1Hepatitis C virus encodes two enveloped glycoproteins, E1 and E2, which 2 are involved in viral attachment and entry into target cells. We have obtained in 3 insect cells infected by recombinant baculovirus a chimeric secreted recombinant 4 protein, E1 341 E2 661, containing the ectodomains of E1 and E2. The described 5 procedure allows the purification of approximately 2 mg of protein from 1 L of 6 culture media. Sedimentation velocity experiments and SDS-PAGE in the absence 7 of reducing agents indicate that the protein has a high tendency to self-associate, the 8 dimer being the main species observed. All the oligomeric forms observed maintain 9 a conformation which is recognized by the conformation-dependent monoclonal 10 antibody H53 directed against the E2 ectodomain. The spectroscopic properties of 11 E1 341 E2 661 are those of a three-dimensionally structured protein. Moreover, the 12 chimeric protein is able to bind to human antibodies present in HCV-positive human 13 sera. Accordingly, this chimeric soluble polypeptide chain may be a valuable tool to 14 study the structure-function relationship of HCV envelope proteins. 15 16
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.
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