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

The fusion domain of the HIV-1 envelope glycoprotein (gp120-gp41) is a cons erved hydrophobic region located at the N-terminus of the transmembrane sub unit (gp41). A prominent feature of this domain is a conserved five-residue "FLGFL" sequence at positions 8-12. Mutation of the highly conserved Phe(1 1) to Val (F11V), presumed not to significantly affect the hydrophobicity a nd the structure of this region, has been shown to decrease the level of sy ncytium 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 pep tide, a 33-residue peptide (WT) identical to the extended fusion domain and its F11V and F11G mutants were synthesized, fluorescently labeled, and stu died with respect to their function, structure, and organization in phospho lipid 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 sh own to be inactive with PC vesicles but able to induce fusion of POPG vesic les which had been preaggragated with Ca2+ or Mg2+, The F11V peptide preser ved 50% activity, and the F11G peptide was virtually inactive, ATR-FTIR spe ctroscopy indicated similar secondary structure of the peptides in multibil ayers that was independent of membrane composition. Furthermore, all the pe ptides 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 > F11 V > WT. Fluorescence studies in the presence of membranes, as well as SDS - PAGE, revealed that the WT and Fl IV peptides self-associate to similar lev els while F11G exhibited a decreased level of self-association. The data su ggest that the FLGFL motif contributes to the functional organization of th e HIV-1 fusion peptide and that the C-terminal domain following the fusion peptide contributes to the membrane fusion process.