Membrane-Anchored HIV-1 N-Heptad Repeat Peptides Are Highly Potent Cell Fusion Inhibitors via an Altered Mode of Action

Peptide inhibitors derived from HIV-gp41 envelope protein play a pivotal role in deciphering the molecular mechanism of HIV-cell fusion. According to accepted models, N-heptad repeat (NHR) peptides can bind two targets in an intermediate fusion conformation, thereby inhibiting progression of the fusion process. In both cases the orientation towards the endogenous intermediate conformation should be important. To test this, we anchored NHR to the cell membrane by conjugating fatty acids with increasing lengths to the N- or C-terminus of N36, as well as to two known N36 mutants; one that cannot bind C-heptad repeat (CHR) but can bind NHR (N36 MUTe,g), and the second cannot bind to either NHR or CHR (N36 MUTa,d). Importantly, the IC50 increased up to 100-fold in a lipopeptide-dependent manner. However, no preferred directionality was observed for the wild type derived lipopeptides, suggesting a planar orientation of the peptides as well as the endogenous NHR region on the cell membrane. Furthermore, based on: (i) specialized analysis of the inhibition curves, (ii) the finding that N36 conjugates reside more on the target cells that occupy the receptors, and (iii) the finding that N36 MUTe,g acts as a monomer both in its soluble form and when anchored to the cell membrane, we suggest that anchoring N36 to the cell changes the inhibitory mode from a trimer which can target both the endogenous NHR and CHR regions, to mainly monomeric lipopetides that target primarily the internal NHR. Besides shedding light on the mode of action of HIV-cell fusion, the similarity between functional regions in the envelopes of other viruses suggests a new approach for developing potent HIV-1 inhibitors

Relative binding of NBD-labeled peptides to cells.

<p>_NBDN36, C16-N36M_NBD, and _NBDN36M-C16 are represented by closed squares, closed triangles, and open triangles, respectively. For comparison, a non-binding peptide _NBDGCN4 (open circles) and the strongly binding peptide C16-_NBDGCN4 (closed circles) were measured.</p

A cartoon illustrating possible modes of inhibition.

<p>The NHR region is denoted by green cylinders, the CHR region is denoted by orange cylinders, and the fusion peptide is denoted by a black line. The C-terminus of the inhibitory N- or C-peptides is represented by a black color. The Pre-fusion conformation is presented on the left of the middle panel. N36 can bind the Pre-fusion conformation in two ways: It can bind the CHR region and inhibit progression into the Post-fusion conformation, or it can interrupt the creation of the central NHR coiled-coil by driving the equilibrium towards the dimeric and monomeric forms (here only the monomeric form is presented for simplicity) thereby preventing progression into the Post-fusion conformation. CHR can only bind the NHR region in the Pre-fusion conformation thereby preventing fusion.</p

Sequences, designations, and IC₅₀ values of the N36 mutated peptides and their lipophilic conjugates.

<p>Sequences, designations, and IC₅₀ values of the N36 mutated peptides and their lipophilic conjugates.</p

The inhibitory oligomeric state of the peptides.

<p>(A) Fusion inhibition curves of the N-terminally conjugated peptides (other data not shown). N36, C8-N36, C12-N36, and C16-N36 are represented by closed squares, diamonds, circles, and triangles, respectively, and the fitted curves are represented by continuous lines. (B) The inhibitory oligomeric state of the peptides. The Hill's coefficient parameter for the different peptides is presented. For each peptide at least four independent experiments were performed and were included in the calculation of the standard deviation.</p

Cell-cell fusion inhibition assay for the N36 peptide and its fatty acid conjugates.

<p>Fusion inhibition (<i>IC₅₀</i> values) induced by the peptides. For each peptide at least four independent experiments were performed and were included in the calculation of the standard deviation.</p

Cell-cell fusion inhibition assay for the N36 mutants as well as their fatty acid conjugates.

<p>(A) Fusion inhibition induced by the N36 MUTe,g peptides. The <i>IC₅₀</i> values of the different peptides are presented. For each peptide at least four independent experiments were performed and were included in the calculation of the standard deviation. (B) The inhibitory oligomeric state of the peptides. The Hill's coefficient parameter for the different peptides is presented. For each peptide at least four independent experiments were performed and were included in the calculation of the standard deviation.</p

CD spectroscopy of the peptides alone, and together with C34.

<p>The peptides were measured at 10 µM in HEPES buffer (5 mM, pH 7.4) or 1% LPC in H₂O (membrane mimetic environment). Left column: The peptide signal alone in buffer solution (open circles) compared to the peptide signal in 1% LPC (closed circles). Middle column: The calculated, non-interacting signal for the N-peptide with C34 (open triangles), compared to the observed experimental signals, obtained following incubation of the two peptides together in buffer solution (closed triangles). Right column: The same experiment was done in LPC. The calculated non-interacting and the experimental signals are represented by open and closed squares, respectively.</p

Sequences and designations of the NBD labeled peptides and their lipophilic conjugates.

<p>Sequences and designations of the NBD labeled peptides and their lipophilic conjugates.</p

A cartoon illustrating a plausible model for the fusion process and its inhibition by the long fatty acid conjugated peptides.

<p>The NHR region is denoted by green cylinders, the CHR region is denoted by orange cylinders, the inhibitory N36 peptide is denoted by blue cylinders (blue and black for the N- and C-terminal of the peptide, respectively), and the fatty acid is denoted by a thick black line. On the left is the conformation immediately after the binding of the receptors and co-receptors, in which the NHR coiled-coil has not been created yet- “loose” pre-fusion. If the NHR region, as well as the N36 peptide, has a planar orientation towards the cell membrane, it can explain the directionality independence of fatty acid conjugation. Here, we show only the interference with the formation of the trimeric coiled-coil since it appears to be the main inhibitory mode of the conjugated N36 peptides.</p