Fusogenic properties of the ectodomains of hepatitis C virus envelope proteins

Abstract

We have used an isolated chimeric protein E1 340 E2 661 that includes the ectodomains of the envelope proteins of hepatitis C virus to study its interaction with model membranes. E1 340 E2 661 has some of the membrane destabilization properties, vesicle aggregation, lipid mixing and the release of internal aqueous content, which have previously been ascribed to fusion proteins. The effects are preferentially produced on vesicles of acidic phospholipids which would indicate the importance of the electrostatic interactions. In fact, an increase of the ionic strength of the buffer induced a considerable decrease of the destabilizing properties. Moreover, fluorescence polarization studies show that the recombinant protein reduces the amplitude of the thermal transition of dimyristoylphosphatidylglycerol vesicles and increases the transition temperature at pH 5.0 in a dose-dependent manner, indicating its insertion into the bilayer. Furthermore, a decrease of the pH induces a conformational change in the protein structure as evidenced by fluorescence of tryptophan residues and 4,4 0 -bis(1-anilinonaphthalene-8-sulfonate). A model for the fusion of hepatitis C virus with the host cell membrane can be postulated. The dissociation of E1E2 dimers would uncover the fusion peptides which can then interact with the polar lipid heads of the outer leaflet of the lipid bilayer and next insert into the hydrophobic moiety producing the destabilization of the bilayer which finally leads to fusion.

High-yield production of a chimeric glycoprotein based on permuted E1 and E2 HCV envelope ectodomains

“…Biochemical, spectroscopical and antigenic characterization of this isolated chimeric polypeptide indicated that E1 341 E2 661 behaved as a properly folded glycoprotein. Moreover, this chimera has been recently used as a tool to study the fusogenic properties of the HCV envelope proteins (Tello et al, 2014). In this report, a novel recombinant chimeric polypeptide, E2 661 E1 340 , has been produced.…”

The deletion of residues 268-292 of E1 impairs the ability of HCV envelope proteins to induce pore formation

“…The regions on both sides of the deletion were amplified separately by PCR, using the plasmid pAcGP67A-E2 661 E1 340 (Tello et al, 2014) as template. After co-purification of both PCR products, E2E1 192-267 and E1 293-340 , they were subjected to a second elongation PCR without primers in order to expand the area.…”

“…Fast-Basic extruder apparatus (Avestin, Inc.) with 100-nm polycarbonate filters (Costar) as previously described (Tello et al, 2014). Lipid concentration was calculated based on their phosphorous content determined according to Barlett (Barlett, 1959).…”

“…The aggregation of phospholipid vesicles induced by the addition of the recombinant proteins was followed by measuring the optical density at 360 nm (OD 360 ) on a Beckman DU-640 spectrophotometer after incubation for 1 h at 37 ºC (Tello et al, 2014). The final phospholipid concentration was 0.14 mM.…”

“…These data suggest that different E1 and E2 regions can cooperate for the fusion process. In this sense, we have recently shown that a recombinant chimeric protein composed of E1 and E2 ectodomains possesses the membrane destabilization properties ascribed to a fusion protein (Tello et al, 2014).…”