Synchronized Retrovirus Fusion in Cells Expressing Alternative Receptor Isoforms Releases the Viral Core into Distinct Sub-cellular Compartments
Cells co-transfected with CFP-Rab5 (cyan) and YFP-Rab7 (green) were allowed to internalize ASLV pseudoviruses labeled with Gag-mKate2 (red) for 40 minutes at 37°C in the presence of 70 mM NH4Cl. (A) Image projections obtained from 30 confocal slices show viruses in a TVA950 cell expressing CFP-Rab5 and YFP-Rab7. Arrowheads show viral particles co-localized with Rab5-positive puncta. A rectangular region of interest, R0I (10×9 µm) encompassing a viral particle (upper left panel) is enlarged and shown in the lower panel. ROI is a confocal slice corresponding to the maximum intensity in Z for the red channel (Gag-mKate2). (B) Same as in panel A, but for TVA800 cell co-transfected with CFP-Rab5 and YFP-Rab7. Arrowheads show viral particles co-localized with Rab7-positive endosomes. A blow up view of ROI encompassing an internalized virus is shown in the lower panel. Co-localization analysis was done by constructing a line histogram (A and B, lower right panels) for a chosen confocal plane that showed a maximum intensity for all channels (ROIs in A and B). The line histogram shows the changes in fluorescence intensities for all three channels along the white lines (lower middle panels) drawn across the viruses and an endosomal compartment in the ROI (10×9 µm). Local maxima for Rab5 and/or Rab7 coinciding with the maximum of the mKate2 signal were counted as co-localization. Scale bars are 10 µm. (C) Summary of analyses of ASLV pseudovirus co-localization with endosomal markers in TVA950 (left) and TVA800 (right) cells.