Accumulation of acidified autophagosomal structures in USUV-infected cells.

<p>(A) Vero cells were transfected with mCherry-GFP-LC3 plasmid and treated with rapamycin, NH₄Cl or infected with USUV SAAR 1776 (MOI of 5 PFU/cell) 24 h post-transfection. Cells were fixed and processed for immunofluorescence (24 h p.i. or after treatment with rapamycin or NH₄Cl) using monoclonal antibody to detect dsRNA and appropriated Alexa Fuor-647 secondary antibodies. Colocalization between GFP and mCherry denotes autophagosomal structures that have not already fused with a lysosomal compartment (phagophores or autophagosomes) whereas mCherry signal without GFP signal denotes autophagosomal acidified organelle that have fused with lysosomes (amphisomes or autolysosomes). GFP, in green; mCherry, in red; dsRNA, in blue. Scale bars: 10 µm. (B) Quantification of the number of fluorescent puncta exhibiting green (GFP) or red (mCherry) fluorescence in cells transfected with mCherry-GFP-LC3 plasmid and treated or infected as in (A). Statistically significant differences between the number of red and green puncta displayed by the cells are denoted by one asterisk (*) for P<0.05 or two asterisks (**) for P<0.005. (C) Quantification of the number of fluorescent puncta displaying only mCherry fluorescence (acidified autophagosomal structures) of cells transfected with mCherry-GFP-LC3 plasmid and treated or infected as in (A). Statistically significant differences between each condition and control cells are denoted by one asterisk (*) for P<0.05 or two asterisks (**) for P<0.005.