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1.
FIG. 1.

FIG. 1. From: Ebola Virus Glycoproteins Induce Global Surface Protein Down-Modulation and Loss of Cell Adherence.

Ebola virus GP expression induces cell rounding. 293T cells were transfected with 20 μg of pAD-TrackCMV containing the coding sequence for EboZ GP (A), EboS GP (B), EboR GP (C), EboC-GP (D), or ASLV-A EnvA (E). GFP expression was observed at 48 h posttransfection using a Nikon TE300 inverted microscope. Fields represent findings from multiple experiments.

Graham Simmons, et al. J Virol. 2002 March;76(5):2518-2528.
2.
FIG. 5.

FIG. 5. From: Ebola Virus Glycoproteins Induce Global Surface Protein Down-Modulation and Loss of Cell Adherence.

Ebola virus GP does not induce cell death. Floating cells (3 × 105 per well) from 293T cells 48 h posttransfection were replated into fresh six-well plates. At 24-h intervals floating cells were counted (•) and then pooled with versine-detached adherent cells, and viable (♦) and nonviable (▪) cell numbers were estimated using trypan blue exclusion. Cells were then adhered to poly-d-lysine-coated plates, and the percentage of Ebola virus GP-positive cells (▴) was quantified using indirect immunofluorescence staining with KZ52. Values represent the geometric means of triplicate samples ± standard errors. Data are representative of three independent experiments.

Graham Simmons, et al. J Virol. 2002 March;76(5):2518-2528.
3.
FIG. 2.

FIG. 2. From: Ebola Virus Glycoproteins Induce Global Surface Protein Down-Modulation and Loss of Cell Adherence.

The cell rounding phenotype correlates with the level of GP expression. (A) 293T cells were transfected with 5, 10, or 15 μg of pcDNA6/V5-His containing the coding sequence for EboZ GP (♦), EboS GP (▪), or EboR GP (▴). The floating cells in the cultures were counted 48 h posttransfection, and results are presented as numbers of floating cells above background levels (8 × 104 cells), calculated from mock-transfected cells. (B) Lysates from cells harvested 48 h posttransfection were subjected to SDS-4 to 15% PAGE, transferred to nitrocellulose, and immunoblotted for the presence of the C-terminal V5 epitope on both the uncleaved precursor (GP0) and processed GP2 using a mouse anti-V5 monoclonal antibody followed by I125-labeled protein A. Data are representative of three independent experiments.

Graham Simmons, et al. J Virol. 2002 March;76(5):2518-2528.
4.
FIG. 3.

FIG. 3. From: Ebola Virus Glycoproteins Induce Global Surface Protein Down-Modulation and Loss of Cell Adherence.

Cell rounding is dependent on O-linked glycosylation-rich domains in GP1. (A) Serial deletions in the C-terminal mucin-like domain of GP1 were made by overlapping extension PCR. Potential O-linked glycosylations (T) were predicted using NetOGlyc, version 2.0. (B) 293T cells were transfected with 30 μg of pcDNA6/V5-His containing the coding sequences for EboZ GP or EboZ GP mucin domain mutants. Floating cells were counted, and values are numbers of floating cells above background levels (2.4 × 105 cells), calculated from mock-transfected cells. Cell lysates from 5 × 106 cells were subjected to SDS-4 to 15% PAGE, and mature-GP2 levels were quantified by phosphorimager analysis following immunoblotting for the presence of the V5 epitope using a mouse anti-V5 monoclonal antibody followed by I125-labeled protein A. GP2 levels are presented as a percentage of EboZ GP2 wild-type expression. Data are representative of three independent experiments.

Graham Simmons, et al. J Virol. 2002 March;76(5):2518-2528.
5.

FIG. 4. From: Ebola Virus Glycoproteins Induce Global Surface Protein Down-Modulation and Loss of Cell Adherence.

Ebola virus GP transduction of human and nonhuman cell lines and primary cell types using adenovirus vectors. (A) Human cell lines U87, 293H, and PMA-pretreated U937 were transduced with the minimum inoculum of adenovirus vectors expressing ASLV-A EnvA, EboR GP, or EboZ GP required to give the highest achievable level of GFP-positive cells (MOIs of 30, 10, and 50, respectively). Cells were monitored at regular intervals for evidence of cell rounding, and representative photographs were taken at 48 h posttransduction. (B) Adenovirus vectors expressing ASLV-A EnvA, EboR GP, or EboZ GP were used to transduce a simian cell line, Vero (MOI, 10), a cat kidney cell line, CCC (MOI, 10), baby hamster kidney cells (BHK; MOI, 50), and a murine cell line expressing the human coxsackievirus/adenovirus receptor, MC57/hCAR (MOI, 50). Cells were monitored for rounding as described for panel A. AGM, African green monkey. (C) Adenovirus vectors expressing ASLV-A EnvA, EboR GP, or EboZ GP were used to transduce low-passage primary HUVEC, HPAEC, CASMC as well as day 8 human primary blood monocyte-derived macrophages (MDM). Similar results were obtained for MDM from three separate donors. An MOI of 10 was used for all primary cell types, apart from MDM, where an MOI of 50 was required. Cells were monitored for rounding as described for panel A. In all cases, control vectors expressing just GFP gave results indistinguishable from those for EnvA (data not shown). M⊘, macrophage.

Graham Simmons, et al. J Virol. 2002 March;76(5):2518-2528.
6.

FIG. 6. From: Ebola Virus Glycoproteins Induce Global Surface Protein Down-Modulation and Loss of Cell Adherence.

Expression of Ebola virus GP induces down-modulation of cell surface molecules. (A) At 48 h after transfection with pCB6-EboZ-GP, -EboR-GP, or -ASLV-A-EnvA, 293T cells were detached from the tissue culture plates, pooled with already-floating cells, and incubated with monoclonal antibodies directed against a number of integrin subunits as well as other common cell surface markers. Flow-cytometric analysis was then performed using secondary antibodies conjugated with fluorescein. Results are presented percentages of the mean channel fluorescence (MCF) compared to that for EnvA-transfected cells. Data are representative of multiple independent experiments. (B) Vero cells transduced at an MOI of 10 with Ad/EnvA Ad/EboR-GP, or EboZ GP were collected at 48 h postinfection. Cells were incubated with monoclonal antibodies to an isotype control, β1-integrin, αv-integrin, MHC-I, or EGFR and a secondary antibody conjugated to Cy5 and assayed by flow cytometry for GFP expression (FL1; y axis) or cell surface markers (FL4; x axis). Dotted lines, estimated MCF of expression for mock-transduced cells (C) HUVEC transduced at an MOI of 10 with Ad/EboZ-GP (solid line), Ad/EboR-GP (dashed line), or Ad/EnvA (dotted line) were collected 24 h postinfection. Cells were incubated with monoclonal antibodies to isotype controls (shaded histograms), β1-integrin, αv-integrin, MHC-I, or PECAM and a secondary antibody conjugated to Cy5 and assayed by flow cytometry. In HUVEC, unlike HPAEC, which showed up-regulation of some markers in response to adenovirus infection, there was no difference between nontransduced and Ad/EnvA-transduced cells for any of the markers (data not shown). Data are representative of three independent experiments.

Graham Simmons, et al. J Virol. 2002 March;76(5):2518-2528.

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