Factors Contributing to the Fusogenic Potency of Foamy Virus Richard M. Epand and Raquel F. Epand 1 Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada Received May 9, 2001 Three model peptides have been studied in an effort to understand the molecular basis for the fusogenic potency of foamy virus. These peptides corresponded to a 23 amino acid helical segment close to the amino terminus, a shortened 17 amino acid, more hydropho- bic homolog of this peptide, and an 18-amino-acid pep- tide close to or within the transmembrane domain. The peptides have a conformation containing both -helical and -structure in aqueous solution but are predominantly -helical in solutions of trifluoroetha- nol, as assessed by circular dichroism. In common with other viruses, the most fusogenic peptide was the one closest to the amino terminus. However, unlike several other fusion peptides that have been studied previously, this peptide did not promote increase neg- ative membrane curvature as assessed by effects of the peptide on lipid polymorphism. Nevertheless, the foamy virus fusion peptide promotes membrane fu- sion, apparently by a mechanism that is independent of changes in membrane curvature. We demonstrate that there is a synergistic action in the promotion of membrane fusion between the peptide from the amino terminal region and the one from the region adjacent to the transmembrane segment. © 2001 Academic Press Key Words: foamy virus; spumavirus; viral fusion peptide; viral membrane fusion; membrane curvature. Segments of viral fusion proteins termed “fusion pep- tides” have been shown to be important in the promo- tion of viral fusion with several different viruses (1–3). Particularly interesting examples to study in this re- gard are foamy viruses. Foamy viruses are retroviruses and are also called spumaretroviruses. These viruses, as indicated by their name, have the characteristic of being highly fusogenic (4). Expression of the envelope, Env protein, in infected cells induces massive syncy- tium formation (5). The sequence of the Env protein from human foamy virus is known (6), although its pathogenicity in humans has not been well estab- lished. As with other Retroviridae, this protein is pro- teolytically cleaved into a surface (SU) and a trans- membrane (TM) subunit. There are a number of features of the sequence of the Env protein of foamy virus that are not typical of other Env proteins. The first segment of hydrophobic residues on the TM seg- ment, after the proteolytic cleavage of Env, is found to begin 11 residues from the amino terminus, rather than at the amino terminus, as is found with many other viral fusion proteins. The other unusual feature of this protein sequence is that the second stretch of hydrophobic amino acids, the putative transmembrane region, is particularly long, being comprised of 36 res- idues and having a Lys residue roughly in the middle of the sequence. The size of this stretch is much longer than a typical transmembrane domain. It has been suggested from mutagenesis experiments, that this re- gion is directly involved in regulating the fusion activ- ity of the virus at a structural level (7). We have inves- tigated the properties and membrane interactions of synthetic peptides corresponding to these two regions of the Env protein of human foamy virus in order to further ascertain the ability of these peptides to per- turb membranes and to promote membrane fusion. The specific peptides chosen were: T18, TAQGIFGTAFS- LLGYLKP-amide; N23, NYAKLRSMGYALTGAVQT- LSQIS-amide; N17, SMGYALTGAVQTLSQIS-amide. T18 corresponds to the amino terminal region of the putative transmembrane domain. This peptide is less hydrophobic than the following 19 amino acids and therefore may protrude from the surface of the mem- brane at the amino terminal side of the transmem- brane segment. The other two peptides correspond to Abbreviations used: DSC, differential scanning calorimetry; DiPoPE, dipalmitoleoylphosphatidylethanolamine; T H , bilayer to hexagonal phase transition temperature; CD, circular dichroism; DOPC, dioleoyl phosphatidylcholine; DOPE, dioleoyl phosphati- dylethanolamine; LUV, large unilamellar vesicles; N-Rh-PE, N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine; N-NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-yl) phosphatidyleth- anolamine. 1 To whom correspondence should be addressed at Department of Biochemistry, McMaster University Health Sciences Centre, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada. Fax: (905) 521-1397. E-mail: epand@McMaster.CA. Biochemical and Biophysical Research Communications 284, 870 – 874 (2001) doi:10.1006/bbrc.2001.5060, available online at http://www.idealibrary.com on 870 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.