Most of the lipid components of hepatitis B surface antigen (HBsAg) can be removed by treatment with the non-ionic non-denaturing detergent beta-D-octyl glucoside (OG) followed by centrifugation through caesium chloride linear density gradients (density 1.15-1.32 g/ml). The conformational changes induced by the elimination of lipids decreased the helical content of HBsAg proteins from 52 to 28% as indicated by c.d. techniques. Measurements of the extent of quenching of protein fluorescence by iodide showed that half of the tryptophan residues which are buried in the native structure of HBsAg particles are brought close to the surface of the molecule by such conformational changes. The antigenic activity, as measured by binding to polyclonal antibodies, was decreased upon removal of lipids. Moreover, the six different antigenic sites recognized by our panel of monoclonal antibodies decreased their capacity to bind to the corresponding antibody when lipids were removed. However, the extent of this decrease differed for the different antibodies. Thus the apparent dependence of antibody binding on the lipid content seemed to indicate a greater involvement of the lipid-protein interaction for some of the epitopes than for others.

One of the first steps in the infective cycle of an enveloped virus consists of the fusion of the viral and cellular membranes. This process is usually achieved as a result of membrane destabilization brought about by a viral fusion peptide located at the amino terminus of one of the viral envelope glycoproteins. Previous sequence similarity studies by Rodrlguez-Crespo et al. (Journal of General Virology 75, 637-639, 1994) have shown that a hydrophobic stretch in the amino-terminal sequence of the S protein of hepatitis B virus shares several characteristics with fusion peptides of retroviruses and paramyxoviruses. A 16 residue peptide with this sequence was synthesized and its interaction with liposomes characterized. This peptide was able to mediate vesicle aggregation, lipid mixing and liposome leakage in a pH dependent manner at concentrations ranging from 3"5 to 52.0 pM. These effects were specific for negatively charged phospholipid vesicles. The peptide was also able to haemolyse erythrocytes. This study supports the notion that the sequence might be important in the initial infective steps of this virus, interacting with the target membranes and bringing about their subsequent destabilization.

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Based on the predicted capacity to interact with membranes at the interface, we have found three regions in the ectodomain of the hepatitis C virus envelope glycoprotein E2 (430-449, 543-560 and 603-624) with the ability to destabilize membranes. Three peptides corresponding to the sequence of these regions have been synthesized and their interaction with liposomes have been characterized. The three peptides were able to insert deeply into the hydrophobic core of negatively charged phospholipids as stated by fluorescence depolarization of the probe 1,6-diphenyl-1,3,5-hexatriene. Peptides E2(430-449) and E2(603-624) were able to induce aggregation of phosphatidylglycerol vesicles in a concentration-dependent manner both at neutral and acidic pH while peptide E2(543-560) did not induce any increase of optical density at 360 nm in the concentration range studied. The three peptides induced lipid mixing and the release of the internal contents in a dose-dependent manner when acidic phospholipids were used. Fourier transformed infrared spectroscopy indicated that the peptides adopted mainly a beta-sheet conformation which is not modified by the presence of acidic phospholipids. Taken together, our results point out to the involvement of these three regions in the fusion mechanism of HCV at the plasma membrane level.

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