Effect of Nonpolar Substitutions of the Conserved Phe 11 in the Fusion Peptide of HIV-1 gp41 on Its Function, Structure, and Organization in Membranes ² Moshe Pritsker, ‡ Joseph Rucker, § Trevor L. Hoffman, § Robert W. Doms, § and Yechiel Shai* ,‡ Department of Biological Chemistry, Weizmann Institute of Science, RehoVot 76100, Israel, and Department of Pathology and Laboratory Medicine, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104 ReceiVed February 1, 1999; ReVised Manuscript ReceiVed May 18, 1999 ABSTRACT: The fusion domain of the HIV-1 envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N-terminus of the transmembrane subunit (gp41). A prominent feature of this domain is a conserved five-residue “FLGFL” sequence at positions 8-12. Mutation of the highly conserved Phe 11 to Val (F11V), presumed not to significantly affect the hydrophobicity and the structure of this region, has been shown to decrease the level of syncytium formation and virus infectivity. Here we show that the substitution of Gly for Phe 11 (F11G) reduces cell-cell fusion activity by 80-90%. To determine the effect of these mutations on the properties of the fusion peptide, a 33-residue peptide (WT) identical to the extended fusion domain and its F11V and F11G mutants were synthesized, fluorescently labeled, and studied with respect to their function, structure, and organization in phospholipid membranes. The WT peptide alone induced fusion of both zwitterionic (PC/Chol) and negatively charged (PS/PC/ Chol and POPG) vesicles, in contrast to a 23-mer fusion peptide lacking the C-terminal domain which has been shown to be inactive with PC vesicles but able to induce fusion of POPG vesicles which had been preaggragated with Ca 2+ or Mg 2+ . The F11V peptide preserved 50% activity, and the F11G peptide was virtually inactive. ATR-FTIR spectroscopy indicated similar secondary structure of the peptides in multibilayers that was independent of membrane composition. Furthermore, all the peptides increased the extent of lipid disorder to a similar extent, but the kinetics of amide II H to D exchange was in the following order: F11G > F11V > WT. Fluorescence studies in the presence of membranes, as well as SDS-PAGE, revealed that the WT and F11V peptides self-associate to similar levels while F11G exhibited a decreased level of self-association. The data suggest that the FLGFL motif contributes to the functional organization of the HIV-1 fusion peptide and that the C-terminal domain following the fusion peptide contributes to the membrane fusion process. Membrane fusion is an essential process for the infectious entry of enveloped viruses into host cells. The envelope glycoprotein gp160 of the human immunodeficiency virus type 1 (HIV-1) contains two noncovalently associated subunits, gp120 and gp41 (1), and mediates the membrane fusion activity of the virus. The outer surface subunit, gp120, contains sites necessary for viral binding to target cells containing CD4 (2) and chemokine receptors (3-5), whereas the transmembrane gp41 is responsible for the fusion process between viral and cell membranes (6). The binding of the gp120 subunit to the CD4 receptor induces a conformational change in the glycoprotein which enables it to interact with a chemokine receptor (7-10). This in turn is thought to result in the exposure of the previously hidden hydrophobic N-terminal domain of gp41, and its penetration into the target cell membrane. This hydrophobic domain, designated the “fusion peptide”, is highly homologous with equivalent domains of other enveloped viruses (11). A prominent feature among the HIV family is the absolutely conserved, five- residue “FLGFL” sequence at positions 8-12. Interestingly, the “FXG” motif is also highly conserved in the fusion peptides of paramyxoviruses (12). Evidence for the role of the fusion peptide domain in mediating membrane fusion comes from mutagenesis studies of intact envelope proteins, as well as from studies with synthetic fusion peptides and their analogues. Site-specific mutations in the fusion peptide domain of various viruses can decrease or increase their activity. For example, polar substitutions in the fusion peptides of HIV-1 (13) and SIV (14) can decrease the level of syncytium formation. Gly to Ala substitutions in the fusion peptide of SIV (14) and the paramixovirus SV5 (15), anticipated to enhance the overall hydrophobicity and R-helix formation, enhanced syncytium formation. On the other hand, mutation in the fusion peptide of HIV-1 of Gly at various positions to Val which enhances R-helix formation decreases the level of syncytium formation and virus infectivity (16). It should be noted that while muta- tions in other regions of envelope proteins can also decrease activity (17, 18) only the fusion peptide is believed to be directly involved in the actual membrane fusion event. Durrer et al. (19), using a radiolabeled photoaffinity reagent, showed that during low pH-induced membrane insertion of influenza ² This work was supported in part by the Basic Research Foundation administered by the Israel Academy of Sciences and Humanities. * To whom correspondence should be addressed: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel. Telephone: 972-8-9342711. Fax: 972-8-9344112. E- mail: bmshai@weizmann.weizmann.ac.il. ‡ Weizmann Institute of Science. § University of Pennsylvania. 11359 Biochemistry 1999, 38, 11359-11371 10.1021/bi990232e CCC: $18.00 © 1999 American Chemical Society Published on Web 08/10/1999