is excluded from the viral transcription complex, simultaneously leading to increased viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of viral RNA synthesis, where VP30 is required to initiate primary viral transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the viral life cycle.
IMPORTANCE The current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that the situation is more complex and that primary transcription as well as the rescue of recombinant Ebola virus also requires the transient phosphorylation of VP30. VP30 encodes six N-proximal serine residues that serve as phosphorylation acceptor sites. The present study shows that the dynamic phosphorylation of serine at position 29 alone is sufficient to activate primary viral transcription. Our results indicate a series of phosphorylation/dephosphorylation events that trigger binding to and release from the nucleocapsid and transcription complex to be essential for the full activity of VP30.
INTRODUCTION
Ebola virus (EBOV) contains a single-stranded negative-sense RNA genome and constitutes, together with Marburg virus and Cueva virus, the family of Filoviridae, order Mononegavirales. Filoviruses cause a severe fever accompanied by gastrointestinal symptoms, vascular leakage, multiorgan failure, and shock (1, 2). The reported case fatality rates can reach 90%. Since there is no antiviral therapy or approved vaccine for human use available, filoviruses are classified as biosafety level 4 (BSL4) pathogens and require handling in specialized laboratories.
The seven genes of the 19-kb EBOV genome encode eight proteins and are flanked by nontranslated trailer and leader regions that contain essential cis-active signals for encapsidation, viral transcription, and replication (3–5). The filamentous EBOV particles are composed of the central nucleocapsid surrounded by a layer of the matrix protein VP40 and a lipid envelope in which the surface glycoprotein, GP, is inserted. The helical nucleocapsid is formed by the viral genomic RNA, which is encapsidated by the nucleoprotein, NP, and two associated proteins, VP35 and VP24 (6). Homooligomers of VP35, together with dimers of L, form the polymerase complex, which is associated with the nucleocapsid through the interaction between VP35 and NP (7–9). The EBOV nucleocapsid is completed by VP30, an essential EBOV-specific transcription factor (5, 10).
After EBOV particles have been endocytosed, the nucleocapsid is released into the cytoplasm and serves as a template for primary transcription, which is carried out by nucleocapsid-associated polymerase L, VP35, and VP30. When a sufficient amount of viral proteins have been translated from the newly synthesized viral mRNA, the replication of full-length antigenomes and genomes occurs (5). Newly replicated genomes serve as templates for transcription and subsequent protein synthesis (secondary transcription). The mechanism that drives the very same viral polymerase complex to either mRNA or genome synthesis, a process that ignores all cis-acting transcription start and stop signals encoded by the genome, is poorly understood. For other Mononegavirales, it is suggested that the amount of NP which encapsidates the nascent genomic and antigenomic RNA plays an important role in activating replication (11–13). It is conceivable that once replication of the viral genome is initiated, transcription and replication run in parallel. Whether transcription complexes can be converted into replication complexes and how replication and transcription can be regulated at all currently is unclear.
Previous studies revealed that EBOV VP30 is essential for transcription initiation and reinitiation and that both functions are regulated by phosphorylation (14, 15). VP30 is phosphorylated at six N-proximal serine residues (S29-S31, S42, S44, and S46) and at threonine 143 and 146 (14, 16). By analysis of the mutation of the N-proximal serine residues to either alanine (mimicking nonphosphorylated VP30) or aspartate (mimicking permanently phosphorylated VP30), it was shown that the presence of nonphosphorylated or weakly phosphorylated VP30 is essential for the protein's function in viral transcription (14, 15, 17). The phosphorylation of VP30 leads to a block of viral transcription and favors viral replication, which is accomplished by L and VP35 alone (15, 17, 18). Recent data showed a correlation of VP30 phosphorylation and its interaction with the polymerase cofactor VP35, suggesting a switch from transcription to replication due to the weakened association with the polymerase complex. It is currently not understood whether the absence of VP30 converts the transcriptase complex into a replicase complex or whether, in the absence of transcription complexes, replicase complexes have preferred access to the NP-RNA templates (17, 18). Together, these results underlined the significance of VP30 dephosphorylation for its function in viral transcription. However, although the nonphosphorylatable VP30 perfectly supported viral transcription, it was impossible to generate a recombinant EBOV encoding VP30 with no N-proximal phosphorylation sites, implicating a critical role for VP30 phosphorylation at certain time points of infection (15).
Here, we characterize the significance of the dynamic phosphorylation of VP30 for its function regarding viral transcription at different time points during the viral life cycle. By employing different phosphorylation mutants of VP30, we show that a reversible phosphorylation/dephosphorylation cycle is critical for the cotransport of VP30 with nucleocapsids and initiation of primary viral transcription. We further demonstrate that phosphorylation of one serine residue, S29, is important and sufficient to support the full activity of VP30 in EBOV-infected cells.
MATERIALS AND METHODS
Cell culture.HEK293 (human embryonic kidney), HUH7 (human hepatoma), and Vero E6 cells were cultivated with Dulbecco's modified Eagle medium completed with penicillium and streptomycin, 5 mM glutamine, and 10% fetal calf serum at 37°C and 5% CO2.
Plasmids.All plasmids coding for wild-type (wt) EBOV proteins (pCAGGS VP30, NP, VP35, L, VP24, VP40, and GP) as well as the EBOV-specific minigenome (pANDY 3E5E) and pCAGGS T7 polymerase have been described earlier (17, 19). The VP30 phosphorylation mutants, VP30_AA and VP30_DD, have been described in references 14 and 17. The cloning of all other pCAGGS VP30 phosphorylation mutants (VP30_SA, VP30_AS, VP30_S29, VP30_S30, VP30_S31, VP30_S42, VP30_S44, VP30_S46, VP30_D29, VP30_A29, VP30_A30, VP30_A31, VP30_A42, VP30_A44, and VP30_A46) were performed with the multisite-directed mutagenesis kit (Agilent) according to the manufacturer's recommendations. All pCAGGS VP30 constructs contain a C-terminal FLAG epitope for immunological detection.
Substitutions for serine residues in VP30 in the full-length cDNA (pAMP rgZEBOV) were introduced via QuikChange multisite-directed mutagenesis by using an intermediate plasmid, pKAN, and subsequently were cloned back into the full-length plasmid.
Detailed cloning strategies and primer sequences are available on request. All constructs have been verified by sequencing.
EBOV-specific trVLP assay.An EBOV-specific transcription-replication-competent virus-like particle (trVLP) assay was performed as described in reference 19. The additional transfection of pGL4 (Promega), encoding a firefly luciferase, was performed for the normalization of transfection efficiency. Reporter activity in producer cells was measured 72 h posttransfection (p.t.) using a Dual-Luciferase assay (Promega). Released trVLPs were purified from supernatant via ultracentrifugation over a 20% sucrose cushion and used for the infection of naive or pretransfected indicator cells. An aliquot of trVLPs was analyzed with respect to the incorporation of VP30 mutants by proteinase K digestion assay as described in reference 20. The activity of proteinase K (PK) was verified by adding the detergent Triton X-100 to the reaction, which destroyed the lipid envelope of the trVLPs, exposing the nucleocapsid to the protease and leading to their complete digestion (data not shown). At 60 h postinfection (p.i.), indicator cells were lysed and a Renilla reporter assay (Promega) was performed. Results obtained with wild-type VP30 (VP30_wt) were set to 100%.
EBOV-specific minigenome assay and treatment with okadaic acid.The EBOV-specific minigenome assay was performed as described in reference 19, except for the expression of structural viral proteins (VP40, VP24, and GP). At 18 h p.t., cells were treated either with 25 nM okadaic acid (dissolved in dimethyl sulfoxide [DMSO]) or DMSO (0.05%) alone. At 48 h p.t., cells were lysed and reporter gene activity was monitored via luciferase assay (pjk, Germany). Results obtained with VP30_AA were set to 100%.
Electrophoresis and Western blot analysis.SDS gel electrophoresis and subsequent Western blot analysis were performed as described in reference 21. VP30 was stained using a polyclonal rabbit anti-VP30 antibody (1:100) followed by a goat anti-rabbit Alexa Fluor 680 antibody. NP staining was carried out via an anti-NP from chicken (1:2,000) and goat anti-chicken Alexa Fluor 680, and tubulin was stained as a control with mouse anti-tubulin (Sigma) and goat anti-mouse Alexa Fluor 680. All secondary antibodies were purchased from Invitrogen (Molecular Probes). The detection of antibodies was performed using the Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA).
Indirect immunofluorescence analysis.HUH7 cells were infected with trVLPs (described above). At 22 h p.i., cells were fixed using 4% paraformaldehyde (PFA) in DMEM. The permeabilization of cells and blocking of unspecific signals were performed as described in references 21 and 22. Antibodies were diluted in blocking buffer containing guinea pig anti-VP30 (1:100) and chicken anti-NP (1:500). All secondary antibodies were diluted 1:100 (Invitrogen and Dianova), and DAPI (4′,6-diamidino-2′-phenylindole) was diluted 1:10,000. Pictures were taken at a magnification of ×100.
Rescue of recombinant EBOV.The rescue of recombinant EBOV (Mayinga; GenBank accession number AF086833) was performed in HuH7 cells via transfection of a full-length EBOV cDNA construct under the control of a T7 promoter (pAMP rg ZEBOV, kindly provided from G. Neumann) in the following setup: 250 ng pAMP rg ZEBOV, 250 ng pCAGGS NP, 125 ng pCAGGS VP35, 1,000 ng pCAGGS L, 75 ng pCAGGS VP30, and 250 ng pCAGGS T7. At 7 days p.t. supernatants were submitted to fresh HuH7 cells, and the development of cytopathic effect (CPE) was monitored after 7 to 12 days. In order to verify the recombinant viruses, viral RNA was extracted from supernatant using a QIAamp viral minikit (Qiagen) according to the manufacturer's protocol. Reverse transcription with ensuing PCR steps was performed using a Titan one-tube reverse transcription-PCR (RT-PCR) (Roche) with EBOV-specific primers. The resulting cDNA was verified via sequencing. All work with recombinant EBOV was performed at the BSL4 laboratory of Philipps University Marburg.
TCID50 analysis.Virus titers were determined by 50% tissue culture infectious dose (TCID50) assay in Vero E6 cells as described in reference 23.
Growth kinetics.Growth kinetics of recombinant ZEBOV were determined in 25-cm2 flasks using HuH7 cells at a multiplicity of infection (MOI) of 0.1 or 0.01. Samples from supernatants were collected as indicated. Viral titers were determined by TCID50 analysis in Vero E6 cells. The experiments were done in triplicate.
RESULTS
Dynamic phosphorylation of VP30 is essential for primary viral transcription.In order to analyze whether dynamic phosphorylation/dephosphorylation of VP30 plays a functional role in the EBOV replication cycle, we used previously described mutants of VP30 in which two phosphorylated N-proximal serine clusters were exchanged for either negatively charged aspartate residues that imitate a completely phosphorylated VP30 (VP30_DD) or uncharged alanine residues mimicking a completely nonphosphorylated VP30 (VP30_AA) (14, 15, 17). In addition, we generated two VP30 mutants that encoded phosphorylatable serine residues in one of the two serine clusters mimicking weakly phosphorylated VP30 (Fig. 1A, VP30_AS and VP30_SA). The ability of the mutants to support viral transcription was tested in an EBOV-specific transcription-replication-competent virus-like particle assay (trVLP assay) (Fig. 1B) (19). This assay measures the transcription/replication of an EBOV-specific minigenome driven by the EBOV polymerase. During replication, the nascent minigenome RNA is encapsidated by NP, resulting in the formation of mininucleocapsids, which in turn serve as a template for replication or transcription. Viral transcription is activated in the presence of VP30, resulting in increased activity of Renilla luciferase (producer cells). Under these conditions, EBOV-specific transcription is boosted by the increasing number of templates formed during replication (24). In the presence of the EBOV proteins VP24, VP40, and GP, the mininucleocapsids also can be released into the supernatant, forming trVLPs that can be purified and used to infect fresh target cells (indicator cells).
Dynamic phosphorylation of VP30 is required for primary viral transcription. (A) Schematic presentation of VP30 phosphorylation mutants. VP30 serine clusters were mutated to either uncharged alanine residues mimicking unphosphorylated VP30 or to aspartate residues mimicking permanently phosphorylated VP30. (B) Infectious virus-like (trVLP) particle assay. Transfection of plasmids encoding all viral proteins and an EBOV-specific minigenome carrying a Renilla luciferase as a reporter gene leads to minigenome transcription and replication in producer cells measurable by reporter assay. VP30 was replaced by the respective VP30 phosphorylation mutant as indicated. Moreover, the expression of all viral proteins induces the generation of trVLPs that resemble wild-type virions but contain a minigenome. The trVLPs are released into the supernatant and concentrated via ultracentrifugation. Infection of indicator cells with purified trVLPs results in primary viral transcription supported by the incorporated nucleocapsid proteins. Reporter gene activity again is measured by reporter assay, here reflecting the early stages of an EBOV infection. (C) Reporter gene activity in producer and indicator cells. Producer cells were lysed 72 h posttransfection, and a luciferase assay was performed. The obtained results (in relative light units) were normalized to the activity of a firefly luciferase (pGL4) that was additionally transfected as a control. VP30_wt was set to 100% (producer cells, black bars). Sixty hours postinfection of naive indicator cells, reporter gene activity was measured and the results, obtained with VP30_wt, were set to 100% (indicator cells, striped bars). (D) Expression of VP30 mutants in producer cells and incorporation into trVLPs. Western blot analysis was performed on cell lysates and probed for the expression of VP30 mutants, NP, and tubulin as a control. An aliquot of the purified trVLPs was analyzed for the incorporation of VP30 via a protease protection assay. SDS-PAGE and Western blot analyses were performed and probed for VP30. Upper bands, untreated trVLPs; middle band, treatment with proteinase K (PK); lower bands, treatment with proteinase K and Triton X-100 (T).
As a first step, we analyzed whether the generated phosphorylation mutants of VP30 supported viral transcription in the producer cells (Fig. 1C, black bars). As described before, when all of the potential phosphorylation sites were replaced by aspartate residues (VP30_DD), VP30 was no longer able to support viral transcription (14, 15, 17). In contrast, VP30_AA, mimicking completely dephosphorylated VP30, and the partially phosphorylated VP30_AS and VP30_SA were able to support viral transcription similar to that of VP30_wt (Fig. 1C). The expression of VP30, mutants of VP30, and NP and cellular tubulin has been verified by Western blotting (Fig. 1D, cells).
We next were interested in how the phosphorylation of VP30 affected primary transcription. For this purpose, target cells were infected with trVLPs, which provide a powerful tool to investigate primary viral transcription, since the limited amount of incorporated viral proteins can promote transcription but is not sufficient to induce replication. This situation is different from that in the producer cells, where the abundance of viral proteins mimics the late stages of an EBOV infection when transcription, replication, assembly, and budding take place simultaneously.
The analysis of reporter gene activity in the indicator cells revealed that VP30_DD was inactive in initiating viral transcription, as in the producer cells (Fig. 1C, striped bars). Unexpectedly, VP30_AA, mimicking a nonphosphorylated VP30, also was not able to support primary viral transcription, although this mutant supported viral transcription in the producer cells (Fig. 1C, black bars). Interestingly, VP30 mutants that still contained phosphorylatable serine residues at the first or the second serine cluster (VP30_AS and VP30_SA) were able to support primary transcription (Fig. 1C).
In order to verify that the VP30 mutants were incorporated into trVLPs and thus were available in the indicator cells, we purified trVLPs from the supernatant of the producer cells and subjected them to a protease protection assay with proteinase K and subsequent Western blotting (Fig. 1D, trVLPs). These experiments demonstrated that NP, VP30, and all phosphorylation mutants of VP30 were incorporated into the generated trVLPs to similar amounts, indicating that this step is phosphorylation independent (Fig. 1D, trVLPs, lanes 2 to 6).
In order to confirm that the incorporated minigenome template was functional in the case of VP30_AA, we employed the trVLPs for the infection of indicator cells that were provided with different amounts of VP30_wt in trans to compensate for inactive VP30_AA (Fig. 2A, right scheme) (19). Reporter gene activity obtained by VP30_wt trVLPs was set to 100% for each condition (Fig. 2A, black bars). As a control for reconstituted viral transcription by VP30_wt in trans, we used trVLPs containing no VP30 for infection. Under those conditions, the presence of VP30_wt resulted in a strong dose-dependent induction of viral transcription (Fig. 2A, striped bars). Compared with trVLPs containing no VP30, infection with VP30_AA containing trVLPs revealed strongly impaired viral transcription independent of the amount of VP30_wt expressed in trans (Fig. 2A, gray bars). Although reporter signals were augmented with increasing amounts of VP30_wt, they did not match levels obtained with VP30_wt trVLPs or trVLPs containing no VP30. The same effect was detected when we provided the indicator cells with different amounts of VP30_AA in trans (Fig. 2B). Although the provision of VP30_AA in trans could rescue primary transcription, reporter gene levels obtained upon infection with VP30_AA trVLPs were 10-fold below levels obtained with trVLPs containing no VP30 (Fig. 2B, gray and striped bars). This effect was not related to differences in the amount of incorporated minigenomes, which was analyzed by quantitative RT-PCR (not shown).
Reporter gene activity in pretransfected indicator cells. (A) Reporter gene activity in VP30_wt pretransfected indicator cells. trVLPs were produced as described in the legend to Fig. 1B and contained either VP30_wt, VP30_AA, or no VP30. Indicator cells were transfected with different amounts of VP30_wt in trans as indicated. Cells were infected with trVLPs, and reporter gene activity was measured 60 h p.i. Results obtained with VP30_wt containing trVLPs were set to 100%. (B) Reporter gene activity in VP30_AA pretransfected indicator cells. trVLPs were produced as described in the legend to Fig. 1B and contained either VP30_wt, VP30_AA, or no VP30. Indicator cells were transfected with different amounts of VP30_AA in trans as indicated. Cells were infected with trVLPs, and reporter gene activity was measured 60 h p.i. Results obtained with VP30_wt containing trVLPs were set to 100%. (C) Immunofluorescence analysis of naive indicator cells infected with trVLPs. trVLPs were produced as described in the legend to Fig. 1B and contained either VP30_wt, VP30_AA, or no VP30. HuH7 indicator cells were infected and fixed for immunofluorescence analysis 22 h postinfection. The staining of NP was achieved with chicken anti-NP and goat anti-chicken Alexa 488 antibodies. VP30 was stained with a guinea pig anti-VP30 and goat anti-guinea pig Alexa 594 antibody. Nuclei were stained using DAPI (4′,6-diamidino-2′-phenylindole). Pictures were taken at ×100 magnification in close proximity of the nucleus (blue).
Previous data revealed a strong phosphorylation-dependent interaction of VP30 with NP (14), which can be monitored by the recruitment of VP30 into NP-induced inclusion bodies that represent places of viral RNA synthesis (25). In contrast, dephosphorylated VP30 (VP30_AA) is not concentrated in NP-induced inclusion bodies (14). In order to test whether VP30_AA is associated with intruding nucleocapsids, we performed immunofluorescence analysis in indicator cells infected with trVLPs containing either VP30_wt, VP30_AA, or no VP30 (Fig. 2C). We analyzed the colocalization of nucleocapsid-induced inclusion bodies and VP30. While we could detect the colocalization of NP and VP30_wt in inclusion bodies next to the nucleus, we were not able to detect the colocalization of VP30_AA with NP, although VP30_AA was incorporated into the trVLPs (Fig. 1D). These data indicate that VP30_AA is not cotransported with the intruding nucleocapsids to the sites of viral transcription due to the missing phosphorylation.
Taken together, these results suggest that phosphorylation is important for the cotransport of VP30 together with the nucleocapsid, which ensures its presence at the sites of viral transcription where dephosphorylated VP30 is essential. Although mutants of VP30 with static charges at the phosphorylation sites are able to activate viral transcription under specific experimental settings, especially when the supply of viral proteins is not a limiting factor (19, 24), the whole range of VP30 activities can be put into effect only if VP30 contains at least some phosphorylatable serine residues in the N-proximal serine clusters. An important difference between the above-described mutants and wild-type VP30 is the ability of the latter to undergo cycles of phosphorylation and dephosphorylation.
The first serine cluster of VP30 is predominantly phosphorylated.We next were interested in determining whether the phosphorylation of either the first or the second serine cluster of VP30 was particularly important for the phosphorylation-mediated regulation of transcription activation. For this purpose, we used okadaic acid (OA), an inhibitor of the phosphatases PP1 and PP2A, which were shown to dephosphorylate VP30 (Fig. 3A) (14). Treatment with OA results in a hyperphosphorylated form of VP30 which is transcriptionally inactive (Fig. 3A, upper) (14). In contrast, mutants of VP30 that are not phosphorylatable (VP30_AA) are not impaired by OA and remain active (Fig. 3A, lower) (14). Since higher concentrations of OA are cytotoxic, the unaffected function of VP30_AA in the presence of OA served as the control. By employing VP30_AS and VP30_SA, we sought to determine whether the phosphorylation of one of the clusters is more important for the transcription activation function of VP30. We used the EBOV-specific minigenome assay and subjected the producer cells to treatment with either DMSO (control) or 25 nM OA (5, 14). The activity of the nonphosphorylatable VP30_AA was set to 100% (Fig. 3B). Upon treatment with OA, the transcriptional support activity of VP30_wt was dramatically reduced, comparable to the activity of VP30_DD (Fig. 3D, gray bars). When VP30_SA or VP30_AS was employed in this setting, reporter gene activity was significantly reduced with VP30_SA, but only a slight reduction was detectable with VP30_AS. Therefore, we concluded that the first serine cluster is phosphorylated predominantly during the viral life cycle, and/or phosphorylation/dephosphorylation of the first cluster is functionally more important.
Phosphorylation of the first serine cluster is important for transcriptional activity. (A, upper) Treatment of VP30_wt with okadaic acid (inhibitor of PP1 and PP2A) results in a hyperphosphorylated VP30 which is transcriptionally inactive. (Lower) VP30 mutants that contain no phosphorylatable serine residues (e.g., VP30_AA) are not (hyper)phosphorylated upon okadaic acid treatment. They stay nonphosphorylated; hence, they are transcriptionally active. (B) Reporter gene activity of VP30 phosphorylation mutants in producer cells upon okadaic acid treatment. Cells were transfected with all components of an EBOV-specific minigenome assay. At 18 h p.t., cells were treated with 25 nM okadaic acid or DMSO (0.05%) as a control. Forty-eight hours posttransfection, cells were lysed and reporter gene activity was measured by luciferase assay. Since the transcriptional activity of VP30_AA is not influenced by okadaic acid treatment, we set levels obtained with VP30_AA to 100%.
Dynamic phosphorylation of serine 29 is sufficient to render VP30 active in primary viral transcription.In order to investigate the role of individual serine residues for the viral life cycle, we generated VP30 mutants that contained only one of the six phosphorylatable serine residues, while all other serines in the clusters were mutated to alanine (Fig. 4A). These mutants were employed to replace VP30_wt in the EBOV-specific trVLP assay. All single-serine VP30 mutants were able to support viral transcription in the producer cells as well as VP30_wt or VP30_AA (Fig. 4B, black bars). The expression of all VP30 mutants in producer cells and their incorporation into trVLPs was verified by Western blot analysis as described above (Fig. 4C). Upon infection of naive indicator cells with the trVLPs containing either of the single-serine VP30 mutants, only VP30_S29 was able to support primary viral transcription, while all other mutants were not (Fig. 3B, striped bars).
Dynamic phosphorylation of VP30 serine 29 is sufficient to activate primary viral transcription. (A) Schematic presentation of VP30 single-serine mutants. (B) Reporter gene activity in producer cells (black bars) and indicator cells (striped bars). VLP assays were performed as described in the legend to Fig. 1B. Producer cells were lysed 72 h posttransfection, and a luciferase assay was performed. VP30_wt levels were set to 100%. Indicator cells were lysed 60 h postinfection with trVLPs. A Renilla luciferase assay was carried out, and results obtained with VP30_wt were set to 100%. (C) Expression of VP30 mutants and NP in producer cells and incorporation into trVLPs. Western blot analysis was performed on cell lysates and probed for the expression of VP30 mutants and NP. An aliquot of the purified trVLPs was analyzed with respect to the incorporation of VP30 and NP via protease protection assay. SDS-PAGE and Western blotting were performed and probed for VP30. Upper bands, untreated trVLPs; middle band, treatment with proteinase K (PK). The activity of the protease was verified by subjecting trVLPs containing VP30_wt to treatment with proteinase K and Triton X-100 (not shown). (D) VLP assay with VP30 phosphorylation mutants as described for panel B. (E) Immunofluorescence analysis of naive indicator cells infected with trVLPs. trVLPs were produced as described in the legend to Fig. 1B and contained either VP30_S29, VP30_D29, or a combination of VP30_D29 and VP30_AA. HuH7 indicator cells were infected and fixed for immunofluorescence analysis 22 h postinfection. The staining of NP was achieved with chicken anti-NP and goat anti-chicken Alexa 488 antibodies. VP30 was stained with a guinea pig anti-VP30 and goat anti-guinea pig Alexa 594 antibody. Nuclei were stained using DAPI. Pictures were taken at ×100 magnification in close proximity to the nucleus (blue).
In order to analyze whether a constant negative charge at position 29 supported VP30 activity in primary viral transcription rather than dynamic phosphorylation, we used VP30_D29, a mutant which contained a negatively charged aspartate at position 29 (Fig. 4A). While VP30_D29 supported viral transcription in producer cells (Fig. 4D, black bars), it failed in supporting primary viral transcription in indicator cells (Fig. 4D, striped bars). To analyze whether it is possible to gain full functionality of VP30 when charged and uncharged phosphorylation sites are simultaneously available in separate molecules, the trVLP assay was performed in the presence of VP30_AA and VP30_D29. Interestingly, this combination could not restore primary viral transcription in indicator cells (Fig. 4D, D29 + AA, striped bars). To test whether this could be related to an inefficient transport of VP30 phosphorylation mutants to the sites of viral RNA synthesis, we performed immunofluorescence analyses in indicator cells staining VP30 and NP (Fig. 4E). While we were able to detect VP30_S29 colocalizing with NP in inclusion bodies like VP30_wt (see Fig. 2C), we were not able to detect a signal for VP30_D29 or for the combination VP30_AA and VP30_D29.
These data indicate that a serine at position 29 is important and sufficient for the phosphorylation-dependent transcriptional support activity of VP30, and phosphorylation/dephosphorylation events need to occur successively in one VP30 molecule.
We then sought to understand whether the phosphorylation of a single serine residue is sufficient to render VP30 inactive. Therefore, the single-serine VP30 mutants described above were tested in a minigenome assay in the presence of the phosphatase inhibitor OA (Fig. 3A and 4A). We did not observe an effect upon the phosphorylation of the single serine residues on reporter gene activity (data not shown), which suggests that the phosphorylation of one phosphoacceptor site is not sufficient to inactivate VP30. We then generated mutants of VP30 that replaced only one serine of the cluster with alanine (Fig. 5). With these single-alanine mutants, we tested whether the absence of phosphorylation at a specific serine could prevent the inactivation of VP30 in the presence of OA. All single-alanine mutants of VP30 were sufficient to support viral transcription (Fig. 5, white bars). As expected, in the presence of OA, reporter gene activity was dramatically decreased with most of the single-alanine VP30 mutants (Fig. 5, gray). Only VP30_A29 and VP30_A31 still were able to support viral transcription in the presence of OA. While the phosphorylation of serine 29 or 31 alone was not sufficient to inactivate VP30 (VP30_S29 and VP30_S31, data not shown), their phosphorylation in concert with the other serine residues contributed significantly to the negative effect of phosphorylation on VP30-mediated viral transcription activation (VP30_A29 and VP30_A31).
Phosphorylation of serine 29 is important for transcriptional activity. (Left) Schematic presentation of VP30 single alanine mutants. (Right) Reporter gene activity of VP30 phosphorylation mutants in producer cells upon okadaic acid treatment (described in the legend to Fig. 3A). Cells were transfected with all components of an EBOV-specific minigenome assay as described in the legend to Fig. 3B. At 18 h p.t., cells were treated with 25 nM okadaic acid or DMSO (0.05%) as a control. At 48 h p.t., cells were lysed and reporter gene activity was measured by luciferase assay. Since the transcriptional activity of VP30_AA is not influenced upon okadaic acid treatment, we set levels obtained with VP30_AA to 100%.
Taken together, these data indicate that the dynamic phosphorylation of VP30 is essential to support the full activity of VP30 in viral transcription, and the phosphate acceptor site S29 as well as S31 are sufficient to mediate this effect.
Phosphorylation of serine 29 is sufficient to rescue recombinant EBOV.Previous studies indicated that the generation of recombinant EBOV was impossible when the VP30 gene did not encode phosphorylatable serines (VP30_AA) (15). This result, in combination with the data presented in Fig. 4 and 5, led us to hypothesize that a single serine residue at position 29 should be sufficient to rescue recombinant EBOV, while a single serine at position 30 should not. To verify this hypothesis, we constructed full-length cDNA clones of the EBOV genome encoding VP30 with a single serine at either position 29 (recEBOV_S29) or position 30 (recEBOV_S30). All other serines within the two clusters were mutated to alanine residues (Fig. 4A). HuH7 cells were transfected with the full-length cDNA along with helper plasmids expressing T7 polymerase, NP, VP35, L, and VP30. Since plasmid-derived VP30_wt can be incorporated into progeny viruses and thus confound the results, in separate experiments we replaced VP30_wt with VP30_S29 or VP30_S30 as helper plasmids. After 7 days, supernatants were transferred to fresh cells (passage 1) to monitor the established infection. The successful recovery of the recombinant virus was evaluated by the development of cytopathic effects (CPE) in passage 1 cells, as well as the sequencing of the isolated viral RNA and detection of viral proteins in the supernatant. We were able to rescue recombinant EBOV_wt and EBOV_S29 using wild-type helper proteins in all three attempts, which was verified by the development of CPE (Fig. 6A and B) as well as the presence of recombinant viral RNA and viral proteins in the supernatant (Fig. 6G, lanes 1 to 6). Additionally, the rescue of recEBOV_S29 in the presence of VP30_S29 as a helper protein also was successful (Fig. 6C and G, lanes 7 to 9). In contrast, we were not able to rescue recEBOV encoding only serine 30 as the phosphorylation site either in the presence of VP30_wt (Fig. 6D and H, lanes 4 to 6) or with VP30_S30 as the helper protein (Fig. 6E and H, lanes 7 to 9). These data show that dynamic phosphorylation at one phosphoacceptor site, serine 29, is necessary and sufficient for the generation of recEBOV_S29, indicating that VP30_S29 is sufficient to support the whole spectrum of VP30 functions necessary for the viral replication cycle.
VP30 serine 29 is sufficient for the generation of a recombinant EBOV. Rescue of recombinant EBOV. Transfection of nucleocapsid proteins together with a full-length EBOV cDNA clone (passage 0) results in viral full-length transcripts and consecutive synthesis of all viral proteins in order to produce recombinant EBOV. Rescues were performed in triplicate using a wild-type full-length cDNA clone and clones carrying serine 29 or serine 30 as the only possible phosphate acceptor site within VP30. Successful rescues were monitored for the development of cytopathic effects (CPE) after passaging the supernatants to fresh cells. (A) CPE development of recEBOV_wt rescued by wild-type nucleocapsid proteins. (B) CPE development of recEBOV_S29 rescued by wild-type nucleocapsid proteins. (C) CPE development of recEBOV_S29 rescued by VP30_S29 and wild-type nucleocapsid proteins. (D) CPE development of recEBOV_S30 rescued by wild-type nucleocapsid proteins. (E) CPE development of recEBOV_S30 rescued by VP30_S30 and wild-type nucleocapsid proteins. (F) Negative control. CPE development of recEBOV_wt rescued by wild-type nucleocapsid proteins without L. (G) Analysis of recEBOV_wt and recEBOV_S29 viral RNA in the supernatant (upper) and viral proteins (NP, VP40, and VP30) in the supernatant by Western blot analysis (lower). (H) Analysis of recEBOV_wt and recEBOV_S30 viral RNA (upper) and viral proteins (NP, VP40, and VP30) in the supernatant by Western blot analysis (lower). (I) Summary of three individual rescue experiments with recEBOV_wt, recEBOV_S29, and recEBOV_S30, respectively.
In order to analyze the growth kinetics of recEBOV_S29, cells were infected at a multiplicity of infection (MOI) of 0.1 (Fig. 7A) or with 0.01 TCID50 per cell (Fig. 7B), and samples of the supernatant were collected from day 0 to day 8 postinfection and subjected to TCID50 and Western blot analysis. RecEBOV_S29 showed slightly reduced titers at an MOI of 0.1 at very early time points compared to recEBOV_wt, which also was reflected by slightly less viral protein expression in cell lysates and supernatants (Fig. 7A). Comparable titers of recEBOV_S29 and recEBOV_wt were observed at later time points (Fig. 7A). The same results were observed at an MOI of 0.01 (Fig. 7B). These data indicate that recEBOV_S29, which contains only one phosphorylation site at serine 29, has growth characteristics similar to those of recEBOV_wt.
Characterization of recombinant EBOV. (A) Growth kinetics of recombinant viruses in HuH7 cells at an MOI of 0.1. Samples of supernatants were collected every day as indicated and analyzed via TCID50 analysis in Vero E6 cells. Aliquots were taken for Western blot analysis from cells and supernatant. (B) Growth kinetics of recombinant viruses in HuH7 cells at an MOI of 0.01. Samples of supernatants were collected every day as indicated and analyzed via TCID50 analysis in Vero E6 cells. Aliquots were taken for Western blot analysis from cells and supernatant.
Taken together, this study illustrated that transcription factor VP30 of EBOV requires sequential phosphorylation and dephosphorylation events at two N-proximal serine clusters to gain its full functionality for primary and secondary transcription. Most sensitive is serine at position 29 in VP30. Even in the absence of other phosphorylatable serine residues, the dynamic phosphorylation of serine 29 is sufficient to mediate all functions of VP30, which was finally demonstrated by a recombinant EBOV expressing VP30 with only serine at position 29.
DISCUSSION
The function of VP30 is to activate EBOV-specific transcription at the first gene of the genome and to support transcription reinitiation at the boundaries of downstream genes (14, 15). The hyperphosphorylation of VP30, which was achieved by inhibiting VP30-recognizing phosphatases, has been shown previously to downregulate the transcriptional support activity of VP30 (14). This effect could be mimicked by a VP30 mutant whose phosphorylated serines were replaced by aspartate residues (VP30_DD) (14). In contrast, a mutant of VP30 with all phosphorylatable serines replaced by alanine supported viral transcription even better than VP30 (Fig. 1C) (14). The present study indicates that the mechanism of how the phosphorylation of VP30 impacts transcription is probably more complex than anticipated. Static charges induced by alanines or aspartates at the phosphorylation sites of VP30 are well tolerated in the producer cells, except when all phosphorylatable serines were exchanged by aspartate residues. In this respect, the inability of the uncharged and nonphosphorylatable VP30_AA to support primary transcription (Fig. 1C, striped bars) was unexpected, as under conditions where viral proteins and templates are abundant, VP30_AA supported viral transcription even better than VP30_wt. Attempts to restore primary transcription either by simultaneous expression of permanently charged or uncharged VP30 mutants or by providing indicator cells with VP30_wt in trans failed (Fig. 2). These findings are supported by previous studies showing that a recombinant EBOV carrying no phosphorylation acceptor sites (VP30_AA) could not be generated (15). The vulnerable early steps of infection, when viral transcription is exclusively dependent on viral proteins associated with the intruding virions, obviously requires a cycle of phosphorylation and dephosphorylation of VP30 to support primary transcription.
We could further demonstrate the significance of serine 29 for the activity of VP30. If there is only one possible phosphate acceptor site present, serine 29 seems to be sufficient to mediate the full transcriptional support activity of VP30, while all other serine residues show only background activity. The fact that the rescue of recombinant EBOV encoding VP30 with serine 29 as a single phosphorylation site exhibited growth kinetics comparable to those of wild-type EBOV supports this idea. Nevertheless, the dynamic phosphorylation of serine 29 is not absolutely essential for primary viral transcription, since VP30_AS also is able to support primary viral transcription in a trVLP assay. Most likely, the preferentially used phosphorylation sites for an unknown cellular kinase(s) lies within the first serine cluster, including serine 29, but cellular kinase(s) might still be able to phosphorylate the second serine cluster if no other serines are available.
Interestingly, trVLPs containing VP30_AA were not able to support primary transcription. This result correlated with the inability of VP30_AA to colocalize with the nucleocapsids in the indicator cells. Therefore, it is suggested that the phosphorylation of VP30 is important for its cotransport with nucleocapsids to the sites of transcription, where VP30 needs to be dephosphorylated to initiate transcription. Support for this scenario comes from previously published data showing that the phosphorylation of VP30 enhances the interaction with NP, and interaction with NP is important for the recruitment of VP30 into newly formed EBOV particles (17). At the same time, primary transcription of trVLPs containing VP30_AA could not be efficiently rescued by trans-complementing large amounts of VP30 compared to those of trVLPs containing no VP30. This suggests that phosphorylation, in addition to ensuring VP30's transport, is necessary for another function of VP30 in the activation of transcription. It is tempting to speculate that the successful initiation of transcription is dependent on the dynamic binding and release of VP30 from the nucleocapsid, which is regulated by the phosphorylation/dephosphorylation of the N-proximal serine clusters.
The phosphorylation of viral proteins is an important regulatory mechanism. Prime examples in the order Mononegavirales are the family of P proteins which are highly phosphorylated, and the phosphorylation of P plays a critical role in the regulation of viral RNA synthesis in complex with the polymerase L and the nucleoprotein N (or NP) (26–32). However, filoviruses represent an exception among the Mononegavirales; VP35, the P protein homologue, is only very weakly phosphorylated, and the potential role of VP35 phosphorylation for viral replication is unclear (5, 33). With VP30, filoviruses encode an additional nucleocapsid-associated protein that acts as a viral transcription factor whose activity is strongly dependent on its phosphorylation (14, 34).
The only other viral protein in the order Mononegavirales with functions similar to those of EBOV VP30 is the M2-1 protein of respiratory syncytial virus (RSV). Like VP30, M2-1 supplements the complex of N, P, and L and was shown to be a phosphorylation-dependent viral transcription factor by acting as an elongation and antitermination factor (35–38). In contrast to EBOV VP30, M2-1s transcriptional support activity is not completely inhibited when it is phosphorylated (39, 40). On the other hand, continuous dephosphorylation mimicked by phospho-ablatant mutation (serine to alanine) did not fully support transcription to M2-1 wild-type levels. In fact, recent data showed that M2-1, like VP30, undergoes a dynamic reversible phosphorylation that is essential for its function in transcription antitermination, presumably by regulating the binding of unknown ligands (40). While M2-1 and VP30 share several similarities in structure and function, there are also differences, and the exact mechanism of how these two proteins support viral transcription still is unclear. It is, however, evident that both proteins use a cycle of phosphorylation/dephosphorylation to accomplish their complete functionality as virus-specific transcription factors.
The results presented in the current study support the following hypothesis concerning the mechanism of VP30-mediated transcription activation (Fig. 8). Upon entry of the virions, the nucleocapsid is released into the cytoplasm of the cell and transported to sites of viral RNA synthesis (steps i and ii). Phosphorylation of VP30 is essential to ensure VP30's close association and cotransport with the nucleocapsid. Nonphosphorylated VP30, in contrast, can easily fall off the nucleocapsid during transport and therefore is not available for primary viral transcription (step iii). For the initiation of viral transcription, VP30 requires a dephosphorylation step (step iv) in order to become fully transcriptionally active, resulting in association with VP35 and L at the transcription start site (step v).
Model of VP30 phosphorylation during viral transcription. (i) Nonphosphorylated and phosphorylated VP30 [marked with a (P)] are incorporated into EBOV particles. (ii) Release of the nucleocapsid into the cytoplasm of the cell and transport to the site of primary transcription. (iii) Phosphorylated VP30 stays tightly bound to the nucleocapsid due to the interaction with NP. Nonphosphorylated VP30 falls off the nucleocapsid during transport and is not available for transcription. (iv) Phosphorylated VP30 is dephosphorylated by cellular phosphatases PP1 and PP2A, resulting in a transcriptionally active VP30. (v) Nonphosphorylated VP30 is integrated via the interaction with VP35 into an active transcription complex.
In conclusion, our data demonstrate that transcription and replication of EBOV seem to be intensely regulated processes that are dependent on the dynamic phosphorylation of VP30.
ACKNOWLEDGMENTS
All work with recombinant Ebola virus was performed at the BSL4 facility of the Philipps University of Marburg. We thank Markus Eickmann, Michael Schmidt, and Gotthard Ludwig for facilitating experiments conducted in the BSL4 laboratory, as well as Astrid Herwig and Jens Dorna for excellent technical assistance.
This work was supported by funding from the Deutsche Forschungsgemeinschaft (DFG) through Collaborative Research Center (CRC) grant CRC 1021, subproject A02 (to N.B. and S.B.), and the German Center for Infection Research (DZIF), section Emerging Infections (to N.B., S.B., and C.L.).
FOOTNOTES
- Received 30 December 2015.
- Accepted 21 February 2016.
- Accepted manuscript posted online 2 March 2016.
- Address correspondence to Stephan Becker, becker{at}staff.uni-marburg.de.
Citation Biedenkopf N, Lier C, Becker S. 2016. Dynamic phosphorylation of VP30 is essential for Ebola virus life cycle. J Virol 90:4914–4925. doi:10.1128/JVI.03257-15.
REFERENCES
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