Fig 1: EBOV VP35 promotes CREB1 phosphorylation at S133 in vitro and in vivo via AKIP1.a HepG2 cells infected with Ad-VP35 or Ad-GFP (MOI = 10) for 48 h were treated with 25 µM FSK or 10 µM H89 for 4 h and then analyzed using immunoblotting. b HepG2 cells expressing GFP-VP35 or GFP were subjected to immunostaining with an anti-pCREB1(S133) antibody (red). c HepG2 cells infected with live EBOV (MOI = 10) for 72 h (upper panel) or transfected with EBOV minigenome (p0) for 48 h (lower panel) were subjected to immunostaining with the indicated antibodies. d HepG2 cells transfected with the AKIP1 siRNA or scrambled (Scr) siRNA were infected with Ad-VP35 or Ad-GFP (MOI = 10) for 36 h. Then, lysates were analyzed using immunoblotting. e Lysates of WT and AKIP1-/- (two independent clones, KO1 and KO2) HepG2 cells infected with Ad-VP35 or Ad-GFP (MOI = 10) were analyzed using immunoblotting. f C57BL/6 N mice were intravenously injected with Ad-VP35 or Ad-null (2 × 109 PFU) twice at an interval of 24 h. Three days after the first infection, the liver (upper panel) and lung (lower panel) tissues were analyzed using immunohistochemical staining with an anti-pCREB1 (S133) antibody. At least two independent replicates were performed in all experiments.
Fig 2: CREB1 is recruited to viral inclusion bodies upon trVLPs or EBOV infection.a, b WT and AKIP1-/- (KO) HepG2 cells were transfected with EBOV minigenome (p0) with or without 1 µM 666-15 or 10 µM H89 for 48 h and then immunostained with anti-VP35 (green) and anti-CREB1 (red) antibodies (a). The cytoplasmic/nuclear distribution of CREB1 in (a) was analyzed by ImageJ software (b). Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The ratio of CREB1 distribution in at least ten cells from two independent assays is presented as the mean ± s.e.m. (n = 10; ***P < 0.001). c, d HepG2 cells infected with live EBOV (MOI = 10) for 72 h were immunostained with anti-CREB1 (red) and anti-VP35 (green) antibodies. Arrows: CREB1 in VIBs. The ratio of cytoplasm/nuclei distributed CREB1 in (c) was analyzed by ImageJ software (d). The ratio of CREB1 distribution in at least ten cells from two independent assays is presented as the mean ± s.e.m. (n = 10; ***P < 0.001). Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. e Lysates of HepG2 cells transfected with the EBOV minigenome (p0) in the presence (upper panel) or absence (lower panel) of the T7 RNA polymerase expression plasmid pCAGGS-T7 were separated on a 25 to 60% (W/V) sucrose gradient. Fractions were collected and analyzed by immunoblotting. f The seventh and eighth fractions from (e, upper panel) were combined and subjected to immunoprecipitation and immunoblotting analysis. g WT and AKIP1-/- (KO) HepG2 cells were transfected with the EBOV minigenome (p0). Cell lysates were subjected to anti-CREB1 (or IgG as a control) immunoprecipitation, and the coprecipitated viral RNA corresponding to 3Le or 5Tr was quantified by qRT-PCR. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The mean ± s.e.m. from three independent assays is presented (ns not significant; **P < 0.01; ***P < 0.001).
Fig 3: EBOV VP35 promotes the transcription of CREB1-directed coagulation-related genes.a HepG2 cells cotransfected with pGL-CRE-Luc, pRL-TK, and the indicated amounts of Flag-VP35 were treated with or without 25 µM FSK for 4 h. The luciferase activity of the cell lysates was analyzed. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. The mean ± s.e.m. from three independent assays is presented (n = 3; ***P < 0.001). b, c HUVECs (b) and WT or AKIP1-/- HepG2 cells (c) infected with Ad-VP35 (MOI = 10) (b) or live EBOV (MOI = 1) (c) for 48 h were treated with or without 10 µM H89 or 1 µM 666-15 for another 24 h. THBD and SERPINB2 mRNA levels were determined by qRT-PCR. Differences between the two groups were evaluated using the two-sided unpaired Student’s t-test. Data were presented as mean ± s.e.m. (n = 3; ***P < 0.001). d WT or Akip1-/- mice were intravenously injected with Ad-VP35 or Ad-null (2 × 109 PFU) twice at an interval of 24 h. Six days post the first infection, the liver tissues were analyzed by immunohistochemistry staining with an anti-Thrombomodulin (TM) antibody. e, f Mice were infected with Ad-VP35 or Ad-null (3 × 109 PFU), treated with 666-15 (2 mg/kg) or solvent and then challenged with or without LPS. The tail bleeding time was determined (n = 8) (e), and a mouse survival curve is shown in (f) (n = 9). Differences between the two groups were evaluated using the two-sided unpaired Student’s t-test (e). Survival curves were analyzed by log-rank test (f). All data from two independent experiments are presented as the means ± s.e.m. (ns not significant; *P < 0.05; **P < 0.01).
Fig 4: The EBOV VP35 associates with AKIP1.a Lysates of HepG2 cells expressing Flag-VP35 or Flag were subjected to anti-Flag immunoprecipitation and analyzed by immunoblotting. b–d, f Lysates of HEK293 cells transfected with the indicated plasmids were subjected to anti-Flag immunoprecipitation and analyzed by immunoblotting. e Lysates of HEK293 cells transfected with the indicated plasmids were treated with/without RNase (the mixture of RNase A and RNase T1) and analyzed using immunoprecipitation and immunoblotting. g HepG2 cells were cotransfected with GFP-VP35 (or GFP vector) and Myc-AKIP1 and immunostained with an anti-AKIP1 antibody (red). At least three independent repeats were performed in all of the experiments.
Fig 5: EBOV VP35 interacts with AKIP1 in the viral inclusion bodies of HepG2 cells infected with EBOV or trVLPs.a HepG2 cells infected with Zaire EBOV (strain Mayinga) (MOI = 10) for 72 h were analyzed by immunostaining with anti-VP35 (green) and anti-AKIP1 (red) antibodies (upper panel) or anti-VP35 antibody only (lower panel). b HepG2 cells infected with live EBOV (MOI = 10) for 72 h or transfected with the EBOV minigenome (p0) for 48 h were subjected to an in situ PLA assay with anti-VP35 and anti-AKIP1 antibodies (red), and immunostaining with an anti-NP antibody (green). Arrows: VP35-AKIP1 complexes in the viral inclusion bodies of EBOV (upper panel) or trVLPs (lower panel). At least three independent repeats were performed in all of the experiments.
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