Regulation of Ebola GP conformation and membrane binding by the chemical environment of the late endosome

Abstract

Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH, Ca2+, and removal of the glycan cap from GP have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Forster resonance energy transfer (smFRET) to elucidate how the chemical environment of the late endosome promotes GP-membrane interaction, thereby facilitating virus entry. We first investigate the role of anionic phospholipids, phosphatidylserine (PS) and bis(monoacylglycero)phosphate (BMP), which are found in the membrane of the late endosome. We find that these lipids enable robust binding of GP to membranes in a pH- and Ca2+-dependent manner. We then identify residues in GP that sense pH and trigger conformational changes that make the fusion loop available for insertion into the membrane. Molecular dynamics (MD) simulations suggest the structural basis for pH-trigger conformational changes. We similarly confirm residues in the fusion loop that mediate GP interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry.

The combination of three CD4-induced antibodies targeting highly conserved Env regions with a small CD4-mimetic achieves potent ADCC activity

“…Labelled pseudovirions previously incubated with 100 µM CJF-III-288 (38), and 50 µg/ml of each 17b and A32 monoclonal antibodies, or with the same concentrations of CJF-III-288, 17b, A32, plus mAb 246D, for 1 hour at room temperature, were immobilized on streptavidin-coated quartz slides and imaged on a custom-built wide-field prism-based TIRF microscope (61, 64). Imaging was performed in phosphate-buffered saline (PBS) pH ∼7.4, containing 1 mM trolox (Sigma-Aldrich, St. Louis, MO, USA), 1 mM cyclooctatetraene (COT; Sigma-Aldrich, St. Louis, MO, USA), 1 mM 4-nitrobenzyl alcohol (NBA; Sigma-Aldrich, St. Louis, MO, USA), 2 mM protocatechuic acid (PCA; Sigma-Aldrich, St. Louis, MO, USA), and 8 nM protocatechuate 3,4-deoxygenase (PCD; Sigma-Aldrich, St. Louis, MO, USA) to stabilize fluorescence and remove molecular oxygen.…”

“…HIV-1 JR-FL Env pseudo-typed virions with a single gp120 domain bearing the non-natural amino acid TCO* (SiChem GmbH, Bremen, Germany) substituting the residue N135 in V1 loop and the insertion of the A1 peptide (GDSLDMLEWSLM) in V4 loop (V4-A1) were generated, purified, and fluorescently labeled as previously described (16) with minor modifications. Briefly, pNL4-3 ΔEnv ΔRT plasmid, and a 20:1 mass ratio of wild-type HIV-1 JR-FL gp160 plasmid to gp160 engineered to have both an amber (TAG) stop codon substituting the N135 residue in V1 loop to introduce the non-natural amino acid TCO*, and V4-A1 peptide, were co-transfected together with the plasmids NESPylRS AF /hU6tRNA Pyl and eRF1-E55D (60) in HEK293T FirB cells (59) and in presence of 0.5 mM TCO* as previously described (61-63). Viruses were collected 48 hours post-transfection and pelleted in DPBS over a 10% sucrose cushion at 25,000 RPM for 2 hours using a SW32Ti rotor (Beckman Coulter Life Sciences, Brea, CA, USA).…”

“…Labelled pseudovirions previously incubated with 100 µM CJF-III-288 (38), and 50 µg/ml of each 17b and A32 monoclonal antibodies, or with the same concentrations of CJF-III-288, 17b, A32, plus mAb 246D, for 1 hour at room temperature, were immobilized on streptavidin-coated quartz slides and imaged on a custom-built wide-field prism-based TIRF microscope (61,64). Imaging was performed in phosphate-buffered saline (PBS) pH ~7.…”

Single-molecule imaging reveals allosteric stimulation of SARS-CoV-2 spike receptor binding domain by host sialic acid

“…The pNL4-3.Luc.R-E- plasmid was obtained from the NIH AIDS reagent program (catalog ARP-3418) ( 86 ). An ochre stop codon was introduced into the tat gene as described ( 87 ). The plasmid encoding the HIV-1 GagPol under the control of the cytomegalovirus promotor has been described previously ( 88 ), while the replication-deficient molecular clone HIV-1 NL4-3 Gag-iGFP ΔEnv ( 89 ) was provided by B. K. Chen (Icahn School of Medicine at Mount Sinai, NY).…”