Article Figures & Data
Figures
- FIG 1
(A) Mean fold increases in viral titers (±SD) of GP variants compared to GP(Mak 82A/544T). 82V, 544I, or both in tandem significantly increase titers (**, P < 0.002; ***, P < 0.001 [by unpaired two-tailed t tests]) (n = 6, from three independent experiments). (B) Representative Western blot of VSV M and EBOV GP indicating similar levels of GP incorporation into VSV for all four GP variants.
- FIG 2
Normalized amounts of VSV-GP variants, in the native or THL-cleaved state, were captured onto ELISA plates using KZ52. Once cleaved, all four GP variants are able to bind NPC1 domain C at equal levels. Means (±SD) of results from two independent experiments with three technical replicates are shown (n = 6).
- FIG 3
Normalized infectivity levels at various concentrations of the viral inhibitor 3.47. Means (±SD) of results from two independent experiments with three technical replicates are shown (n = 6). At 3.47 concentrations of >2.5 and >12.5 μM, respectively, Makona and Mayinga VSV-GP variants bearing 82V are significantly more infectious than their 82A counterparts (P < 0.001 [by multiple t tests with the Holm-Šídák correction for multiple testing]).
- FIG 4
(A) Normalized ELISA signal of KZ52 binding to native VSV-GP variants following heating. Means (±SD) of results from two independent experiments with three technical replicates (n = 6) are shown. (B) Normalized ELISA signal of KZ52 binding to THL-cleaved VSV-GP variants following heating. Means (±SD) of results from two independent experiments with three technical replicates (n = 6) are shown. (C) Tm values for native VSV-GP variants, derived by nonlinear regression analysis of ELISA signals. Statistical significance was determined by one-way ANOVA with Tukey correction for multiple testing (*, P < 0.033; **, P < 0.002; ***, P < 0.001) (D) Tm values for THL-cleaved VSV-GP variants, derived by nonlinear regression analysis of ELISA signals. Statistical significance was determined by one-way ANOVA with Tukey correction for multiple testing (*, P < 0.033; **, P < 0.002; ***, P < 0.001).
- FIG 5
(A) Kinetics of viral fusion triggering. (B) Time of viral colocalization with NPC1. Data represent the means and SD from three independent experiments. ns, not significant. (C) Time interval between viral colocalization with NPC1 and the onset of lipid mixing. Data represent the means and SD from three independent experiments (**, P < 0.01 [by one-way ANOVA with a post hoc Tukey test]). (D) Total percentage of virions undergoing GP-mediated lipid mixing. Data represent the means and SD from three independent experiments. (E) Total percentage of virions colocalized with Rab5 at the time of lipid mixing (*, P < 0.05 [by Student’s t test]). Data represent the means and SD from three independent experiments.
- FIG 6
(A) Normalized infectivity levels at various concentrations of the CatB inhibitor CA074. Means (±SD) of results from two independent experiments with three technical replicates (n = 6) are shown. At concentrations of CA074 of >15 μM, VSV-GP(May 82V/544I) is significantly more infectious than all other GP variants (P < 0.001 [by multiple t tests with the Holm-Šídák correction for multiple testing]). (B) Kinetics of viral fusion triggering for VSV-GP(May 82V/544I) and VSV-GP(May 82A/544I) in U2OS CatL KO cells. (C) WT and CatL KO U2OS cells were infected with VSV-GP variants, and infectivity was scored by automatic counting of GFP-positive cells at 14 h postinfection. Groups were compared by one-way ANOVA with Šídák correction for multiple testing in order to determine statistical significance (*, P < 0.033; **, P < 0.002; ***, P < 0.001). On CatL KO cells, viruses carrying 82V variants have higher relative infectivity than those carrying 82A. Means (±SD) of results from nine trials from three independent experiments with three technical replicates are shown.
- FIG 7
(A) Representative Western blots of GP1 CatL cleavage products analyzed by SDS-PAGE following PNGase F treatment. With increased incubation times (0 to 3 h), GP variants bearing 82V were converted to two distinct products of approximately 18K and 12K, respectively. (B) Representative Western blots of GP1 THL cleavage products analyzed by SDS-PAGE following PNGase F treatment. No overt differences in proteolysis were observed between the GP variants. (C) Representative Western blot of CatL-treated and native VSV-GP(May 82V/544I), with or without PNGase F treatment, showing the glycosylation-dependent migration of GP (black arrows), GP18K (open arrows), and GP12K (white arrow).
- FIG 8
(A and B) Normalized ELISA signals of ADI-15878 binding to native (A) and CatL-treated (B) VSV-GP variants following heating. Means (±SD) of results from two independent experiments with three technical replicates (n = 6) are shown. (C and D) Tm values for native (C) and CatL-treated (D) VSV-GP variants, derived by nonlinear regression analysis of ELISA signals. Statistical significance was determined by one-way ANOVA with Tukey correction for multiple testing (ns, not significant [P > 0.05]). (E) Relative infectivity of native and CatL-treated VSV-GP variants. Statistical significance was determined by one-way ANOVA with Šídák correction for multiple testing. Means (±SD) of results from two independent experiments with three technical replicates (n = 6) are shown.
