ASLV-A pseudovirus fusion with endosomes results in iCherry release and YFP dequenching.

<p>(A, C) Micrographs are showing consecutive snapshots of ASLV-A pseudovirus entry into CV1 cells expressing TVA950. Viruses were co-labeled with HIV-1 Gag-iCherry and YFP-Vpr. The release of viral content (iCherry) into the cytosol is detected by the loss of red signal at t ∼280 s (A, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071002#pone.0071002.s007" target="_blank">movie S2</a>) and ∼250 s (C). The pseudovirus in panel C did not fully release its content, as better seen on the lower image panel showing only the iCherry signal. The time (min:sec) elapsed after raising the temperature is overlaid on all images. Scale bars are 2.5 µm (A) and 3 µm (C). (B, D) The intensity profiles for red (iCherry) and green (YFP) signals from the particles shown in panels A and C were obtained by single particle tracking using the YFP-Vpr channel. In both panels, iCherry release coincides with the stepwise increase in the YFP-Vpr fluorescence. The incomplete release of iCherry and the slow decrease in the YFP signal are apparent in panel D. The YFP signal decay can be caused by YFP-Vpr dissociation from the viral capsid.