Journal: Cell reports
Article Title: Molecular basis for the increased fusion activity of the Ebola virus glycoprotein epidemic variant A82V: Insights from simulations and experiments
doi: 10.1016/j.celrep.2025.115521
Figure Lengend Snippet: (A) Schematic of the smFRET assay in which pseudovirions formed with the HIV-1 core are immobilized to enable imaging with TIRF microscopy. (B) A GP CL monomer with the sites of fluorophore attachment indicated (donor, green; acceptor, red. PDB: 5JQ3 with glycan cap removed to reflect GP CL ). (C) Example smFRET trajectories from individual GP CL trimers on pseudovirions with A82 (top) or V82 (bottom) GP CL . The experimental trajectories are shown in blue, overlaid with the idealized trajectory resulting from fitting to the 5-state HMM (red). The prefusion conformation is indicated with a gray bar, along with the observed intermediate conformations. (D) FRET histograms for A82 GP CL smFRET trajectories acquired under the indicated conditions. Histograms reflect the average of three independent groups of trajectories; error bars represent the standard error. Overlaid on the histograms are four Gaussian distributions (gray) for the four non-zero FRET states, with means determined through HMM analysis of the individual smFRET trajectories. The sum of the Gaussians is highlighted by the red line. The 0.8-FRET prefusion conformation and the 0.05-FRET state are indicated. The 0-FRET state has been removed from the histograms to facilitate visualization of the 0.05-FRET state. N indicates the number of smFRET trajectories used to compile each histogram. (E) FRET histograms for V82 GP CL as in (D). (F) Violin plots displaying the occupancy distribution in the 0.8-FRET prefusion conformation for each GP CL population imaged under the given conditions. Horizontal lines indicate the population mean occupancies; the gray circles and whiskers indicate the medians and quantiles, respectively. p values were determined by one-way ANOVA and multiple comparison test (ns, p > 0.05). (G) Violin plots displaying the occupancy distribution in the 0.05-FRET state, displayed as in (F). Numeric data are presented in . (H and I) Histograms of dwell times in the 0.8-FRET prefusion conformation extracted from the HMM analysis of smFRET trajectories (blue circles) acquired for (H) A82 and (I) V82 GP CL , under the indicated conditions. Histograms are displayed with logarithmically spaced bins to assist in visualization of the two time constants. Histograms were fit to double exponential functions A fast e x p t / t fast + A slow e x p t / t slow , where A fast and A slow are amplitudes, and t fast and t slow are the corresponding time constants. Double exponential fits are overlaid in solid red lines with the individual exponentials in dashed red lines. The two time constants are indicated with 95% confidence intervals in parentheses. (J) The amplitudes, A fast and A slow , determined in the exponential fitting in (A) and (B). Bars reflect the fitted values with error bars indicating the 95% confidence intervals. p values are indicated (ns, p > 0.05), determined by one-way ANOVA and multiple comparison test.
Article Snippet: Projection of the simulations into the eigenspace formed by the first three principal components and subsequent clustering using Gaussian mixture models was performed in Matlab.
Techniques: Smfret Assay, Imaging, Microscopy, Glycoproteomics, Comparison