1. Volume 69, Issue 1, Pages 136-145.e6 (January 2018)
The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30 
Thomas Kruse, Nadine Biedenkopf, Emil Peter Thrane Hertz, Erik Dietzel, Gertrud Stalmann, Blanca López-Méndez, Norman E. Davey, Jakob Nilsson, Stephan Becker 
Molecular Cell 
Volume 69, Issue 1, Pages e6 (January 2018)
DOI: /j.molcel
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2. Molecular Cell 2018 69, 136-145.e6DOI: (10.1016/j.molcel.2017.11.034)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Ebola Virus NP Contains a Functional and Important PP2A-B56-Binding Site
(A) Schematic of the EBOV NP with N-terminal domain (NTD), C-terminal domain (CTD), and the intrinsically disordered region (IDR) indicated. The position, sequence, and distance in amino acids of the LxxIxE and PPxPxY motifs in EBOV, MARV, and LLOV are indicated below.
(B) KD values obtained by ITC measurements with full-length recombinant B56α and the indicated NP peptides.
(C) The indicated myc-tagged NP constructs were transfected into a stable HeLa cell line expressing YFP-tagged B56α, and B56α-interacting proteins were isolated using a YFP affinity resin. The purifications were analyzed by western blotting for the indicated proteins (representative of 3 independent experiments).
(D) The indicated myc-tagged versions of NP and YFP-tagged versions of B56α were expressed in HeLa cells, and the cells were fixed and analyzed by immunofluorescence. B56α was visualized using a YFP antibody (green), NP using a myc antibody (red), and DNA was stained with DAPI (blue). Scale bar, 5 μm.
(E) Quantification of B56α intensity (YFP signal) in NP inclusions (myc signal) from images as in (D). The z stacks with 200 nm apart through the cell were projected in a single plane and quantified using SoftWorx. The B56α signal was normalized to the signal from NP, and at least 20 NP inclusions from 5 different cells were quantified and individual values represented by dots. The mean and SD are indicated.
(F) HEK293 cells were transfected with plasmids encoding all viral proteins (VP30, VP35, polymerase L, VP24, VP40, GP, and NPwt or NPΔB56) and a reporter minigenome encoding luciferase flanked by viral regulatory elements under the control of T7 promoter, T7 polymerase, and a firefly-encoding plasmid (pGL4) for normalization (transcription and replication-competent [tr]VLP assay). At 72 hr after transfection, luciferase activity was measured, reporting on both viral transcription and replication. The luciferase values from NPwt-transfected cells was set to 100%. The mean and SD from 3 independent experiments are indicated.
(G and H) HEK293 cells were transfected with plasmids encoding viral nucleocapsid proteins (VP30, VP35, polymerase L, and NPwt or NPΔB56) and a reporter minigenome encoding luciferase flanked by viral regulatory elements under the control of T7 promoter, T7 polymerase, and a firefly-encoding plasmid (pGL4) for normalization (minigenome assay). RNA was isolated and was analyzed by 2-step strand-specific qRT-PCR to measure the amount of viral cRNA/mRNA (G) and the amount of viral genomic RNA (H). The samples containing NPwt were set to 100%. The mean and SD from 3 independent experiments are indicated.
(I) trVLPs from the cells in (F) were used to infect fresh indicator cells, and luciferase activity was measured 60 hr after infection. The luciferase for NPwt was set to 100%. The mean and SD from 3 independent experiments are indicated.
(J) As in (I) but indicator cells were either pre-transfected with NPwt or NPΔB56 24 hr prior to infection with trVLPs containing either NPwt or NPΔB56. Luciferase activity was measured 60 hr later, and luciferase activity of NPwt trVLPs was set to 100%. The mean and SD from 3 independent experiments are indicated.
∗∗∗p < 0.001; n.s., not significant (unpaired t test). See also Figures S1 and S2.
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4. Figure 2
PP2A-B56 Bound to NP Controls VP30 Dephosphorylation to Activate Transcription
(A) HEK293 cells were transfected with the indicated mutants of NP and VP30. At 48 hr after transfection, luciferase activity was measured, and the luciferase values from NPwt-transfected cells were set to 100%. The mean and SD from 3 independent experiments are indicated.
(B and C) RNA samples from (A) were analyzed by 2-step strand-specific qRT-PCR to measure the amount of viral cRNA/mRNA (B) and the amount of viral genomic RNA (C). The samples containing NPwt were set to 100%. Data points from Figures 1G and 1H are included here for comparison to conditions with VP306A. All data points arise from the same experiment. The mean and SD from 3 independent experiments are indicated.
(D) HeLa cells were transfected with plasmids expressing the indicated proteins, and 48 hr later a total cell extract was made and analyzed by western blotting for the indicated proteins and VP30 Ser29 phosphorylation (VP30 pSer29).
(E) The level of VP30 Ser29 phosphorylation was quantified using LI-COR technology normalized to total VP30 levels obtained by probing for myc. The mean and SD from 3 independent experiments are indicated.
(F and G) Cells treated as in (D) but analyzed by immunofluorescence microscopy (F). The level of VP30 Ser29 phosphorylation was quantified in NP inclusions and normalized to the total level of VP30 obtained from the myc signal (G). At least 30 NP inclusions from 5 different cells were quantified and individual values are indicated. The mean and SD are indicated. Scale bar, 5 μm.
(H and I) The indicated purified recombinant MBP-VP30 (8–272) proteins were phosphorylated by PKA in the presence of radioactive ATP (H). The phosphorylated MBP-VP30 (8–272) wild-type (WT) protein was incubated with purified PP2A-B56 and either 5 μM GST-NPwt 533–643 or GST-NPΔB56 533–643 and analyzed at the indicated time points (I). The intensity from autoradiograms of phosphorylated MBP-VP30 (8–272) (It) was normalized to the value prior to the addition of PP2A-B56 (I0), and the data were fitted to models of exponential decay. Data points represent mean and SD of 2 independent experiments. The experiment was conducted four times with comparable results. Difference between slopes was tested using extra sum of squares F-test.
ND, not determined; ∗∗∗p < 0.001; n.s., not significant (unpaired t test). See also Figure S2.
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5. Figure 3 NP Scaffolds the Dephosphorylation of VP30
(A) HeLa cells stably expressing inducible YFP (Ctrl) or YFP-B56α were transfected with myc-tagged VP30 (lanes 1 and 2) alone or myc-tagged VP30 together with myc-tagged NP (lanes 3–6); 48 hr after transfection, B56α complexes were captured on a YFP affinity resin and analyzed by western blotting. Representative of 3 independent experiments.
(B and C) HeLa cells were transfected with the indicated plasmids; 48 hr later a total cell extract was made and analyzed by western blotting for the indicated proteins and VP30 Ser29 phosphorylation (VP30 pSer29) (B) and quantified using LI-COR technology (C). The level of Ser29 phosphorylation was normalized to total VP30 levels obtained by probing for myc. The mean and SD from 3 independent experiments are indicated.
(D) HeLa cells transfected with plasmids encoding the indicated myc-tagged VP30 proteins and YFP-NPΔVP30 were analyzed by immunofluorescence for VP30 Ser29 phosphorylation and total VP30 levels (myc). Scale bar, 5 μm.
(E) The level of VP30 Ser29 phosphorylation was quantified in NP inclusions and normalized to the total level of VP30 obtained from the myc signal in (D). At least 25 NP inclusions from 5 different cells were quantified and individual values are indicated. The median and interquartile range are indicated.
(F and G) HeLa cells transfected with plasmids encoding myc-tagged VP30 and the indicated NP proteins either tagged with myc or YFP. At 48 hr after transfection, a total cell extract was made and analyzed for VP30 Ser29 phosphorylation and total VP30 levels by western blotting (F). The level of VP30 Ser29 phosphorylation was normalized to the total amount of VP30 and the level of phosphorylation set to 1 in the absence of NP protein (G). The mean and SD from 3 independent experiments are indicated.
∗∗∗p < (unpaired t test). See also Figure S3.
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6. Figure 4
A PP2A-B56 Inhibitor Blocks Ebola Virus Transcription and Proliferation in Cells
(A) KD values of B56α binding to the indicated peptides measured by ITC.
(B) HeLa cells were transfected with plasmids encoding the YFP-LxxIxE B56 inhibitor or the control inhibitor YFP-AxxAxA, and a YFP affinity resin was used to purify the inhibitor from cells. The purifications were analyzed by western blotting. Representative of 3 independent experiments.
(C and D) HeLa cells were transfected with plasmids encoding the indicated proteins, and total lysates were analyzed for VP30 Ser29 phosphorylation and the indicated proteins 48 hr after transfection (C). The western blots were quantified using LI-COR technology, and the VP30 Ser29 phosphorylation intensity was normalized to total VP30 level. The mean and SD from 3 independent experiments are indicated (D).
(E and F) HeLa cells transfected with plasmids encoding the indicated proteins were analyzed by immunofluorescence microscopy for VP30 expression and VP30 Ser29 phosphorylation as well as expression of the YFP-LxxIxE B56 inhibitor, the YFP-AxxAxA control inhibitor (YFP signal), and NP (E). At least 30 NP inclusions from 5 different cells were quantified and individual values are indicated (F). The mean and SD are indicated. Scale bar, 5 μm.
(G) HEK293 cells were transfected with plasmids encoding viral nucleocapsid proteins (VP30, VP35, polymerase L, and NP and VP30 mutants as indicated) and a reporter minigenome encoding luciferase flanked by viral regulatory elements under the control of T7 promoter, T7 polymerase, and a firefly-encoding plasmid (pGL4) for normalization (minigenome assay). Increasing plasmid amounts of the YFP-LxxIxE B56 inhibitor or YFP-AxxAxA control inhibitor were co-transfected as indicated, and luciferase activity was measured and represented as the fold reduction compared to omission of B56 inhibitor. The mean and SD from 3 independent experiments are indicated.
(H and I) HEK293 cell lines stably expressing inducible YFP-LxxIxE B56 inhibitor or YFP-AxxAxA control inhibitor were induced with doxycycline and 3 hr later infected with recEBOVwt or recEBOVS29 as the control. After 1 hr the inoculum was removed and 24 hr later the supernatant was analyzed for (H) infectious virus particles (TCID50) and the (I) presence of viral genomes by qRT-PCR. Individual data points from 5 (TCID50) or 3 (qRT-PCR) independent experiments are plotted with mean and SD indicated. The cells were also analyzed by western blotting for the presence of NP (Figure S4D).
ND, not determined; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; n.s., not significant (unpaired t test). See also Figure S4.
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