Figure 1.
Live cell imaging of individual HIV-1 assembly sites.
HeLa cell transfected with a mixture of pCHIV and pCHIVeGFP imaged at 25 h hpt (A) in WF and (B) in TIRF mode. The scale bar represents 5 µm. The arrow and the arrow head indicate the two individual punctae analyzed in Figure 2A and B respectively. (C), Normalized histograms of fluorescence intensities from Gag punctae on the cell surface (upper panel, shown in hatched bars with different colors representing different cells) and released particles that adhered to the coverslip after cell retraction during a live-cell imaging experiment (lower panel, hatched bars). For comparison, the fluorescence intensity distribution of a purified particle preparation is shown in grey bars in both panels (the normalized area of the histogram has been scaled by a factor of 2 for clarity).
Figure 2.
The three phases of HIV-1 assembly.
(A–B) Intensity traces of two individual Gag.eGFP clusters indicated with an arrow (A) and arrow head (B) in Figure 1 recorded in TIRF (black line) and WF (grey line) modes are shown in the top panels. Three phases, separated here by dashed lines, can be observed: Phase I) a rapid rise in fluorescence intensity, Phase II) a plateau region with fluctuations in fluorescence intensity, and Phase III) a decay of the fluorescence signal. The middle panels show the intensity traces of the local background. The bottom panel displays a plot of the corresponding instantaneous velocities of the particles. In A, an abrupt increase in instantaneous velocity is observed concomitantly with the onset of Phase III. (C) Trajectory of the individual Gag.eGFP cluster from panel A color-coded according to the three different phases. The scale bar represents 1 µm. (D) A mean-square displacement analysis of the trajectory shown in panel C indicates the random character of motion in the different phases and shows a change by two orders of magnitude in diffusion coefficient between the phases I+II and III.
Figure 3.
Average kinetics of phases I and III.
The rates of processes involved in HIVeGFP assembly determined from single cells using TIRF microscopy. Shown are the averaged data (black line), the standard deviation of the individual traces (grey halo) and the fit of the average data to a saturating (Phase I, left panels) or decaying (Phase III, right panels) exponential (red line). Data are shown for 125 traces from a cell expressing wt HIV-1 (A), 89 traces from a ESCRT-recruitment defective variant displaying diminished release (B) and 69 traces from a maturation defective variant carrying a mutation in the PR active site (C). As the measurements were performed with different camera settings, the fluorescence intensities have been normalized to allow a direct comparison between experiments.
Figure 4.
Rates of Gag assembly versus time.
Assembly rates were measured at different time intervals after appearance of the first assembly sites. (A) Average fluorescence intensity traces of clusters which appeared between 0 and 25 minutes (upper panel) and 40–60 minutes (lower panel) after the onset of virus production on the surface of a single cell. (B) background in WF and TIRF channels (upper panel) indicating the concentration of Gag in the cytosol and plasma membrane respectively and ratio of the fluorescent background in TIRF and WF (lower panel). The grey halo in the lower panel shows the standard deviation of the data. (C) Rates of assembly versus time of first detection for three individual cells (left panel) and histogram summarizing all rates from the same three cells (right panel). The black line shows a log-normal fit yielding an average rate of (5.5±0.5)×10−3 s−1.
Table 1.
Kinetic rates of Gag assembly for phase I and phase III.
Figure 5.
Recruitment of Gag to the budding site.
(A) Scheme of the photoconversion experiment designed to determine the source of Gag molecules recruited to an assembly site. Recruitment can either occur from the surrounding plasma membrane (I) or directly from the cytosol (II). By calculating the ratio of fluorescence intensities in green and red channels at the assembly site with respect to the same ratio calculated from the local background, the origin of the recruited Gag molecules could be determined. (B–C) Live-cell images of a HeLa cell transfected with a mixture of pCHIV andpCHIVmEosFP before photoconversion (B) and directly after photoconversion (C). The scale bar signifies 5 µm. (D) Intensity trajectory from an individual Gag cluster that appeared 26 s after photoconversion. (E) The average fluorescence intensity with 488 nm excitation (green line) and with 561 nm excitation (red line) of the plasma membrane measured in the vicinity of 23 assembly sites that were detectable before photoconversion as a function of time after photoconversion. The standard deviation of the individual traces is given as a halo. (F) The ratio of fluorescence intensity after green excitation to the fluorescence signal after red excitation normalized to the same ratio determined from the local background measured for 102 different assembly sites in five cells.
Figure 6.
Comparison of the dynamics of eGFP-GPI and HIV-1 Gag during viral assembly.
(A) Live-cell images of mCherry-labeled Gag in wide field (left panel) and TIRF (middle panel) as well as eGFP-GPI imaged in TIRF (right panel); the scale bar represents 10 µm. (B) The fluorescence intensity of eGFP-GPI observed in the green channel at the location of the tracked HIVmCherry assembly site (black line) and the background signal in the red channel derived from a region surrounding the tracked particle (grey line). The fluorescence intensity from the eGFP channel is divided by a factor of 4.5. Fluctuations of the plasma membrane in the TIRF evanescent field were detectable in both traces, validating usage of the Gag background signal as an indicator of membrane fluctuations in our analyses. (C) The background GPI signal (grey) increased over time indicating the delivery of new eGFP-GPI to the membrane. No enhancement of the eGFP-GPI signal (blue) was found at the individual HIV budding site, whereas Gag (red) increased.