Inhibition of HIV-1 Envelope Glycoprotein-mediated Cell Fusion by a DL-Amino Acid-containing Fusion Peptide

Abstract

The N-terminal fusion peptide (FP) of human immunodeficiency virus-1 (HIV-1) is a potent inhibitor of cellcell fusion, possibly because of its ability to recognize the corresponding segments inside the fusion complex within the membrane. Here we show that a fusion peptide in which the highly conserved Ile 4 , Phe 8 , Phe 11 , and Ala 14 were replaced by their D-enantiomers (IFFA) is a potent inhibitor of cell-cell fusion. Fourier transform infrared spectroscopy confirmed that despite these drastic modifications, the peptide preserved most of its structure within the membrane. Fluorescence energy transfer studies demonstrated that the diastereomeric peptide interacted with the wild type FP, suggesting this segment as the target site for inhibition of membrane fusion. This is further supported by the similar localization of the wild type and IFFA FPs to microdomains in T cells and the preferred partitioning into ordered regions within sphingomyelin/phosphatidyl-choline/cholesterol giant vesicles. These studies provide insight into the mechanism of molecular recognition within the membrane milieu and may serve in designing novel HIV entry inhibitors.Specific fusion proteins, located on the surface of viral membranes, mediate membrane fusion (1). The envelope glycoprotein gp160 from HIV, 1 containing two non-covalently associated subunits, gp120 and gp41 (2), mediates the membrane fusion activity of the virus. The binding of the gp120 subunit to target cell receptors (3-7) induces a conformational change in the glycoprotein, which results in the exposure of a previously hidden hydrophobic N-terminal stretch of gp41, designated the "fusion peptide" (8, 9). Evidence supporting the role of the FP domain in mediating membrane fusion came from studies with intact envelope proteins (10 -12), as well as synthetic peptides used in model and biological systems (13-25). However, the molecular mechanism of membrane fusion is still poorly understood despite extensive studies done in both biological and model systems. Nevertheless, the results of many studies suggest common motifs for the diverse biological and model fusion reactions (13,26).Virus-induced membrane fusion is highly sensitive to single amino acid mutations in the FP domain (27,28). This can lead to the conclusion that the structure of FPs is a crucial parameter in the fusion process. Indeed, several studies suggest that the structure of FPs plays a major role in their activities (12,29). To distinguish between the effects of structure and hydrophobicity, we compared an all-L-amino acid FP with its enantiomer composed entirely of D-amino acids (25). Both had the same activity in liposome fusion assays, although having mirror image structures (25). Furthermore, the FP of HIV-1 was shown to inhibit cell-cell fusion (30 -32). Cumulative evidence suggests that the mechanism of inhibition is through interaction with the corresponding region in the intact gp41 (23,25). This inhibitory activity of the peptide is chirality-independent, eliminating the possibility of intera...

HIV-1 fusion peptide targets the TCR and inhibits antigen-specific T cell activation

“…We have recently reported that the 33-aa FP inserts into microdomains on T cells (15). Moreover, this FP showed a higher affinity toward ordered membrane domains in vitro (15).…”

“…We have recently reported that the 33-aa FP inserts into microdomains on T cells (15). Moreover, this FP showed a higher affinity toward ordered membrane domains in vitro (15). Assembly of the TCR and the CD4 molecules as well as other key molecules is required for complete T cell activation (16,17).…”

Current Peptide HIV Type-1 Fusion Inhibitors

“…Therefore, FP has a dual role to promote and inhibit fusion. This property of the HIV-1 FP makes it unsuitable for further development of the FP peptides as a therapeutic agent [46].…”

“…Kliger et al [45] found another 33 amino acid FP that could inhibit fusion at a two-order magnitude lower concentration than that of the 22 amino acid peptide. Later, Gerber et al [46] introduced d-amino acids into the peptides. A 33 amino acid peptide was synthesized in which four highly conserved amino acids, Ile4, Phe8, Phe11 and Ala14, were replaced by the respective d-enantiomers.…”

Targeting Cellular Cofactors in HIV Therapy

“…The optimized peptide variant VIR-576 had promising results in phase I/II clinical studies, which were save and revealed a dose-dependent decrease in viral load [92,93]. In a different approach the fusion peptide could be effectively inhibited in vitro by a variant of the natural fusion peptide containing four inserted D-amino acids [94].…”