Structures of vesicular stomatitis virus glycoprotein G alone and bound to a neutralizing antibody
Fig 5
Residues D241 and D243 are essential for the interaction of 8G5F11 with VSV G and for neutralization.
(A) Schematic description of the binding assays used to assess the capacity of VSV G WT and VSV G alanine mutants to bind CR domains and 8G5F11 mAb or Fab. (B) Surface expression test for VSV G WT and VSV G alanine mutants by measuring their ability to bind the conformational probe (CR3-GST labelled by and ATTO550 fluorophore). The histogram indicates the mean fluorescence intensity of ATTO550 positive cells for each G construct. Three independent experiments were performed. Error bars represent the standard deviation. (C) Antibody recognition assay for VSV G WT and mutants G by measuring their ability to bind 8G5F11 mAb (left panel) or Fab (right panel). Statistically significant differences with WT are indicated by stars (**p < 0.005, ***p < 0.0005). (D) Schematic description of the neutralization assay using VSVΔG/G pseudotypes. (E) Incorporation of VSV G WT and VSV G alanine mutants in VSVΔG viral particles, assessed using a polyclonal anti-VSV G and an anti-VSV M antibodies. (F) Neutralization of VSVΔG/GWT, VSVΔG/GD241A, and VSVΔG/GD243A by mAb (left panel) or Fab 8G5F11 (right panel). VSV pseudotypes were preincubated with increasing concentrations of mAb or Fab. At 16 hours p.i., the percentage of infected cells was determined by counting the number of cells expressing eGFP using flow cytometry. This was used to calculate the infectious viral titer. Data depict the mean with standard error for experiments performed in triplicate. Average IC50 are indicated.