Fusion-competent state induced by a C-terminal HIV-1 fusion peptide in cholesterol-rich membranes

Abstract

The replicative cycle of the Human Immunodeficiency Virus type-1 begins after fusion of the viral and target-cell membranes. The envelope glycoprotein gp41 transmembrane subunit contains conserved hydrophobic domains that engage and perturb the merging lipid bilayers. In this work, we have characterized the fusion-committed state generated in vesicles by CpreTM, a synthetic peptide derived from the sequence connecting the membrane-proximal external region (MPER) and the transmembrane domain (TMD) of gp41. Pre-loading cholesterol-rich vesicles with CpreTM rendered them competent for subsequent lipid-mixing with fluorescently-labeled target vesicles. Highlighting the physiological relevance of the lasting fusion-competent state, the broadly neutralizing antibody 4E10 bound to the CpreTM-primed vesicles and inhibited lipid-mixing. Heterotypic fusion assays disclosed dependence on the lipid composition of the vesicles that acted either as virus or cell membrane surrogates. Lipid-mixing exhibited above all a critical dependence on the cholesterol content in those experiments. We infer that the fusion-competent state described herein resembles bona-fide perturbations generated by the pre-hairpin MPER-TMD connection within the viral membrane.

Efficient Fusion at Neutral pH by Human Immunodeficiency Virus gp41 Trimers Containing the Fusion Peptide and Transmembrane Domains

“…There is also correlation to vesicle fusion observed at neutral pH with short FP (gp41 512−534 ) or MPER/TM (gp41 671−693 ) peptides that have large positive charges from non-native lysines appended to increase their aqueous solubility. 70,71 For the study presented here, large ectodomain constructs with FP and/or TM segments induced significant fusion at neutral pH of both neutral and anionic vesicles, which reflects expected physiologic conditions of HIV/host cell fusion. 51 Fusion under physiologic conditions for the study presented here but not earlier studies may be due to inclusion of more hydrophobic segments in the study presented here, and also stock solutions with predominant trimer in the study presented here versus monomer in previous studies.…”

“…A higher extent at low pH with neutral versus anionic vesicles correlates with weakened bulk protein/vesicle electrostatic attraction that may allow for greater protein and lipid conformational flexibilities. There is also correlation to vesicle fusion observed at neutral pH with short FP (gp41 512–534 ) or MPER/TM (gp41 671–693 ) peptides that have large positive charges from non-native lysines appended to increase their aqueous solubility. , …”

“…There is also correlation to vesicle fusion observed at neutral pH with short FP (gp41 512–534 ) or MPER/TM (gp41 671–693 ) peptides that have large positive charges from non-native lysines appended to increase aqueous solubility. 70, 71…”

Deficient Endoplasmic Reticulum-Mitochondrial Phosphatidylserine Transfer Causes Liver Disease

“…The mixture was subsequently overlaid by 200 mL of TBS 1X containing 27% sucrose and then by 150 mL of TBS 1X containing 17% sucrose. After centrifugation at 84000 rpm for 3h at 4 C, fractions were collected from top to bottom with a Hamilton syringe, and each fraction was analyzed by western blotting as reported (Apellá niz and Nieva, 2015).…”

Small Mismatches in Fatty Acyl Tail Lengths Can Effect Non Steroidal Anti-Inflammatory Drug Induced Membrane Fusion

“…Membrane fusion occurs through a series of steps, each step associated with different energy barriers . This process involves close apposition of the participating membrane bilayers, followed by subsequent mixing of their inner and outer leaflet monolayers resulting in pore formation thereby, leading to overall internal content mixing. , Proteins , and peptides , are the most common class of fusogenic agents which are responsible for lowering the energy barriers associated with fusion, by bringing about conformational reorganizations in their structures. This advantage is not shared by the small molecules.…”