The Polar Region Consecutive to the HIV Fusion Peptide Participates in Membrane Fusion

Abstract

The fusion peptide of HIV-1 gp41 is formed by the 16 N-terminal residues of the protein. This 16-amino acid peptide, in common with several other viral fusion peptides, caused a reduction in the bilayer to hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (T(H)), suggesting its ability to promote negative curvature in membranes. Surprisingly, an elongated peptide corresponding to the 33 N-terminal amino acids raised T(H), although it was more potent than the 16-amino acid fusion peptide in inducing lipid mixing with large unilamellar liposomes of 1:1:1 dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine/choleste rol. The 17-amino acid C-terminal fragment of the peptide can induce membrane fusion by itself, if it is anchored to a membrane by palmitoylation of the amino terminus, indicating that the additional 17 hydrophilic amino acids contribute to the fusogenic potency of the peptide. This is not solely a consequence of the palmitoylation, as a random peptide with the same amino acid composition with a palmitoyl anchor was less potent in promoting membrane fusion and palmitic acid itself had no fusogenic activity. The 16-amino acid N-terminal fusion peptide and the longer 33-amino acid peptide were labeled with NBD. Fluorescence binding studies indicate that both peptides bind to the membrane with similar affinities, indicating that the increased fusogenic activity of the longer peptide was not a consequence of a greater extent of membrane partitioning. We also determined the secondary structure of the peptides using FTIR spectroscopy. We find that the amino-terminal fusion peptide is inserted into the membrane as a beta-sheet and the 17 C-terminal amino acids lie on the surface of the membrane, adopting an alpha-helical conformation. It was further demonstrated with the use of rhodamine-labeled peptides that the 33-amino acid peptide self-associated in the membrane while the 16-amino acid N-terminal peptide did not. Thus, the 16-amino acid N-terminal fusion peptide of HIV inserts into the membrane and, like other viral fusion peptides, lowers T(H). In addition, the 17 consecutive amino acids enhance the fusogenic activity of the fusion peptide presumably by promoting its self-association.

Synthesis, Enhanced Fusogenicity, and Solid State NMR Measurements of Cross-Linked HIV-1 Fusion Peptides

“…In addition, there was approximately the same NMR signal per scan for the FPmn and FPdm samples (cf. In related studies, other investigators have studied the lipid binding and fusogenicities of peptides that contained between 16 and 70 of the N-terminal residues of gp41 (7,10). Longer peptides had higher fusogenicities, but at 0.1 µM peptide concentration there were negligible differences in the lipid affinities of the different peptides.…”

“…The interaction between fusion peptides and lipids is believed to be one key event in initiating membrane fusion (5). Recent studies suggest that fusion protein regions other than the fusion peptide also interact with membranes and play a role in fusion (6)(7)(8)(9)(10).…”

Cell-Cell Membrane Fusion Induced by p15 Fusion-associated Small Transmembrane (FAST) Protein Requires a Novel Fusion Peptide Motif Containing a Myristoylated Polyproline Type II Helix

“…Although liposome aggregation is a priori for peptide-induced lipid mixing, the p10 and p14 FAST protein FPs induce both events concurrently with no lag time in lipid mixing (30,31). The same situation applies to enveloped virus FPs, where peptide addition leads to lipid mixing with little (Ͻ20 s) to no lag (47)(48)(49). Lipid mixing induced by the p15 ectodomain is therefore a biphasic reaction, where the rate-limiting step is peptidemembrane interactions that trigger liposome aggregation followed by additional interactions that drive lipid mixing.…”

“…6). In contrast, the 80 -100% lipid mixing induced by the HIV FP occurs within 20 -30 s at a peptide:lipid ratio of 1:10, and ϳ40% lipid mixing occurs in Ͻ150 s at a peptide:lipid ratio of 1:60 (36,49,50). Thus, the p15 ectodomain FP has a lower specific activity in lipid mixing assays compared with the FP of HIV, and to the FPs of influenza virus and Sendai virus (48,51).…”

Phosphatase‐Triggered Fusogenic Liposomes for Cytoplasmic Delivery of Cell‐Impermeable Compounds

“…13,19 As a result phosphatases have been used for decades as a trigger mechanism for the activation of pro-drugs whose water solubility is increased by the attachment of a phosphate. 20,21 We selected the well characterized HIV gp41 N-terminus fusion peptide 7 , [22][23][24] as a template for designing triggerable fusogenic phosphopeptides. Phosphate groups were placed in nonconserved positions at the N and C termini [25][26][27] of a 22 residue peptide in order to disrupt membrane insertion and increase sequence polarity and trigger control.…”