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.
Fig 2.
L2 translocation requires endosome acidification and cyclophilin activity.
(A) Representative translocation blot from asynchronous HaCaT-GFP-BAP cells infected with 400 ng L1/ml of L2-BirA pseudovirions for indicated times. (B) Infection levels in HaCaT GFP-BAP cells infected with 300 ng L1/ml of L2-BirA or L2-HA PsV for the indicated times. Infection levels are expressed relative to the 16 hour time point, which is set at 100%. (C) Infection and translocation in HaCaT GFP-BAP cells infected with L2-BirA PsV in the presence of vehicle, the cyclophilin inhibitor cyclosporin A (CsA), or the endosomal acidification inhibitors bafilomycin A (BafA) and ammonium chloride (NH4Cl). Infection levels are expressed relative to vehicle-treated cells, which are set at 100%. All infection values represent mean percent infection (±SEM, n = 2), normalized to GAPDH.
Fig 3.
L2 translocation requires furin and γ-secretase activity.
(A) Infection and (B) translocation of HaCaT GFP-BAP cells infected with L2-BirA PsV in the presence of varying amounts of furin enriched media. Infection values are expressed relative to 0% furin enrichment, which is set at 100% infection. (C) Infection and translocation in HaCaT GFP-BAP cells infected with L2-BirA PsV in the presence of vehicle, furin inhibitors dRVKR and hexa-D-arginine (Hexa) or γ-secretase inhibitors DAPT and XXI. Infection values are expressed relative to vehicle-treated cells, which are set at 100%. (D) Infection and translocation in HaCaT GFP-BAP cells infected with wt or mutant L2-BirA PsV. Infection values are expressed relative to wt L2-BirA infected cells, which are set at 100% (E) Percent infection and translocation in HaCaT GFP-BAP cells infected with L2-BirA PsV 40 hours post-treatment with scramble or nicastrin siRNAs. Infection values are expressed relative to scramble-treated cells, which are set at 100%. All infection values represent mean percent infection (±SEM, n = 2–3), normalized to GAPDH.
Fig 4.
Cell cycle arrest blocks L2 translocation.
(A) Diagram depicting where the inhibitors used in this study block cell cycle progression: aphidicolin (Aphi), hydroxyurea (HU), purvalanol A (PurA), kbNB 142–70 (kbNB), or monastrol (Mon). (B) Percent infection and (C) translocation in HaCaT GFP-BAP cells infected with L2-BirA in the presence of various cell cycle inhibitors. Infection values represent mean percent infection (±SEM, n = 3), normalized to GAPDH and expressed relative to vehicle-treated cells, which are set at 100%.
Fig 5.
Cell cycle arrest traps vDNA and L2 in the TGN.
(A) Representative image slices (0.35 μm thick) of HaCaT cells infected with wt PsV containing EdU-labeled DNA. Scale bar represents 10 μm. After fixation the cells were stained with Alexa Fluor 488 azide to visualize vDNA (green), p230 for the TGN (red) and DAPI to visualize nuclei (blue). (B) Representative image slices (0.35 μm thick) of HaCaT cells infected with wt L2-3xFTHA PsV and stained for FLAG (green), the TGN marker TGN46 (red) and DAPI to visualize nuclei (blue).
Fig 6.
Mitotic progression is sufficient for L2 translocation to occur.
(A) Schematic of the aphidicolin washout assay. (B) Representative translocation blot in the presence of various drugs post-aphidicolin washout. (C) Infection levels in the presence of various drugs post-aphidicolin washout. Values represent mean percent infection (±SEM, n = 3), normalized to GAPDH and expressed relative to vehicle-treated cells, which are set at 100%.
Fig 7.
Time course of translocation in synchronized cells.
HaCaT GFP-BAP cells infected with L2-BirA in the presence of aphidicolin or hydroxyurea to arrest cells at the G1/S transition. At 12 hours post-infection, the inhibitors were washed out and the cells were processed for SDS-PAGE at the indicated time points. (A, C) Representative translocation and phospho-H3 western blots. (B, D) Densitometry values represent mean GFP-biotin or phospho-H3, normalized to total GFP and expressed relative to the 0 hour time point (±SEM, n = 3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as compared to the 0 hour time point. Blue asterisks denote significant differences in GFP-biotin levels; red asterisks denote significant differences in phospho-H3 levels.
Fig 8.
vDNA relocalizes from the TGN to condensed chromosomes during mitosis.
HaCaT cells were infected with wt PsV containing EdU-labeled DNA in the presence of aphidicolin. At 24 hours post-infection, the aphidicolin was washed out and cells were fixed immediately or at 9 hours post-release. Cells were stained with AlexaFluor-488 azide (green) to visualize vDNA, p230 (red) to visualize the TGN, the centriole component pericentrin (magenta), and DAPI (blue). Individual channels and the merged images are representative image slices (0.35 μm).
Table 1.
Pharmacological inhibitors used in this study.