Y623H increases <i>Hs</i>ACE2 binding and RBD availability by scVSV-Rs3367-CoV.
<p><b>(a)</b> Genome-normalized amounts of scVSV particles bearing spikes of WIV1-CoV, Rs3367-CoV, Rs3367-CoV(Y623H), or Rs3367-CoV(N1167D) were diluted with 3-fold dilutions onto ELISA plates precoated with soluble <i>Hs</i>ACE2, followed by a spike-specific mAb, and i...
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2025
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| Summary: | <p><b>(a)</b> Genome-normalized amounts of scVSV particles bearing spikes of WIV1-CoV, Rs3367-CoV, Rs3367-CoV(Y623H), or Rs3367-CoV(N1167D) were diluted with 3-fold dilutions onto ELISA plates precoated with soluble <i>Hs</i>ACE2, followed by a spike-specific mAb, and incubation with an anti-human HRP-conjugated secondary antibody (average±SD, n = 6-8 from 3-4 independent experiments). A range of 2.4 × 10<sup>7</sup> to 9.9 × 10<sup>4</sup> viral GEQ was used. Groups were compared against WT with two-way ANOVA with Tukey’s correction for multiple comparisons, ns p > 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. <b>(b)</b> Pre-titrated amounts of scVSV-WIV1-CoV, scVSV-Rs3367-CoV, scVSV-Rs3367-CoV(Y623H), or scVSV-Rs3367-CoV(N1167D) were incubated with serial 3-fold dilutions of ADG-2 mAb, starting at 2.5 µM, for 1 h at 37˚C. Virus:ADG-2 mixtures were applied to monolayers of DBT-9 cells overexpressing <i>Rs</i>ACE2. At 16-18 hours post-infection, cells were fixed, and infected cells were scored by eGFP expression (average±SD, n = 9 from 3 independent experiments). Relative infectivity (%) was calculated by normalizing to a no-mAb control for each virus. AUC values were calculated for each curve, and groups were compared by one-way ANOVA with Dunnett’s post hoc test, ns p > 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Only the statistically significant comparisons are shown. <b>(c-d)</b> 293T cells were transfected with plasmids expressing either WIV1-CoV or Rs3367-CoV spikes and harvested 24 h post-transfection. <b>(c)</b> Cells were incubated with 50 nM of soluble <i>Hs</i>ACE2 and bound protein was detected with Strep-Tactin XT PE. Binding was assessed by flow cytometry (average±SD, n = 5 from 3 independent experiments). <b>(d)</b> Cells were immunostained for cell surface expression by ADG-2, followed by a fluorescent secondary antibody, and analyzed using flow cytometry (average±SD, n = 5 from 3 independent experiments). WIV1-CoV vs. Rs3367-CoV were compared with unpaired t-test with Welch’s correction, ns p > 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.</p> |
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