Fig 1.
C1q-mediated ADE of EBOV GP-mediated infection.
(a) Schematic representation of C1q-mediated ADE. (b) HEK293 cells were infected with VSVΔG-EBOV GP or VSVΔG-VSV G pretreated with C1q (20 μg/ml), ZGP12/1.1 (1 μg/ml), or combination of C1q (20 μg/ml) and either CTR-IgG (1 μg/ml) or ZGP12/1.1 (1 μg/ml). (c) VSVΔG-EBOV GP was incubated with ZGP12/1.1 (1 μg/ml) or CTR-IgG (1 μg/ml) together with the indicated concentrations of C1q and inoculated to HEK293 cells. (d) Vero E6 cells were infected with VSVΔG-EBOV GP pretreated with C1q (20 μg/ml), ZGP12/1.1 (1 μg/ml), or a combination of C1q (20 μg/ml) and either CTR-IgG (1 μg/ml) or ZGP12/1.1 (1 μg/ml). Virus titers were determined as IUs by counting GFP-positive cells. The mean and standard deviation of three independent experiments are shown. Statistical analysis was performed using Student’s t-test (*p < 0.05).
Fig 2.
The effect of signaling pathway inhibitors on C1q-mediated ADE of VSVΔG-EBOV GP infection.
(a, b) HEK293 cells were treated with the indicated concentrations of BMS-777607, IWP-2, LGK-974, R788, PP2, UNC2250, LFM-A13, LY294002, or manumycin A and infected with VSVΔG-EBOV GP preincubated with CTR-IgG (1 μg/ml) or ZGP12/1.1 (1 μg/ml) and C1q (20 μg/ml). (c, d) HEK293 cells were treated with the indicated concentrations of each inhibitor and infected with VSVΔG-VSV G. The relative percentage of infectivity was determined as described in Methods. The mean and standard deviation of three independent experiments are shown. Statistical analysis was performed using Student’s t-test (*p < 0.05). In panels c and d, statistical significances were evaluated for the comparison to control (DMSO- or ethanol-treated) cells.
Fig 3.
Cell viability in the presence of each inhibitor.
HEK293 cells were treated with the indicated concentrations of each inhibitor. At 24 h after treatment, cell viability was determined using trypan blue staining.
Fig 4.
Expression of C1q receptors and contribution to C1q-mediated ADE.
(a) The presence of C1q receptors LPR1, CD93, gC1qR and CD35 was confirmed by Western blot analysis of HEK293 and Vero E6 cell lysates. (b) HEK293 cells were treated with the indicated concentrations of anti-gC1qR antibody and infected with VSVΔG-EBOV GP preincubated with CTR-IgG (1 μg/ml) or ZGP12/1.1 (1 μg/ml) and C1q (20 μg/ml). The means and standard deviations of three independent experiments are shown. Statistical analysis was performed using Student’s t-test (*p < 0.05).
Fig 5.
Enhanced VLP attachment to the cell surface in the presence of ZGP12/1.1 and C1q.
(a, b, c) Fluorescent images of attachment and trafficking to late endosomes of VLPs. (d, e, f, g) Quantified fluorescent signals of VLPs, Rab7, and Dx10. HEK293 cells expressing eGFP-Rab7 (a, b) or HEK293 cells (c) were inoculated DiI-labeled VLPs mixed with PBS (Untreated), or treated with C1q (20 μg/ml) alone, ZGP12/1.1 (10 μg/ml) alone, CTR-IgG (10 μg/ml) and C1q, or ZGP12/1.1 and C1q. After adsorption, cells were incubated with Alexa647-labeled Dx10 (0.5 mg/ml) for 2 h at 37˚C (c). VLPs (red) on the cell surface at 0 h (a, d), VLPs (red) and eGFP-Rab7 (green) in the cytoplasm at 2 h (b, e, f), and VLPs (red) and Alexa647-labeled Dx10 (green) in the cytoplasm at 2 h (c, g) after adsorption were monitored by confocal laser scanning microscopy. Top panels show merged image and bottom panels show VLPs (b, c). Scale bars represent 10 μm (a, b, c). Nuclei of cells are visualized with DAPI (blue). The number of VLPs on the cell surface (d) and incorporated into the cells (e), the colocalization of VLPs (DiI) and eGFP-Rab7 signals (f), and the colocalization of VLP (DiI) and Dx10 (Alexa647) signals (g) were quantified and percentages of DiI-labeled VLPs that colocalized with eGFP-Rab7 (f) and Alexa647 labeled Dx10 (g) were determined as described in Methods. The mean and standard deviation of three independent experiments are shown. Statistical analysis was performed using Student’s t-test (*p < 0.05).