The fusion-related properties of segments p9, p3, p4, and p9 ؉ p2 surrounding the p2 phospholipid-binding domain of the protein G (pG) of the salmonid rhabdovirus of viral hemorrhagic septicemia (VHS) (Nuñ ez, E., Fernandez, A. M., Estepa, A., Gonzalez-Ros, J. M., Gavilanes, F., and Coll, J. M. (1998) Virology 243, 322-330; Estepa, A., and Coll, J. M. (1996) Virology 216, 60 -70), have been studied at neutral and fusion (low) pH values by using its derived peptides. Cell-to-cell fusion, translocation of phosphatidylserine, and inhibition of fusion of pG-transfected cells defined the p9 ؉ p2 (fragment 11, sequence 56 -110) as a fragment with higher specific activity for anionic phospholipid aggregation than the previously reported p2. While fragment 11, p2, and p3 showed interactions with anionic phospholipids, p9 and p4 showed no interactions with any phospholipids. When added to a cell monolayer model at low pH, fragment 11 induced pH-dependent cell-to-cell fusion and translocated phosphatidylserine from the inner to the outer leaflet of the membrane. At low pH and in the presence of anionic phospholipids, fragment 11 showed more than 80% ␤-sheet conformation (IR and CD spectroscopies). Finally, anti-fragment 11 antibodies inhibited low pH-dependent pG-transfected cell-to-cell fusion. All of the data support the conclusion that fragment 11 is a primary determinant of some of the viral cell fusion events in VHSV.

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.

Sequence homology between the amino-terminal region of the S protein of hepatitis B Virus (HBV) and known fusion peptides from retroviruses and paramyxoviruses led us to propose that this region might be equally involved in the initial infective steps of hepadnaviruses. In fact, we showed that a synthetic peptide corresponding to the N-terminus region of the S protein of HBV had membrane-interacting properties and was able to induce liposome fusion adopting an extended (beta-sheet) conformation (Rodríguez-Crespo et al., 1996, 1995). We describe herein studies on the interaction of peptides derived from the N-terminal region of the S protein of duck (DHBV: Met-Ser-Gly-Thr-Phe-Gly-Gly-Ile-Leu-Ala-Gly-Leu-Ile-Gly-Leu-Leu) and woodchuck hepatitis B viruses (WHV: Met-Ser-Pro-Ser-Ser-Leu-Leu-Gly-Leu-Leu-Ala-Gly-Leu-Gln-Val-Val) with liposomes. These peptides were able to induce to a different extent aggregation, lipid mixing, and leakage of internal aqueous contents from both neutral and negatively charged phospholipid vesicles in a concentration-dependent and pH-independent manner. Fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene-labeled vesicles indicated that both peptides become inserted into the hydrophobic core of the lipid bilayer. Circular dichroism studies indicated that the DHBV peptide adopts an extended conformation in the presence of lipids, whereas the WHV peptide displays a high content of alpha-helical conformation. Therefore, these results extend our previous findings obtained for human hepatitis B virus to other members of the hepadnavirus family and suggest that this region of the S protein is important in the initial steps of the infective cycle.

Previous studies mapped a p2 domain (aa 82-109) which binds phosphatidylserine (PS) (Estepa and Coll, 1996a) and contains three contiguous hydrophobic amino acid heptad repeats followed by a positively charged stretch (Coll, 1995b) in the glycoprotein G of the viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus. Anti-p2 antibodies inhibited low-pH VHSV-induced fusion (Estepa and Coll, 1997) and low-pH PS binding to VHSV (Estepa and Coll, 1996a). We report here further studies on the interaction of the synthetic peptide p2 with phospholipid vesicles. The synthetic p2 peptide was able to mediate aggregation, lipid mixing, and leakage of contents only with negatively charged phospholipid vesicles and in a concentration-dependent manner. As shown by its effect on lipid phase transitions deduced from data with fluorescence polarization and differential scanning calorimetry, the p2 peptide becomes inserted into the hydrophobic negatively charged phospholipid vesicle bilayers. In addition, data based on circular dichroism showed that the p2 peptide folds as a structure with a high content of beta-sheets stabilized by interaction with anionic phospholipids. These studies are potentially relevant to viral fusion in VHSV.

The gene coding for the major capsid protein of feline immunodeficiency virus (FIV) has been cloned into the expression vector pQE60, which allows protein purification by affinity chromatography on a nitrilotriacetic acid/Ni/agarose column. The gene was expressed in Escherichia coli and the resultant soluble protein (FIV-rp24) purified to electrophoretic homogeneity. The amino-acid composition of the recombinant protein is almost identical to that predicted from the DNA sequence. This protein has two tryptophan residues at positions 40 and 126 that have been replaced by phenylalanine by site-directed mutagenesis to obtain two single mutants and a double mutant. Circular dichroism and fluorescence spectroscopy were employed to study the structural features of FIV-rp24 protein and its tryptophan mutants. The analysis of the CD spectra indicated that a-helix is the major secondary structural element (48±52%) and that the overall three-dimensional structure is not modified by the mutations. The fluorescence emission spectra showed that both tryptophan residues occupy a highly hydrophobic environment. Moreover, the different tyrosine fluorescence intensities of wild-type and mutant proteins are indicative of the existence of resonance energy transfer processes to nearby tryptophan. The individual contributions of each tryptophan residue to the spectroscopic properties of the wild-type protein were obtained from the spectra of all these proteins. Thermal denaturation studies indicate that the two tryptophan residues do not contribute equally to the stabilization of the three-dimensional structure.