FIG. 7.

Fusogenicity of WT NiV-F and the CT mutants inversely correlates with the avidity of F-G interactions. (A) Western blot analysis of co-IP F₀ and F₁ (top part a), immunoprecipitated G (bottom part c), and the relative amounts of F₀ and F₁ present in total cell lysate (middle part b). Cell lysates of 293T cells transfected with WT NiV-G and NiV-F or the indicated CT mutants were immunoprecipitated with rabbit anti-NiV-G specific antisera. The top and middle parts were blotted with mouse anti-AU1 to detect NiV-F, and the bottom part was blotted with mouse anti-HA to detect NiV-G. (B) A coimmunoprecipitation experiment identical to that in panel A was performed with tagged and untagged NiV-F (F and F_NA, respectively) but with a rabbit anti-F₂ peptide Ab for detection. Parts a, b, and c are as in panel A. (C) Relative avidities of NiV-F-NiV-G interactions for WT NiV-F and the indicated CT mutants. The amounts of co-IP NiV fusion proteins in panel A were quantified by densitometry as described in the text, with a VersaDoc Imaging System (Bio-Rad). The avidity of F-G interactions is represented by the ratio of the amount of NiV-F protein co-IP with anti-NiV-G antiserum to the relative amount of NiV-F expressed in cell lysates (parts a and b, respectively). The data presented are averages ± standard errors from three experiments. (D) Avidity of the F-G interactions from panel C plotted against the fusion/CSE ratios from Table 1. Pearson correlation analysis was performed with GraphPad PRISM. (E) The avidities of F-G interactions for the multiple N-glycan mutants previously reported by Aguilar et al. (3) were overlaid with the datum points from panel C and plotted together against their respective fusogenic indexes. CT mutants and N-glycan mutants are represented by closed and open symbols, respectively. Pearson correlation analysis was performed with GraphPad PRISM.