Figure 5: JUNV buds at CD9-enriched sites.

JUNV-A647 viral particles stained with either a CD9 antibody conjugated to PE (a) or CTB conjugated to FITC (b) were analysed by flow virometry and plotted as a function of Alexa 647 GPC antibody staining. (c) JUNV particles stained with a GPC antibody coupled to an Alexa Fluor 647 as in Fig. 1g, were adsorbed on glow-discharged glass coverslip. Samples were blocked in 0.5% BSA in PBS for 30 min, incubated with mouse anti-CD9 antibody coupled to PE for 30 min and then washed extensively in 0.5% BSA in PBS. Coverslips were mounted on slides and imaged by spinning disk confocal microscopy. The micrograph represents CD9-PE (red) and JUNV-A647 (cyan) and the red channel was shifted by six pixels up to better appreciate colocalization of the two fluorophores. Over 500 JUNV-A647 particles were counted and Pearson correlation with CD9-PE was evaluated at 0.77±0.1. Scale bar, 5 μm. (d) Vero cells infected with JUNV for 24 h were fixed, permeabilized and stained with a CD9 antibody conjugated to PE and the GPC-specific GD01 antibody conjugated to Alexa Fluor 488. Images were acquired by spinning disk confocal microscopy and represent the plasma membrane of an infected cell. The images show CD9 staining (upper panel), GPC staining (middle panel) and the overlay of the two channels (bottom panel). The pink arrows highlight GPC positive spots, likely corresponding to budding events that are enriched in CD9. Scale bar, 5 μm.