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Results: 6

1.
Fig 6

Fig 6. From: Filoviruses Require Endosomal Cysteine Proteases for Entry but Exhibit Distinct Protease Preferences.

Analysis of cathepsin B dependence of EBOV-1995 and BDBV. (A) VSVGFP particles pseudotyped with GP from EBOV-May containing E47 or T544 from EBOV-1995 were prepared, and dependence on cathepsin B was analyzed. Vero cells were incubated in the presence of CA074 (80 μM), E-64 (300 μM), or the vehicle for 3 h prior to the addition of VSVGFP bearing ΔMuc GP from EBOV-May D47E or I544T or wild-type EBOV-May GP. Infection was measured as described in the legend to Fig. 1A. Data are means ± SD (n = 3) Shown is a representative of three independent experiments. (B) Effect of cathepsin B selective inhibitor CA074 on infection by BDBV GP. Vero cells were incubated in the presence of increasing concentrations of CA074 (0 to 80 μM) or the vehicle for 3 h prior to the addition of VSVluc particles bearing ΔMuc GP from EBOV-May (Z), BDBV (B), or RESTV (R). After 6 h, cells were lysed and relative luminescence (RLU) was measured. Data are presented as follows: (virus-encoded luciferase activity of cells treated with CA074)/(luciferase activity of cells treated with vehicle) × 100%. Shown are representatives of three independent experiments. n/a, not applicable.

John Misasi, et al. J Virol. 2012 March;86(6):3284-3292.
2.
Fig 2

Fig 2. From: Filoviruses Require Endosomal Cysteine Proteases for Entry but Exhibit Distinct Protease Preferences.

Function of protease-cleaved ebolavirus GPs. (A) VSV particles bearing GPs from EBOV-May (Z), CIEBOV (CI), SUDV (S), and RESTV (R) were incubated with chymotrypsin (CHY) or reaction buffer alone for 1 h. Virus particles were deglycosylated with PNGase F and analyzed by immunoblot assay using rabbit anti-GP1 antibody. (B) Untreated CHO cells expressing NPC1 or treated with E-64d (300 μM) and NPC−/− CHO cells were challenged with VSV particles encoding luciferase (VSVluc) and bearing GPs from uncleaved or chymotrypsin-cleaved ΔMuc GP from EBOV-May, CIEBOV, SUDV, RESTV, or VSV. After 16 h, infection was measured in RLU. Data are means ± SD (n = 3). (C) SUDVΔTM GP was cleaved with chymotrypsin, and binding to LE/LY membranes from CHO cells expressing NPC1 (right panel) or lacking NPC1 (middle panel) was determined as described in Materials and Methods. Bound proteins were analyzed by immunoblot assay for GP1. Uncleaved SUDVΔTM GP and thermolysin (Thl)-cleaved EBOV-MayΔTM GP were included as controls. Input proteins are shown in the left panel.

John Misasi, et al. J Virol. 2012 March;86(6):3284-3292.
3.
Fig 1

Fig 1. From: Filoviruses Require Endosomal Cysteine Proteases for Entry but Exhibit Distinct Protease Preferences.

Endosomal cysteine proteases are host factors for filoviruses. (A) Vero cells were incubated in the presence of E-64 (cysteine protease inhibitor, 300 μM) or vehicle (1% DMSO) for 4 h prior to being challenged with VSV particles encoding GFP (VSVGFP) and bearing GPs from EBOV-May (Z), CIEBOV (CI), SUDV (S), RESTV (R), MARV (M), or VSV (G). After 24 h, infectivity (IU/ml) was determined by manually counting GFP-positive cells using fluorescence microscopy. Data are means ± standard deviations (SD; n = 3). Shown is a representative of four independent experiments. (B) Vero cells were incubated in the presence of E-64d (cysteine protease inhibitor, 300 μM) or vehicle for 4 h prior to being challenged with the filovirus EBOV-May or SUDV (MOI, 0.1). After 3 days, the titer was determined by quantitative RT-PCR. Data are means of three wells ± SD (n = 3). (C) Vero cells were incubated in the presence of E-64d (300 μM) or vehicle for 4 h prior to being challenged with the filovirus EBOV or MARV (MOI, 0.2). After 72 h, copies of the viral L gene per cell were determined by quantitative RT-PCR. Data are means of three wells ± SD (n = 3).

John Misasi, et al. J Virol. 2012 March;86(6):3284-3292.
4.
Fig 5

Fig 5. From: Filoviruses Require Endosomal Cysteine Proteases for Entry but Exhibit Distinct Protease Preferences.

Comparison of cathepsin B requirements for EBOV and RESTV. (A) Virus determinants of cathepsin B dependence. Vero cells were incubated in the presence of CA074 (80 μM), E-64 (300 μM), or the vehicle (1% DMSO) for 3 h prior to the addition of VSVGFP particles bearing ΔMuc EBOV-May GP, RESTV GP, EBOV-May GP1/RESTV GP2, or RESTV GP1/EBOV-May GP2. Infectivity was determined as described in the legend to Fig. 1A. Data are means ± SD (n = 3). Shown are representatives of three independent experiments. (B) Schematic of EBOV-May GP and locations of amino acid residues previously shown to mediate resistance to CA074. Inverted triangles identify residues that mediate resistance. Boxes identify differences in amino acids between EBOV-May and RESTV. The positions of the receptor-binding domain (RBD), the β-13-14 disordered loop, the fusion loop (fl), and heptad repeats 1 and 2 (hr1, hr2) are indicated. Residue numbering relative to EBOV GP. (C and D) Analysis of the effects of D47E and I584L on cathepsin B dependence. The effects of reciprocal substitutions of residues (D47/E48 and I584/L585) that differ between EBOV-May and RESTV GP were measured in native and chimeric GPs from panel A. Infectivity was determined as described in the legend to Fig. 1A. Data are means ± SD (n = 3). Shown are representatives of three independent experiments. n/a, not applicable.

John Misasi, et al. J Virol. 2012 March;86(6):3284-3292.
5.
Fig 3

Fig 3. From: Filoviruses Require Endosomal Cysteine Proteases for Entry but Exhibit Distinct Protease Preferences.

Cathepsin (Cat) B is not an essential host factor for all filoviruses. (A, B) Effect of cathepsin B selective inhibitor CA074 on cathepsin B and L activities (A) and on infectivity (B) by VSVGFP particles bearing filovirus GPs. Vero cells were incubated in the presence of increasing concentrations of CA074 (0 to 80 μM) or vehicle for 4 h prior to cell lysis or the addition of VSVGFP particles bearing GPs from MARV (M) or ΔMuc GP from EBOV-May (Z), CIEBOV (CI), SUDV (S), or RESTV (R). (A) Cathepsin B and L activities were determined by fluorogenic substrates (n = 2). (B) After 24 h, GFP-positive cells were manually counted using fluorescence microscopy. Infectivities from each of two replicates are shown and are representative of four independent experiments. Data presented were determined as follows: (infection [IU/ml] of CA074-treated cells)/(infection of vehicle-treated cells) × 100%. (C) Wild-type and cathepsin B-deficient (cathepsin B−/− cathepsin L+/+) MEF cells were infected with VSVGFP particles bearing GP from MARV (M) or ΔMuc GP from EBOV-May (Z), CIEBOV (CI), SUDV (S), or RESTV (R) or VSV (G). After 24 h, infectivity was determined as described for panel B. Data presented were determined as follows: (infection [IU/ml] of cathepsin B-deficient cells)/(infection of wild-type MEF cells) × 100%. Data are means ± SD (n = 3). Shown is a representative of three independent experiments. (D) Wild-type and cathepsin B-deficient MEF cells were transfected with expression plasmids encoding mouse cathepsin B, mouse cathepsin L, or a sham plasmid. After 24 h, cells were exposed to VSVGFP particles as described above. After 24 h, the percentage of cells infected was determined using flow cytometry. Data presented were determined as follows: (percent infection of transfected cathepsin B-deficient MEF cells)/(percent infection of wild-type MEF cells) × 100%. Data are means ± SD (n = 3). Shown is a representative of three independent experiments.

John Misasi, et al. J Virol. 2012 March;86(6):3284-3292.
6.
Fig 4

Fig 4. From: Filoviruses Require Endosomal Cysteine Proteases for Entry but Exhibit Distinct Protease Preferences.

Cathepsin (Cat) L is a host factor for Reston ebolavirus and Marburg virus. (A, B) Effect of the inhibitor FYdmk on cathepsin L and B activities (A) and on the infectivity (B) of VSVGFP particles bearing filovirus GPs. Vero cells were incubated in the presence of increasing concentrations of FYdmk (0 to 10 μM) or vehicle for 4 h prior to cell lysis or the addition of VSVGFP particles bearing GP from MARV (M) or ΔMuc GP from EBOV-May (Z), CIEBOV(CI), SUDV(S), or RESTV (R). (A) Cathepsin B and L activities were determined by fluorogenic substrates (n = 2). (B) After 24 h, GFP-positive cells were manually counted using fluorescence microscopy. Infectivities from two replicates are shown and are representative of four independent experiments. Data presented were determined as follows: (infection [IU/ml] of FYdmk-treated cells)/(infection of vehicle-treated cells) × 100%. (C) Wild-type and cathepsin B/cathepsin L-deficient (cathepsin B−/− cathepsin L−/−) MEF cells were infected with VSVGFP particles bearing GP from MARV (M) or ΔMuc GP from EBOV-May (Z), CIEBOV(CI), SUDV(S), or RESTV (R) or with VSV (G). Infectivity was determined as described in the legend to Fig. 3C. Data are means ± SD (n = 3). Shown is a representative of three independent experiments. (D) Cathepsin B/cathepsin L-deficient MEF cells were transfected with expression plasmids encoding mouse cathepsin B, mouse cathepsin L, both cathepsins B and L, or the vector alone. After 2 days, cells were exposed to VSVluc bearing GP from MARV (M) or ΔMuc GP from EBOV-May (Z), CIEBOV(CI), SUDV(S), or RESTV(R) or to VSV (G). Twenty-four hours later, infectivity was determined by measuring the RLU in cell lysates. Data presented were determined as follows: virus-encoded RLU in cathepsin-transfected cells/RLU in vector-transfected cells. Data are means ± SD (n = 6). Shown is a representative of four independent experiments.

John Misasi, et al. J Virol. 2012 March;86(6):3284-3292.

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