Figure 1.
Labeling of EnvA-pseudotyped virus with a fluorescent pH-sensor and monitoring virus entry into acidic endosomes.
(A) A diagram of the EcpH-TM construct that consists of the Ecliptic pHluorin (EcpH) flanked by the FLAG-tag and the transmembrane (TM) domain of ICAM-1. (B) Illustration of EnvA-pseudotyped viruses co-labeled with EcpH-TM (green) and HIV-1 Gag-mCherry (red) markers at alkaline (left) and acidic (right) pH. (C) Image of pseudoviruses co-labeled with EcpH-TM and HIV-1 Gag-mCherry. Viruses pseudotyped with EnvA were immobilized on a poly-lysine-coated coverslip and visualized as described in the Materials and Methods. (D) Images of single EnvA-pseudotyped virus entry into acidic endosomes. Disappearance of the EcpH-TM signal (green) from two individual particles (arrow and arrowhead) marks the virus delivery into acidic compartments. The Gag-mCherry-labeled viral core (red) is not considerably quenched by low pH. Time intervals (in seconds) from shifting cells to 37°C are shown. See also Video S1. (E) Integrated EcpH and mCherry signals from multiple cell-associated virions as a function of incubation time at 37°C with cells expressing TVA800, TVA950 and with parental receptor-deficient CV-1 cells. Cells were grown on coverslips, allowed to bind double-labeled pseudoviruses in the cold and washed to remove unbound viruses. Virus uptake was synchronously triggered by transferring cells into an imaging chamber pre-equilibrated at 37°C and visualized for 1 hr. Image fields encompassing multiple cells with several hundred viruses were analyzed by identifying double-labeled puncta and calculating the changes in the sums of green and red fluorescence from these puncta over time. The plots were obtained by averaging the data from 6–9 independent imaging experiments with 800H, 950L (blue line) and parental CV-1 cells and from 2 experiments with 950H cells. Error bars are not shown for visual clarity. Open and gray circles are the fractions of virions that remained unquenched at a given time point, as determined by single particle analysis for 950H and 800H cells, respectively. The waiting times from raising the temperature to the loss of EcpH fluorescence were measured for single particles, ranked and plotted as a fraction of green fluorescent virions over time. The rate constants for EcpH quenching in 800H and 950H cells determined by curve fitting the single particle quenching data are shown on the graph, along with the fractional loss of the EcpH fluorescence by the end of experiment (in parentheses). Note the slight differences in the ensemble and single particle quenching kinetics. The single EcpH quenching data were used hereafter to compare with the rates of productive endocytosis (Figure 2) and of single virus fusion (Figure 5).
Figure 2.
The time courses of productive EnvA-pseudotyped virus entry into cells expressing alternative receptors.
(A, B) The kinetics of EcpH-TM quenching (cyan circles), productive virus uptake (black circles) and progression through low pH-dependent steps of fusion (red circles) for 950H and 800H cells, respectively. Data are normalized to the fusion signal or the extent of EcpH quenching at the last time point. Viruses were pre-bound to cells in the cold, and fusion was initiated by quickly raising the temperature. The EcpH quenching data were re-plotted from Figure 1E as a fraction of quenched virions at a given time point. Virus escape from fusion inhibitors, R99 (50 µg/ml, black circles) or NH4Cl (70 mM, red circles), was measured by adding the inhibitors at indicated times after shifting to 37°C. After the last time point (90 min), cells were chilled to 12°C to stop fusion, and the resulting fluorescence signal was measured after the overnight incubation at this temperature, using the BlaM assay. Data points are means of at least three independent measurements. Error bars are SEM. The kinetics of EcpH quenching and of the virus escape from inhibitors were identical in 800H cells (P>0.997), but appeared different in 950H cells (P<0.001). This difference is likely due to a 1–2 min delay in the image acquisition after moving cells into a pre-warmed buffer due to the need to find a proper image field and test the autofocus function. This delay was more evident for the faster internalizing 950H cells (panel A).
Figure 3.
EcpH quenching patterns for individual EnvA-pseudotyped virus particles.
Changes in fluorescence intensities of single virions entering 800H (A, C, E) and 950H (B, D, F) cells are shown. Single particles were tracked, using the relatively stable mCherry signal (red), and the mean fluorescence intensities of both EcpH (green) and mCherry were plotted. The instantaneous velocities of particles are shown by blue lines. (E, F) Examples of possible pseudovirus recycling events manifested by the consecutive quenching and dequenching of the EcpH fluorescence. The transient recovery of green signal likely occurs due to recycling to the cell surface or as a result of re-entry of into pH-neutral compartments. (G) Images of the transient EcpH dequenching event shown in panel F. To aid clarity, only the time interval encompassing the reversible dequenching event is represented in F and G, and an arbitrary chosen time point preceding the first EcpH decay was set to zero. See also the corresponding Video S2.
Figure 4.
Detection of fusion between single EnvA-pseudotyped viruses and endosomal membranes.
(A) EnvA-bearing pseudoviruses co-labeled with DiD (red) and NC-eGFP (green) were pre-bound to 950H cells in the cold and allowed to enter and fuse by shifting to 37°C. A double-labeled virus (arrow) underwent quick retrograde movement (light blue trajectory) and released its NC-eGFP marker near the cell nucleus, which was labeled with Hoechst-33342 (blue). See also the corresponding Video S3. (B) The loss of NC-eGFP signal from individual virions was blocked in the presence of 70 mM of NH4Cl, but not in the presence of 0.1 mg/ml of R99 peptide in both 800H and 950H cells (open bars). The NC-eGFP decay events that lasted less than 5 min (“fast” events) were virtually abrogated by R99 (dark cyan bars), whereas the “slow” events were still observed. The bars represent the NC-eGFP disappearance events per experiment normalized to the total number of double-labeled particles bound to cells. Error bars are SEM from at least three or more independent experiments. (C) Histograms of the time required for the complete loss of the NC-eGFP fluorescence from viruses in the presence (light cyan bars) and in the absence (blue bars) of R99. Inset: examples of “fast” and “slow” NC-eGFP decay events without and with the peptide inhibitor, respectively.
Figure 5.
Analyses of EnvA-driven fusion with permissive cells.
EnvA-pseudotyped viruses were co-labeled with NC-eGFP (green) and DiD (red), and single particle tracking was performed using the red channel. (A) Representative examples of EnvA-mediated fusion with 950H cells without (left) and with (right) significant particle motility associated with the NC-eGFP release. Red arrow marks the increase in red signal due to the DiD fluorescence dequenching. (B) Virus fusion with 800H cells without and with prior quick movement. The particles' trajectories are shown on lower panels. Red crosses mark the beginning of trajectories, while cyan circles mark the segment where the NC-eGFP signal was lost. (C) The kinetics of virus fusion with 800H cells (blue), 950H (red), and 950L (green) cells. The time intervals from shifting to 37°C and the onset of the NC-eGFP release (fusion) from individual particles were measured, ranked and plotted as cumulative probability distributions after proper normalization. The final time points were normalized according to the probability of fusion with different cells determined by single virus imaging: 0.16 for 950H, 0.04 for 800H and 0.06 for 950L cells (shown in parentheses). The resulting plots were fitted with single exponential functions (solid lines), and the obtained exponential coefficients are shown by each plot. (D) Comparison of the EcpH quenching kinetics (solid lines) and the single virus fusion kinetics (triangles). Shown are the EnvA-mediated fusion data from panel C along with the EcpH quenching data re-plotted from Figure 2 (triangles and solid lines, respectively). Symbols in panels C and D are colored identically. To aid comparison, all measurements were normalized to the respective signals at the last time point. (E) EnvA-pseudotyped virus infectivity in CV-1 cells expressing alternative receptors. Cells expressing a reduced level of TVA950 are designated 950L. In these experiments, pseudoviruses consisting of EnvA and the core of MLV encoding β-Gal were used. TVA800- or TVA950-expressing cells were incubated with the same amount of viral inoculum in the cold to allow virus binding, washed and incubated for 1.5 hr at 37°C to allow infection. Viral fusion was stopped by adding R99 peptide (50 µg/ml), and cells were maintained for 2 days prior to determining the infectious titer, using a β-Gal assay. A representative experiment with triplicate measurements is shown. Error bars are standard deviations. Asterisk indicates a significant difference in virus infectivity in 800H and 950H cells (P<0.047).
Figure 6.
Analysis of the rate of NC-eGFP release from single viruses fusing with endosomes.
(A) The NC-eGFP release from representative EnvA-pseudotyped virus particles upon treatment with 0.1 mg/ml saponin (cyan circles) and as a result of fusion with a 950H cell (green circles). Exponential fits (solid black lines) of the decaying green fluorescence and the obtained decay rates are shown. (B) Similar to panel A, but for the virus fusion with 800H cells in the absence (blue circles) or in the presence (open circles) of 0.1 mg/ml R99 peptide. (C) Half-times (T50) of the NC-eGFP release in 950H (red bars), 950L (gray bars) and 800H (blue bars) cells. The T50 values were determined from the exponential decay coefficients, as shown in panels A and B. The distribution of T50 for saponin-formed lytic pores in single virions is shown by an open bar.
Figure 7.
Transient openings of fusion pores formed by EnvA in endosomes.
The NC-eGFP release profiles (green circles) in 800H (A, B) and 950L (C) cells was determined by single particle tracking, using the red DiD signal as reference (not shown for clarity). The decay in the total eGFP fluorescence of a single particle was normalized to the green signal prior to fusion (at arbitrary time = 0). After smoothing the traces (gray lines), the effective permeability (P) of a fusion pore was calculated, using the equation P = (dF(t)/dt)/F(t), where F(t) is the normalized eGFP fluorescence intensity of a virion as a function of time. The obtained pore permeability traces (blue lines) show reversible opening and closure of fusion pores.
Figure 8.
Lipid mixing step precedes the release of the viral content into the cytosol.
(A, B) Single EnvA-pseudotyped particles labeled with NC-eGFP and DiD were tracked and the total fluorescence intensities of these markers were plotted (green and red circles, respectively). Blue traces are the instantaneous velocities of viral particles. DiD dequenching is manifested in the increased red fluorescence intensity. The decrease of the DiD fluorescence after dequenching is likely caused by membrane trafficking that removes the lipid dye from a recipient endosome. (C) The distribution of lag times between the increase in DiD intensity (TDiD) and the onset of NC-eGFP release (TGFP).
Figure 9.
Schematic illustration of ASLV entry and fusion through alternative receptors.
The viral membrane (DiD, red) marker redistributes to the limiting membrane of an acidic endosome as a result of hemifusion. Transition from dark red to light red color illustrates the dequenching of the DiD fluorescence upon its dilution within the membrane of a recipient endosome. Subsequent formation of a small fusion pore permits the release of a viral content marker (green) into the cytosol. The ASLV entry/fusion pathway via the transmembrane TVA950 receptor is shown by the left series of arrows, while its entry through TVA800 is depicted on the right. The relative rates of ASLV endocytosis and of intermediate steps of fusion are indicated by the width of arrows. The transient opening of small fusion pores seen in TVA800-expressing cells is schematically shown as the reversal to a hemifusion stage. The question mark reflects the lack of data of the rate of transition from low pH-activation to hemifusion, as well as the inability to detect DiD dequenching in cells expressing TVA800. The slow transition from hemifusion to a small pore in 950H cells is illustrated by a thin arrow.