1 2 dipalmitoyl sn glycero 3 phosphocholine dppc

The peptides were synthesized by Fmoc solid-phase synthesis using a commercial CEM Liberty peptide synthesizer. The synthesis was carried out from the C-terminus to the N-terminus on the Rink amide MBHA resin, thus producing C-terminally amidated peptides. The peptides were purified by cold ether precipitation eight times to reach the purity of >95%, followed by lyophilization for 2 days. The peptide solutions were prepared by dissolving the lyophilized peptide powders in Milli-Q water (Millipore Reagent Water System, USA) and their pH was adjusted to the desirable range using sodium hydroxide. All the chemicals, reagents and organic solvents were sourced from Merck (Sigma Aldrich), UK, with analytical grade. Rink amide-methylbenzhydrylamine hydrochloride salt (MBHA) resin, and 9-fluorenyl-methoxycarbonyl (Fmoc) protected amino acids were bought from GL Biochem Ltd (Shanghai, China).1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) were purchased from Avanti Polar Lipids (USA). The Dulbecco's Modified Eagle Medium (DMEM), Phosphate buffered saline (PBS), Fetal bovine serum (FBS), Trypsin, Penicillin and streptomycin were sourced from GIBCO (Thermo Fisher Scientific, UK). JC-1 mitochondrial probe (Invitrogen™) was sourced from Invitrogen (Thermo Fisher Scientific, UK).

Incorporation of the HIV glycoproteins into vesicles and subsequent bilayer formation was achieved via reconstitution of the glycoproteins into lipid vesicles followed by extrusion resulting in unilamellar vesicles. Either HIV gp160 (ab73770, Abcam, UK) or gp41 (ab49070, Abcam, UK), depending on the experiment, were diluted in ultrapure water to a working concentration of 20µg mL -1 . A 3:1 molar ratio of 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) (Avanti Polar Lipids) were dissolved in chloroform followed by drying under nitrogen. The dried lipid film was then re-suspended in ultrapure water to give a final concentration of 1mg mL -1 . DOPC/DPPC vesicles were then subjected to 10 freeze-thaw cycles. The relevant glycoprotein (gp160 or gp41) was then added to the vesicle solution giving a protein concentration of 1µg mL -1 . Following this the vesicles were extruded at 45°C ten times through a polycarbonate membrane (pore size 100nm) by means of a mini extruder (Avanti Polar Lipids).