We developed a microfluidic device that allows, for the first time, the kinetic investigation of the fusion of single vaccinia virions (VACVs) with single cells initiated by a fast low-pH trigger

Dittrich, PS

Kuhn, P

Mercer, J

Schmidt, FI

UCL Discovery

Supplementary Figure 1. EV internalization by electron microscopy. Internalization of IHD-J F13-GFP EVs by HeLa cells was analyzed by transmission electron microscopy and immunogold labeling of GFP as described in figure 2E and F. Panels B, D, and F represent magnified views of virus particles in panels A, C, and E, respectively. Arrows in D highlight the two viral membranes of internalized EVs.  Supplementary Figure 2. MV internalization by flow cytometry. IHD-J EGFP-A5 MVs (MOI 75) were bound to HeLa cells on ice for 1 h and cells subsequently incubated at 0° or 37°C for 30 min. To detect internalized MVs, bound virions were removed and cells detached with trypsin (int.); to quantify total cell-associated virions, cells were detached with EDTA (total). Cells were fixed, and green fluorescence quantified by flow cytometry. Representative histograms of untreated samples are shown in (A); green fluorescence intensity from three independent experiments was quantified and the average of measured geometric means of internalized (B) and total (C) MVs is displayed ±SEM.   Supplementary Figure 3. EV infection in presence of neutralized MVs. WRΔA34R GFP EVs were incubated with 7D11 for 1 h at 37°C (MOI 2). EVs and increasing amounts of neutralized MVs of the same strain were added to HeLa cells and incubated at 37°C for 30 min. Cells were washed, incubated in full medium, and harvested 4 h p.i.; infection was quantified as in figure 4. Experiments were performed three times independently and normalized to untreated samples, mean ±SEM is shown.  Supplementary Figure 4. EVs in dextran-containing macropinosomes. HeLa cells were preincubated with full (A) or serum-free medium (B) for 4 h. IHD-J F13-GFP EVs in 0.2% BSA/RPMI were bound to cells on ice for 1h (MOI 25) and incubated for 30 min at 37°C in presence of 0.5 mg/mL 10 kDa dextran AF 594 (A), or dextran and 25 μg/mL transferrin AF 647 (B). Images were recorded by confocal microscopy and representative maximum projections of Z-stacks are shown. Arrowheads highlight EV particles in dextran-containing vesicles. Scale bars = 5 μm.  Supplementary Figure 5. Intracellular accumulation of EVs in presence of BafA. HeLa cells were infected with IHD-J mCherry-A5 F13-GFP EVs (MOI 25) in the presence of 5 μg/mL ActD without (A) or with (B) 25 nM BafA. Particles were bound to pretreated cells on ice for 1 h, washed with PBS, and incubated in full medium with drugs for 3h. Bound EV particles were stained with VMC-20 (anti-B5) under non-permeabilizing conditions. Images were recorded by confocal microscopy and representative maximum projections of Z-stacks are shown. Arrows in the inset of B highlight internalized EV particles not accessible to VMC-20 staining. A bound EV (open arrowhead), and a free membrane (closed arrowhead) are visualized as well. Scale bars = 10 μm.  Supplementary Figure 6. Examplary images of core release assay. Additional examplary images of core release assay quantification in figure 8E. HeLa cells were infected with IHD-J EGFP-A5 7D11-treated EVs (MOI 8 after 7D11 incubation) (A), MVs (MOI 45) (B-D), or 7D11-treated MVs (equivalent to MOI 45 before neutralization) (E) in the presence of 5 μg/mL ActD and 25 nM BafA where indicated. Particles were bound to pretreated cells on ice for 1 h, washed with PBS, and fixed after binding (A-B) or after incubation in full medium with drugs for 3h (C-E). L1 (Mab 7D11) and actin were stained and images were recorded by confocal microscopy. Representative maximum projections of Z-stacks are shown. Arrows in insets highlight released viral cores, closed arrowheads mark viral particles stained for the MV membrane marker L1.  Scale bars = 10 μm.   A BCMiNuPMPMPMCoresFEDSupplementary Figure 1Supplementary Figure 2A MV internalization - HistogramB Fluorescence intensity of internalized MVs05101520253035404550mock MV mock MV0ºC 37ºCGeoMean of Fluorescence Intensity (a. u.)0102030405060708090100mock MV mock MV0ºC 37ºCGeoMean of Fluorescence Intensity (a. u.)C Fluorescence intensity of total MVsFL1-H - Fluorescence Intensity (a.u.)Count020406080100120MOI 1 MOI 5 MOI 25untreated + neutralized MVs % infected cells (relative)Supplementary Figure 3    EV Infection in presence of neutralized MVsSupplementary Figure 4AHeLa30 minBHeLa (starved)30 min                  Dextran     Dextran                          F13-GFP                 F13-GFP                          Transferrin                                 Dextran                                         F13-GFP                                Transferrin                Dextran                F13-GFP   Supplementary Figure 5AEV3 hActDBEV3 hActD +BafAmCherry-A5  F13-GFP                 anti-B5             merge   Supplementary Figure 6Actin        EGFP-A5                anti-L1              merge   AEV+7D110 hActDBMV0 hActDCMV3 hActDDMV3 hActD +BafAEMV+7D113 hActD

Fusion of vaccinia virus particles with single cells - A kinetic study facilitated by microfluidic technology

http://discovery.ucl.ac.uk/1455693/1/2011_Schmidt_EMBOJ_SOM.pdf

Fusion of vaccinia virus particles with single cells - A kinetic study facilitated by microfluidic technology

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