3.3 binding to the hydrophobic central pocket of LAMP1 D1.

(A) Docking of 3.3 (yellow) in the predicted structure of LAMP1 D1. Residues within the hydrophobic pocket predicted to contact 3.3 are colored orange. Residues on the surface at the edge of the pocket predicted to contact the diphenyl group of 3.3 are colored red. (B) Mutational analysis of predicted 3.3 contacts with LAMP1 D1 on cross-linking by photoclick cholesterol and by 1519. Purified LAMP1 D1 containing the indicated mutations were incubated with photoclick cholesterol (top) or 1519 (bottom) for 1h at 37°C prior to UV irradiation and click chemistry with AF 488 azide. Labeled protein was detected by immunoblot with an anti-AF 488 antibody. Input LAMP1 D1 was detected using an anti-His antibody. (C) Mutational analysis of predicted 3.3 contacts with LAMP1 D1 on transduction by MLV LASV GP and 3.3 inhibition. 293T LAMP1 KO cells transfected with the indicated LAMP1 mutants were incubated with the indicated concentrations of 3.3 for 1h before challenge with MLV pseudotyped with LASV GP and encoding GFP. Virus transduction is reported as % of GFP-positive cells relative to cells exposed to DMSO vehicle alone. Inset shows the MLV LASV GP transduction efficiency relative to that on cells transfected with wild-type (WT) LAMP1. Data are mean ± SD (n = 3). The IC50 of the WT curve differs significantly from all other curves (p p < 0.001). Statistical analyses were performed using one-way ANOVA with Tukey post-hoc analysis.