FIG 5.

HeV G N-glycan mutants modulate G-F interactions. (A) The G-F interactions of WT HeV F and WT or mutant HeV G were determined by immunoprecipitating HeV G from transfected 293T cells using μMACS anti-HA antibody-coated MicroBeads. Total cell lysates (lysate) and coimmunoprecipitated proteins (IP: αHA) were separated by 10% SDS-PAGE and immunoblotted with HeV F (IB: αAU1)- and HeV G (IB: αHA)-specific antibodies. Cell lysates from cells transfected with F or G only served as the respective negative or positive controls. Actin was detected as an internal loading control for lysate samples, and IgG (light chain) for immunoprecipitated proteins (IP). (B) Fusion indices described in Table 1 are plotted next to the avidities of G-F interactions, which were measured as the ratio of the amount of coimmunoprecipitated F (IP) to the amount of total F (F₀ and F₁) present in the cell lysate (F IP/F lysate). The relative ratios were measured by normalizing the values for mutant HeV G to those for WT HeV G (set to 1). The individual amounts were measured by densitometry using ImageLab software. The data represent the average results ± SEM from three independent experiments. (C) Individual data (lysate versus IP) used to calculate the avidities of G-F interactions are shown.