10 páginas, 8 figuras, 3 tablas.We have identified a region within the ectodomain of the fusogenic human immunodeficiency virus type 1 (HIV-1) gp41, different from the fusion peptide, that interacts strongly with membranes. This conserved sequence, which immediately precedes the transmembrane anchor, is not highly hydrophobic according to the Kyte-Doolittle hydropathy prediction algorithm, yet it shows a high tendency to partition into the membrane interface, as revealed by the Wimley-White interfacial hydrophobicity scale. We have investigated here the membrane effects induced by NH2-DKWASLWNWFNITNWLWYIK-CONH2 (HIVc), the membrane interfacepartitioning region at the C terminus of the gp41 ectodomain, in comparison to those caused by NH2- AVGIGALFLGFLGAAGSTMGARS-CONH2 (HIVn), the fusion peptide at the N terminus of the subunit. Both HIVc and HIVn were seen to induce membrane fusion and permeabilization, although lower doses of HIVc were required for comparable effects to be detected. Experiments in which equimolar mixtures of HIVc and HIVn were used indicated that both peptides may act in a cooperative way. Peptide-membrane and peptide-peptide interactions underlying those effects were further confirmed by analyzing the changes in fluorescence of peptide Trp residues. Replacement of the first three Trp residues by Ala, known to render a defective gp41 phenotype unable to mediate both cell-cell fusion and virus entry, also abrogated the HIVc ability to induce membrane fusion or form complexes with HIVn but not its ability to associate with vesicles. Hydropathy analysis indicated that the presence of two membrane-partitioning stretches separated by a collapsible intervening sequence is a common structural motif among other viral envelope proteins. Moreover, sequences with membrane surfaceresiding residues preceding the transmembrane anchor appeared to be a common feature in viral fusion proteins of several virus families. According to our experimental results, such a feature might be related to their fusogenic function.T.S. and A.A. are recipients of predoctoral fellowships from the Basque Government. This work was supported by DGCYT (grant PB96-0171), the Basque Government (PI 96-46; EX-1998-28; PI-1998- 32), and the University of the Basque Country (UPV 042.310-EA085/ 97; UPV 042.310-G03/98). Support to W.R.G. includes senior fellowship 1F32AI08549 and grant 1R01DE10862 from the National Institutes of Health of the United States.Peer reviewed