Fig 1.
Robust EBOV GP-mediated virus-liposome lipid mixing requires acidic pH, Ca2+, and NPC1.
Fluorescence dequenching reports on lipid mixing between labeled GPCL-containing pseudovirions and unlabeled liposomes. Fluorescence dequenching data acquired at the indicated pH are presented as the percentage of dequenching seen upon addition of 1% triton X-100, which solubilizes the viral membrane leading to maximal dequenching. Data are shown for liposomes without (yellow) or with (orange) sNPC1-C and in the additional presence of 0.5 mM CaCl2 (blue). The data are displayed as the average of 4 independent measurements, with error bars reflecting the standard deviation. All dequenching data acquired in the presence of sNPC1-C and Ca2+ are fit to the exponential function A(1-e-kt) with fitting parameters at pH7: A = 0.038 ± 0.001, k = 0.06 ± 0.02; pH6: A = 0.072 ± 0.002, k = 0.023 ± 0.003; and pH5.2: A = 0.224 ± 0.006, k = 0.011 ± 0.001. The error bars in the fits represent 95% confidence intervals. All fits had R2 > 0.9. Fits were not well determined for the data lacking Ca2+ or sNPC1-C because of the limited observation window. Numeric lipid mixing data are provided in S1 Data. EBOV, Ebola virus; GP, EBOV envelope glycoprotein; NPC1, Niemann-Pick C1; sNPC1-C, soluble domain C of NPC1.
Fig 2.
smFRET imaging assay for visualization of EBOV GP conformational changes related to membrane fusion.
(A) Experimental setup of smFRET imaging assay. (B) Structural model of the prefusion GPΔmuc trimer with one protomer labeled with donor and acceptor fluorophores (model based on PDB 5JQ3; Materials and methods). Unlabeled protomers within the trimer are shown in gray. GP2 (light blue) wraps around GP1 (maroon), with the FL residing in a hydrophobic cleft at the protomer interface and contacting the neighboring protomer. MD simulation indicates a time-averaged distance between the fluorophores of approximately 35 Å, consistent with a high-FRET state. (C) Representative example of fluorescence (donor, green; acceptor, red) and smFRET (blue) trajectories acquired from an individual GPΔmuc molecule, indicating a predominant high-FRET state at pH 7. Fluorophore photobleaching occurs at approximately 8 s. (D) Contour plot and FRET histogram indicating the predominant high-FRET state across the population of GPΔmuc molecules at pH 7. The loss of FRET signal over time shown in the contour plot arises because of photobleaching of the fluorophores. Overlaid on the FRET histogram are Gaussian fits of the 3 observed FRET states identified through HMM (Materials and methods). N indicates the number of FRET traces compiled into each contour plot and histogram. EBOV, Ebola virus; FL, fusion loop; FRET, Förster resonance energy transfer; GP, EBOV envelope glycoprotein; GPΔmuc, GP with the mucin-like domain deleted; HMM, hidden Markov modeling; MD, molecular dynamics; smFRET, single-molecule Förster resonance energy transfer.
Fig 3.
Ca2+, acidic pH, glycan cap removal, and NPC1 binding destabilize the prefusion conformation of GP.
(A) Contour plots displaying the FRET distribution from the population of individual GPΔmuc molecules, at the indicated pH, and in the absence and presence of 0.5 mM CaCl2. Also shown for each contour plot is the corresponding histogram, overlaid with Gaussian fits for each of the 3 FRET states. N indicates the number of FRET traces compiled into each contour plot and histogram. Shown at the right are the occupancies in the high- (blue), intermediate- (orange), and low-FRET (yellow) states, which are displayed as the average of 3 independent groups of traces, with error bars reflecting the standard error. Occupancies in the 3 FRET states are normalized such that their sum equals 100%. (B) The same data for GPCL and (C) GPCL bound to sNPC1-C. FRET, Förster resonance energy transfer; GP, EBOV envelope glycoprotein; GPΔmuc, GP with the mucin-like domain deleted; NPC1, Niemann-Pick C1; sNPC1-C, soluble domain C of NPC1.
Fig 4.
NPC1 binding induces transition to an irreversible GP conformation.
(A) Contour plots and FRET histograms displaying the FRET distribution across the population of observed GPΔmuc molecules under the indicated conditions, either at pH 7, pH 4.5, or at pH 7 after 10-min exposure to pH 4.5 as indicated by the timeline at the top. N indicates the number of FRET traces compiled into each contour plot and histogram. Also shown at the right are the occupancies in high and low FRET under the indicated conditions, which are displayed as the average of 3 independent groups of traces, with error bars reflecting the standard error. (B) The same data for GPCL and (C) GPCL with sNPC1. FRET, Förster resonance energy transfer; GP, EBOV envelope glycoprotein; GPΔmuc, GP with the mucin-like domain deleted; NPC1, Niemann-Pick C1.
Fig 5.
Mechanistic model of GP-mediated membrane fusion.
The present observations are consistent with a model in which acidic pH and Ca2+ shift the prefusion conformational equilibrium of GP in favor of a conformation optimal for NPC1 binding. In this conformation, the GP2 N terminus and FL have moved out of the prefusion cleft to a position distal to the viral membrane. NPC1 then triggers irreversible transition to the postfusion conformation necessary for virus-liposome lipid mixing. The intermediate FRET state may represent an additional on-pathway intermediate or a conformation not relevant to the mechanism of viral fusion. FL, fusion loop; FRET, Förster resonance energy transfer; GP, EBOV envelope glycoprotein; NPC1, Niemann-Pick C1.