Synchronized Retrovirus Fusion in Cells Expressing Alternative Receptor Isoforms Releases the Viral Core into Distinct Sub-cellular Compartments
(A) Micrograph showing single ASLV pseudoviruses co-labeled with Gag-GFP (green) and DiD (red) following the incubation with cells expressing TVA800 for 40 min at 37°C in isotonic HBSS supplemented with 70 mM NH4Cl (left panel). Removal of NH4Cl through perfusion with HBSS caused a marked decrease in the GFP signal, but not in DiD fluorescence (middle). Upon returning to NH4Cl (right), the GFP fluorescence of fusion-incompetent particles fully recovered (#1, arrowhead), whereas the signal from fused particles remained undetectable (#2, arrow). The viral core release into the cytosol is manifested in spatial separation of green and red puncta (#3, double arrowhead). Cell nuclei are labeled with Hoescht (blue). Scale bar is 15 µm. (B–G) ASLV pseudoviruses co-labeled with Gag-GFP and DiD were internalized by CV-1 cells expressing TVA950 (B, D, F) or TVA800 (C, E, G) in the presence of NH4Cl, and virus-endosome fusion was initiated by perfusion with HBSS, as described in Materials and Methods. (B, C) The fluorescence intensity profiles corresponding to particles that failed to fuse following the arrest/release protocol, as evidenced by complete recovery of the GFP signal. (D, E) Examples of complete loss of the GFP marker from virions. (F, G) Partial release of the content marker. Cells were initially perfused with 70 mM NH4Cl in HBSS (white thick horizontal bars at the top of each graph) followed by perfusion with plain HBSS for 2 min (black horizontal bars) and returned to NH4Cl. The mean intensities of GFP and DiD fluorescence from single particles are shown (green and red circles, respectively). Pink lines show the changes in the intraviral pH upon removal of NH4Cl (for details, see Figures. S1 and S2). Kymographs illustrating the time-dependent changes in the mean GFP and DID signals (overlaid) are also shown above each plot. An irreversible transition from yellow (co-localized GFP and DiD signals) to red (DiD only) manifests the release of the GFP-based content marker into the cytosol. Diagrams of the fusion outcomes (reversible GFP quenching by acidic pH, full and partial release of the content marker) for respective panels are shown on the right.