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).