Cholesterol serves critical roles in enveloped virus fusion by modulating membrane properties. The glycoprotein (GP) of Ebola virus (EBOV) promotes fusion in the endosome, a process that requires the endosomal cholesterol transporter NPC1. However, the role of cholesterol in EBOV fusion is unclear. Here we show that cholesterol in GP-containing membranes enhances fusion and the membrane-proximal external region and transmembrane (MPER/TM) domain of GP interacts with cholesterol via several glycine residues in the GP2 TM domain, notably G660. Compared to wild-type (WT) counterparts, a G660L mutation caused a more open angle between MPER and TM domains in an MPER/TM construct, higher probability of stalling at hemifusion for GP2 proteoliposomes and lower cell entry of virus-like particles (VLPs). VLPs with depleted cholesterol show reduced cell entry, and VLPs produced under cholesterol-lowering statin conditions show less frequent entry than respective controls. We propose that cholesterol–TM interactions affect structural features of GP2, thereby facilitating fusion and cell entry.
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The code of programs used to collect and analyze the single-particle fusion data is available from the authors upon request. Source data are provided with this paper.
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This work was supported by NIH grants R01 AI030557 (to L.K.T.) and R01 AI114776 (to J.M.W.) and by the Human Frontiers Science Program grant RGP0055/2015 (to L.K.T.). D.A.N. was supported by the NIH training grant T32 GM080186.
The authors declare no competing interests.
Peer review information Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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4–16% polyacrylaminde gel of EBOV MPER/TM in DPC micelle and q = 0.5 DMPC/DHPC bicelle, stained with Coomassie Blue.
Extended Data Fig. 2 Attenuation of amide 1H NMR peak intensities at increasing concentrations of the nitroxide free-radical cholesterol analog 3β-doxyl-5α-cholestane.
Titration of the paramagnetic cholesterol analog into an EBOV MPER/TM q = 0.5 DMPC/DHPC bicelle sample. Amide proton intensity ratios between bicelles with 1 (red), 3 (green), 5 (cyan), 10 (blue) mol% 3β-doxyl-5α-cholestane (relative to DMPC) and cholesterol analog free bicelles are plotted.
Extended Data Fig. 3 Secondary structure and polypeptide backbone dynamics of EBOV WT and G660L MPER/TM in DMPC/DHPC bicelles.
a) Cα chemical shift index of WT (green) and G660L mutant (purple). Both show a helix-break-helix motif (see also Fig. 3). b–d) Backbone dynamics measurements of WT (green) and G660L mutant (purple) showing that the N-terminus is flexible and the TM domain is rigid in both constructs. b) Heteronuclear 15N-NOEs. c) 15N T1 spin-lattice and d) 15N T2 spin-spin relaxation times. All measurements were carried out at 45 °C, in pH 5.5 buffer, and in q=0.5 bicelles.
Extended Data Fig. 4 Distance distribution obtained using DEER on double-MTSL-labeled EBOV MPER/TM and G660L in POPC liposomes.
a) Background-corrected DEER data for WT (green) and G660L (purple) EBOV MPER/TM in POPC liposomes. b) Distance distributions obtained by a best fit to the data in (a). As seen with the bicelle data (Fig. 3), the addition of the G660L mutation causes a shift towards longer distance elements consistent with an opening of the MPER/TM angle. Measurements were performed at pH 5.5.
Liposomes (5 µM, 79:20:1 POPC:Chol:Rh-DOPE, 50 nm diameter) were added to SLBs (80:20 POPC:Chol) containing GP2 (lipid:protein 1000) at time 0 and the fluorescence within in the TIRF field was recorded. The average fluorescence intensities were determined from initial frames and used to determine the density of liposomes on the SLB. Binding was determined as a function of pH and also assessed for the fusion-deficient LIAA mutant at pH 5.5.
Extended Data Fig. 6 SDS-PAGE gels of crosslinked WT and G660L GP2 in POPC:POPG (85:15) proteoliposomes.
Samples of WT and G660L GP2 proteoliposomes (each with ~10 μg GP2) were incubated with 10 mM DTSSP for the indicated times at room temperature. After quenching, the samples were run, at the same time, on parallel SDS-PAGE gels, after which proteins were visualized by silver staining. The positions of the monomeric (M), dimeric (D) and trimeric (T) forms of GP2 are indicated with arrows.
Extended Data Fig. 7 Western blot of VLPs produced from untreated HEK293T cells (WT) or HEK293T cells treated with 4 μM lovastatin.
1, 2, and 5 μg of each type of VLP was applied to the gel. After probing for EBOV GP and VP40 (see Methods), the relative amounts of GP to VP40 were calculated for each lane. When normalized to WT VLPs, the ratio of GP:VP40 in Statin VLPs was 1.1 ± 0.07 that in WT VLPs based on analysis of all lanes. The ratio was 0.86 ± 0.07 based on analysis of the last 2 lanes of each gel. For comparison, VLPs produced in cells treated with 20 or 50 μM lovastatin (statin) showed reduced GP incorporation (see ref. 41).
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Lee, J., Kreutzberger, A.J.B., Odongo, L. et al. Ebola virus glycoprotein interacts with cholesterol to enhance membrane fusion and cell entry. Nat Struct Mol Biol 28, 181–189 (2021). https://doi.org/10.1038/s41594-020-00548-4