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

We have obtained a chimeric protein containing the ectodomains of hepatitis C virus (HCV) envelope proteins but lacking the region 268-292 of E1. All its structural properties are coincident with those of the corresponding full length chimera. The deleted and entire chimeras were compared in terms of their membrane destabilizing properties. No differences were found in their ability to induce vesicle aggregation and lipid mixing but the deleted chimera showed a reduced capacity to promote leakage. The role of the deletion was also studied by obtaining HCV pseudoparticles (HCVpp). Both E1 and E2, and also the E1 deleted mutant, were incorporated into HCVpp to a similar level. However, HCVpp containing the E1 deleted protein are almost unable to infect Huh7 cells. These results point to the involvement of the region 268-292 in the formation of pores in the membrane necessary for the complete fusion of the membranes.

Production and characterization of the ectodomain of E2 envelope glycoprotein of hepatitis C virus folded in the presence of full-length E1 glycoprotein.

Hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, are involved in the first steps of virus infection. The E2 ectodomain can be produced as an isolated form (E2661). However, there is some concern about its proper conformation and the role that E1 can play as a chaperone for the folding of E2. In order to verify this fact we have expressed a chimeric protein (E1tmbE2) based on the full-length E1 sequence followed by the E2 ectodomain using the baculovirus-insect cells system. The E2 ectodomain is folded in the presence of the E1, proteolytically processed by cellular proteases and secreted to cell culture media (E2661p), while the E1 protein is retained into the cell due to its transmembrane sequence. The purification of E2661p from culture media was facilitated by a His tag introduced in its amino terminus. Both E2661 and E2661p glycoproteins shared very similar structural features, monitored by spectroscopic and antigenic studies. Moreover, their functional properties, tested by means of CD81 binding, were almost indistinguishable, indicating that the E2 ectodomain constitutes an independent folding unit.

Fusogenic properties of the ectodomains of hepatitis C virus envelope proteins.

We have used an isolated chimeric protein E1340 E2661 that includes the ectodomains of the envelope proteins of hepatitis C virus to study its interaction with model membranes. E1340 E2661 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'-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.