Fig. 3: Targeted mutagenesis of gB 526EHV528 disrupts gB/gH-gL dependent membrane fusion and viral replication.
From: Targeted mutagenesis of the herpesvirus fusogen central helix captures transition states
a The predicted structures for the WT and mutant VZV gB DIII central helices by AlphaFold263, 64 and the mutant structures aligned to WT gB using Matchmaker62 are shown; WT gB: yellow, alanine mutants: green, proline mutants: red. b Flow cytometry analysis of CHO-DSP1 cells transfected with vectors expressing WT gB or gB proline and alanine mutants, permeabilized, and immunostained with Cy5-conjugated mAb 93k; nonspecific antigen control: gray, WT gB: blue, mutant gB: orange. Gating strategy is provided in Supplementary Fig. 4a. c Frequency of gB-expressing cells detected by mAb 93k and median florescence intensity (MFI) of mAb 93k binding normalized to WT gB (mean ± SEM). n = 3 independent experiments for WT gB and proline mutants, and n = 2 independent experiments for alanine mutants. Statistical differences were evaluated by one-way ANOVA, Dunnett’s multiple comparisons test (**, p = 0.0014; ***, p = 0.0004; ****, p < 0.0001). d Cell-cell fusion measured by the stable reporter fusion assay (SRFA) using CHO-DSP1 cells transfected with vectors expressing WT gB or gB mutants together with gH-gL expression vectors, mixed with MeWo-DSP2 reporter cells. Fusion efficiency was normalized to that mediated by gB[WT]/gH-gL (%). Data from n = 8 independent experiments is shown in Box (25-75 percentile) and Whisker (10-90 percentile) plots; horizontal bar is the median and the + indicates the mean. Statistical differences were analyzed by one-way ANOVA, Dunnett’s multiple comparisons test (***, p = 0.0005; ****, p < 0.0001). e Plaque sizes in MeWo cells infected with pOka (WT), pOka-TKGFP-gB[E526P], pOka-TKGFP-gB[E526A], pOka-TKGFP-gB[H527A], or pOka-TKGFP-gB[V528A] at 4 dpi; lethal mutant viruses pOka-TKGFP-gB[H527P] and pOka-TKGFP-gB[V528P] are indicated by N/A (not available). Comparison of 45 plaques per virus (mm2) from n = 3 independent experiments is shown by Box (25-75 percentile) and Whisker (10-90 percentile) plots; dots are outliers, horizontal bar is the median and the + indicates the mean. Statistical differences between plaque sizes of WT and mutant viruses were evaluated by one-way ANOVA, Dunnett’s multiple comparisons test (****, p < 0.0001). f Immunohistochemistry staining of VZV plaques in MeWo cells; Representative examples for each virus plaque recorded 45 times from n = 3 independent experiments are shown. Scale bar = 1 mm. Source data are provided as a Source Data file.
