Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle

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.

FIG 1

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).

FIG 2

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).

FIG 3

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%.

FIG 4

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).

FIG 5

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%.

FIG 6

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.

FIG 7

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.

FIG 8

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.