Figure 1.
Models for fusion-mediated release of an HIV-1 content marker produced upon virus maturation.
Shown are fully (A, B) and partially (C) matured particles originating from an immature HIV-1 virion (lower left). (A) A virus with mature “leaky” capsid fully releases the content marker (iGFP, green) through a fusion pore. (B) A sealed mature capsid may release iGFP after separating from the viral envelope and uncoating. An extreme case when all iGFP molecules are trapped within the capsid is illustrated. A more likely outcome of maturation is entrapment of a fraction of iGFP in the capsid. (C) A partially matured particle contains free iGFP that is released through a fusion pore, whereas the remnants of immature capsid (Gag-iGFP) are not released. Illustrated are the virus fusion events with the plasma membrane, whereupon the viral membrane marker (red) disappears as a result of dilution.
Figure 2.
The fraction of a releasable GFP-based content marker correlates with the extent of HIV-1 maturation.
(A) Lysis of pseudoviruses co-labeled with Gag-iGFP (content marker, green) and the Cherry-ICAM-1 chimera (membrane marker, red). Pseudoviruses immobilized on a coverslip (left) were exposed to saponin at 37°C (0.1 mg/ml in HBSS, right). Scale bar is 8 µm. (B) The extent and kinetics of HIV-1 pseudovirus lysis. Gag-iGFP-labeled viruses produced in the presence of escalating doses of saquinavir (SQV) were immobilized onto coverslips and imaged. After the first three images, the acquisition was paused, saponin was added at the final concentration of 0.1 mg/ml, and imaging was continued for ∼5 min at 37°C. The total fluorescence of all iGFP-positive particles as a function of time was calculated for each experiment and the average fluorescence profile from 4–5 independent experiments were plotted (error bars are SEM). The fluorescence profile for 40 nM SQV-treated viruses is the mean from 2 independent experiments. (C) Pseudoviruses co-labeled with Gag-iGFP and DiD exhibit different degrees of iGFP release upon addition of saponin, from full or partial loss of content to no iGFP release. More than 200 particles were tracked for each SQV concentration. Red brackets show the total percentage of full and partial release events. Green circles show the fraction of free iGFP relative to the total amount of fluorescent protein obtained by densitometry analysis of bands shown in panel D. (D) The effect of escalating doses of SQV on HIV-1 Gag and Gag-iGFP cleavage. Pseudoviruses produced in the presence of indicated concentrations of SQV were analyzed by SDS-PAGE and blotted with antibodies to HIV-1 Gag/p24 (top panel) or with anti-GFP antibodies (bottom panel). The four intermediate bands on the GFP blot that run between Gag-iGFP and iGFP, likely correspond to MA-GFP-CA-NC, MA-GFP-CA, GFP-CA-NC and GFP-CA.
Figure 3.
Free iCherry is released through a pore in the viral membrane, while the saponin-resistant content marker is retained under conditions expected to destabilize the capsid.
(A) Content release from single pseudoviruses co-labeled with YFP-Vpr (capsid marker) and HIV-1 Gag-iCherry (content marker). Particles were adhered to a coverslip and imaged for 40 min at 37°C. Saponin was added after the third image frame (vertical dashed line). Normalized mean fluorescence intensities of iCherry and YFP from representative particles over time are plotted. Note the two distinct groupings in the graph where the YFP traces are shifted upward relative to the iCherry traces for visual clarity. The gray trace shows a particle that did not release its content marker and the blue trace corresponds to a typical particle that fully released iCherry upon addition of saponin before the imaging was resumed (the corresponding YFP traces are not shown for clarity). Red and dark pink lines show lytic events exhibiting different degrees of iCherry release; the corresponding YFP traces (green and dark green, respectively) are also shown. Double-arrows mark the onset of lysis and the corresponding increase in the YFP signal for the two representative partially lysed particles. Note that ∼10% of particles did not exhibit detectable changes in YFP fluorescence at the time of iCherry release (not shown). The lack of YFP dequenching is likely due to the slightly elevated membrane permeability, which equilibrates the pH across the viral membrane prior to addition of saponin. (B) Images of coverslip-immobilized pseudoviruses co-labeled with Gag-iCherry (red) and EcpH-ICAM-1 (green) before and after lysis with saponin. Saponin (0.1 mg/ml) was added together with 10 µM PF74 in phosphate buffer and the resulting lysis was imaged for 40 min at 37°C. Scale bar is 8 µm. (C) Single particle analysis of the lysis experiment illustrated in panel B. The number of Cherry-positive particles over time quickly decreased after adding saponin and PF74 (arrow) and stabilized after ∼3 min. Inset: Mean intensities of iCherry (red and pink traces) and EcpH (green and cyan traces) fluorescence as a function of time after addition of saponin (arrow) are plotted for representative full iCherry release (red) and partial release (pink) events. A slow decrease of the EcpH-ICAM-1 signal over time in panels B and C is likely related to disruption of the viral membrane upon prolonged exposure to saponin.
Figure 4.
Detection of HIV-1 fusion by content release and intraviral pH-sensing assays.
HIV-1 pseudoviruses bearing HXB2 Env and co-labeled with YFP-Vpr and Gag-iCherry were spinoculated onto CV-1/CD4/CXCR4 cells in the cold. Cells were washed and imaged at 37°C. (A) Images of single virus fusion leading to loss of the iCherry signal (red). Time stamps (in min:sec) indicate the time after shifting the cells to 37°C (see also movie S1). Scale bar is 2 µm. Cartoons above and below the image panel illustrate that this labeling approach does not discern between virus-endosomes (top) and virus-plasma membrane (bottom) fusion. (B) Mean fluorescence intensities of YFP and iCherry signals for the particle shown in panel A. Virus-cell fusion results in simultaneous YFP fluorescence dequenching and loss of the iCherry signal (arrow). (C) An example of a particle that fails to fuse. The slow decrease in the YFP fluorescence is caused by acidification of the virus’ interior, which most likely reflects the pH drop in endosomal compartments. (D) The kinetics of HIV-1 fusion with cells measured as the distribution of waiting times from raising the temperature to loss of iCherry (dark red circles). For comparison, the kinetic of ASLV-A Env-mediated fusion of HIV-1-based particles with CV1 cells expressing TVA950 is also shown (open circles).
Figure 5.
ASLV-A pseudovirus fusion with endosomes results in iCherry release and YFP dequenching.
(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 movie S2) 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.
Figure 6.
A gain-of-signal assay for detecting the synchronized fusion of NH4Cl-arrested ASLV-A pseudoviruses.
ASLV-A pseudoviruses carrying HIV-1 Gag-iCherry and YFP-Vpr were allowed to enter CV-1 cells stably expressing TVA950 in the presence of NH4Cl. Virus fusion was then initiated by replacing NH4Cl with HBSS, thereby quickly acidifying the endosomal and intraviral pH. (A) Depiction of the NH4Cl arrest-release protocol for synchronized ASLV-A fusion. NH4Cl blocks the ASLV-A fusion and traps YFP-Vpr/Gag-iCherry labeled pseudoviruses in endosomes by raising the endosomal pH (left). Removal of NH4Cl results in acidification of endosomal lumen and of viral interior, as evidenced by quenching of the YFP-Vpr signal (middle). Subsequent acid-mediated fusion with an endosome results in iCherry release and YFP-Vpr dequenching caused by re-neutralization of the intraviral pH through a fusion pore that connect the virus interior to the cytoplasm (right). (B) Snapshots of ASLV-A pseudovirus fusion triggered by removal of NH4Cl (see also movie S3). Particles co-labeled with Gag-iCherry (red) and YFP-Vpr (green) were pre-bound to cells expressing TVA950 in the cold and incubated for at 37°C for 40 min in HBSS supplemented with 70 mM NH4Cl. The first micrograph is taken prior to removal of NH4Cl (t = 0 s). The second micrograph (t = 100 s) is taken shortly after substituting NH4Cl with HBSS, which results in YFP quenching due to acidification of the virus interior. The third micrograph shows pseudoviruses exhibiting the loss of iCherry and concomitant appearance of bright YFP signal caused by virus fusion (marked with white circles), while the endosomal and intraviral pH still remain acidic. The fourth micrograph shows the fluorescence pattern after returning to NH4Cl, which re-neutralizes the endosomal/intraviral pH and causes dequenching of the YFP signal from particles that failed to undergo fusion. The particle marked by an arrow exhibited a delayed release of iCherry relative to YFP dequenching, apparently due to slow dilation of a nascent fusion pore. Scale bar 14 µm. (C) Images of a single ASLV-A pseudovirus (dashed circle) fusing after removal of NH4Cl. The initial drop in the YFP signal is caused by acidification of the virus interior, whereas the loss of iCherry during the HBSS perfusion (while the intraviral pH is still acidic) corresponds to virus-endosome fusion (see movie S4). A non-fusing particle (arrow) exhibits reversible changes in YFP but not iCherry fluorescence in response to HBSS/NH4Cl perfusion. Scale bar is 8 µm. (D-F) The fluorescence intensity profiles for the fusing (D) and non-fusing (F) particles from panel C. The fusion event occurring during HBSS perfusion (block arrow) is manifested in the iCherry release (red) and concomitant dequenching of YFP fluorescence (green). Changes in the intraviral and the cytosolic pH during the removal/addition of NH4Cl determined in separate experiments are shown by blue and pink lines, respectively (E). The double-arrow in panel E shows the predicted pH difference between the intraviral and cytosolic compartments at the time of fusion shown in panels C and D.
Figure 7.
Temporal correlation between YFP dequenching and complete and partial release of iCherry upon synchronized ASLV-A pseudovirus fusion.
ASLV-A fusion was arrested with NH4Cl and synchronously triggered by removing the weak base. (A) ASLV-A fusion results in complete release of iCherry and biphasic recovery of the YFP signal during HBSS perfusion. (B) Transient closure of a fusion pore followed by a complete loss of iCherry and recovery of the YFP signal. Pore closure was manifested in transient cessation of the drop in iCherry fluorescence and of YFP dequenching, which resumed upon pore reopening. (C) ASLV-A fusion leading to a partial release of iCherry. (D) Transient pore closure with partial release of content. The steady iCherry signal following the partial release events shown in panels B and D shows the lack of post-fusion content release. The slowly decreasing YFP signal after fusion could reflect the YFP-Vpr dissociation from the viral core. Vertical dashed lines indicate the onset and the end of HBSS perfusion. Blue lines in panels B and D illustrate the predicted profiles of flickering fusion pores.
Figure 8.
Synchronized ASLV-A Env-mediated fusion of immature HIV-1 particles can be detected by YFP dequenching.
(A, B) Time-lapse images of single ASLV-A pseudovirus fusion manifested in YFP dequenching without loss of iCherry (marked by a dashed circle, see also movie S5). Scale bar is 7 µm. (B) Mean fluorescence intensity traces of YFP-Vpr (green circles) and Gag-iCherry (red circles) obtained by tacking the particle shown in panel A. Accompanying changes in the cytosolic pH are shown by a pink line. Vertical dashed lines indicate the onset and the end of HBSS perfusion. The blue trace schematically illustrates the predicted profile of a fusion pore. (C) The rates of synchronized single ASLV-A pseudovirus fusion triggered by removal of NH4Cl (the HBSS perfusion interval corresponds to the blue strip on the graph). The endosomal pH is shown by a pink line, and the time when the pH reaches a threshold value of 6.3 (dashed lines) and designated t = 0. Dark pink circles show the distribution of waiting times to single fusion events following the acidification of endosomal compartments below pH 6.3, as measured by simultaneous loss of the iCherry and recovery of the YFP signal. The rate of fusion events exhibiting YFP dequenching without loss of iCherry is shown by green triangles.