Fig 1. Development of the BirA/GFP-BAP assay for detecting membrane penetration by L2.

(A) Schematic of the BirA translocation assay. L2-BirA is physically separated from its substrate (GFP-BAP) while in the lumen of endocytic vesicles. Following translocation across the limiting membrane, L2-BirA can biotinylate cytosolic GFP-BAP. (B) Brightfield and epifluorescent imaging of HaCaT GFP-BAP cells. (C) Coomassie staining of wt-L2 and L2-BirA PsV. Molecular weights are shown in kilodaltons. (D) Transmission electron microscopy imaging of L2-3xFLAGTHA or L2-BirA viral particles. Scale bar represents 100 nm. (E) In vitro biotinylation of reduced wt L2-BirA or R9,12K L2-BirA particles incubated with MBP-BAP, ATP, and biotin for the indicated times prior to processing by SDS-PAGE/Western blotting. (F) Titration of infectivity and translocation of L2-BirA in HaCaT GFP-BAP cells. Graph shows percent infectivity, relative to the 200 ng L1/ml concentration, which is set at 100%. GFP-biotin and total GFP were analysed by Western blotting with neutravidin and anti-GFP staining respectively. (G) Infectivity of wt and L2-BirA pseudovirions in HaCaT GFP-BAP cells at an equal MOI, expressed relative to wt, which is set at 100%. All infection values represent mean percent infection (±SEM, n = 2–3), normalized to GAPDH.