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Synchronized Retrovirus Fusion in Cells Expressing Alternative Receptor Isoforms Releases the Viral Core into Distinct Sub-cellular Compartments

Figure 2

Spatial separation of the GFP-tagged viral cores from membranes as a result of fusion.

(A) Spatial separation of the GFP- and DiD-tagged puncta following the NH4Cl arrest/release protocol. Double-labeled viruses were internalized by TVA950 (top panel) and TVA800 (bottom panel) cells in the presence of NH4Cl. Fusion was triggered by replacing NH4Cl with HBSS for 2 min (black horizontal bars) through local perfusion, and cells were returned to NH4Cl. Following the initial decay in the GFP fluorescence due to a partial release of the content marker, co-labeled particles split in two and continued to drift apart. The GFP signal partially recovered during the HBSS perfusion in TVA950 cells, but not TVA800 cells where recovery occurred only after returning to NH4Cl. Scale bar is 0.15 ┬Ám. (B, C) Separation of green (Gag-GFP) and red (DiD) puncta in NH4Cl-arrested TVA950 (B, video S3) and TVA800 (C, video S4) cells during perfusion with HBSS. The graphs show changes of the mean intensity of green and red signals for single particles shown in panel A. A drop in the red signal during the HBSS perfusion (marked by a blue asterisk) occurs due to the separation of formerly co-localized red and green puncta. The changes in cytosolic (B, E) and endosomal (C, F) pH during the HBSS perfusion measured in separate experiments (for details, see Figure S2) are shown by pink lines. (D) Kinetics of spatial separation of SVPs and the viral membrane triggered by a 2 min-perfusion with HBSS (black horizontal bar and vertical dashed lines). Cumulative probabilities of core release as a function of time are plotted for TVA800 (filled circles) and TVA950 (open circles) cells. (E, F) Late spatial separation events occurring at the end or after the first HBSS pulse (horizontal black bars above each plot). The SVP release in a TVA950 cell (E, asterisk) was followed by partial recovery of the Gag-GFP fluorescence during the second HBSS perfusion, which paralleled the increase in the cytosolic pH (pink line). A spike in the DiD signal at the beginning of the second HBSS pulse in panel D occurred due to a transient overlap between separated puncta. The late SVP release events in TVA800 cells (F) did not exhibit the Gag-GFP signal recovery in the course of HBSS perfusion. The two-dimensional trajectories of the recipient endosomes (red) and the GFP-tagged sub-viral particles (green) obtained by single particle tracking are shown under for each respective panel. The beginning and the end of trajectories are marked by triangles and circles, respectively. A diagram showing the viral content and core release (spatial separation from the membrane) as a result of fusion are shown on the right.

Figure 2