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Published as doi: 10.1096/fj.07-8582com.
 (The FASEB Journal. 2007;21:3677-3684.)
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Demonstrating the C-terminal boundary of the HIV 1 fusion conformation in a dynamic ongoing fusion process and implication for fusion inhibition

Yael Wexler-Cohen and Yechiel Shai1

Department of Biological Chemistry, the Weizmann Institute of Science, Rehovot, Israel

1Correspondence: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100 Israel. E-mail: yechiel.shai{at}weizmann.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The core complex is a structure involved in the fusion mechanism of many viruses, as well as in intracellular vesicle fusion. A powerful approach for studying the dynamic stages of HIV-1-cell fusion utilizes DP178, a core complex inhibitory peptide derived from the known sequence of the virus. Strikingly, we show that fatty acids can replace the entire C-terminal region of DP178, known to play a crucial role in the activity of the peptide. The inhibitory activity correlated with the length of the fatty acid, with the direction of fatty acid attachment (N- or C-terminus) and, as envisioned by a new triple staining assay, with the concentration of the peptides on cells. Our findings indicate, for the first time, the C-terminal boundary of the endogenous core structure in situ and establish that the C-terminal region of DP178 functions mainly as an anchor to the cell membrane. Apart from the mechanistic implications, such short lipopeptides provide new, promising fusion inhibitors. Because the fusion mechanism of HIV-1 is shared by other pathogen-enveloped viruses and by intracellular vesicle fusion, our results might influence the research and therapeutic efforts in these systems as well.—Wexler-Cohen, Y. and Shai, Y. Demonstrating the C-terminal boundary of the HIV 1 fusion conformation in a dynamic ongoing fusion process and implication for fusion inhibition.


Key Words: HIV-1 inhibition • lipopeptides • coiled coil • T-20 • virus entry • envelope protein • heptad repeat • peptide-membrane interaction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HIV-1, LIKE OTHER ENVELOPED VIRUSES UTILIZES a protein embedded in its membrane, termed envelope protein (ENV), to facilitate the fusion process (1 , 2) . The ENV glycoprotein is organized as trimers on the membrane of the virus and is composed of two noncovalently associated subunits. The surface subunit (SU), gp120, mediates host tropism (reviewed by Clapham and McKnight (3) ), whereas the transmembrane subunit (TM), gp41, is responsible for the actual fusion event (reviewed by Chan and Kim (4) ). The extracellular part of gp41, (see Fig. 1 )is composed of several functional regions, including the fusion peptide (FP), the N-terminal heptad repeat (NHR), the C-terminal heptad repeat (CHR), and the pretransmembrane (PTM) domain (Fig. 1 A).


Figure 1
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  		Figure 1. A) A schematic representation of the gp41 ectodomain. Internal regions are specified by abbreviations: FP-fusion peptide, NHR- N-terminal heptad repeat, CHR- C-terminal heptad repeat, and PTM- pretransmembrane region. The location of the DP178 peptide is marked as well as its sequence numbering. B) A model for the HIV-1 fusion mechanism. In the native state, gp41 is presumed to be sheltered by gp120. After binding, the cell receptors (and coreceptors, not illustrated here for simplicity reasons), both subunits undergo major conformational changes leading to the prehairpin conformation. In this conformation, gp41 is extended, leading to the insertion of the FP into the membrane of the host cell. Folding into the hairpin conformation juxtaposes the viral and host membranes, resulting in membrane fusion.

The ability of the virus to fuse its own membrane with that of the hosting cell is due to conversion among three identified ENV conformations (Fig. 1B ). Initially, the envelope subunits are in a metastable native conformation (5) , in which gp41 is considered to be sheltered by gp120. Binding of gp120 to specific cell receptors involves conformational changes of both subunits, resulting in the Pre-Hairpin conformation (5 , 6) , in which gp41 is exposed and extended, leading to insertion of the FP into the host cell membrane (7) . Additional conformational changes produce the hairpin conformation (3 , 8) , where a trimeric central coiled-coil is created by three NHR regions. Three CHR regions are packed in an antiparallel manner into conserved hydrophobic grooves exposed on the surface of the central NHR coiled-coil. A complex representing this structure has been resolved by X-ray crystallography (9 , 10) and is designated as the "six helix bundle" or "core" structure. Similar bundles are created in intracellular vesicle fusion by SNARE proteins demonstrating a common mechanism in diverse systems (11 , 12) . Since the crystal structure of the core was based on peptides, the precise endogenous sequence responsible for its creation is not known.

Inhibition of HIV-1-mediated fusion has been demonstrated by several C-peptides: peptides that originate from the endogenous CHR sequence of gp41 (5 , 13 , 14) . One of these is the recently FDA-approved anti-HIV-1 drug, DP178 (15) . It is a 36-amino acid peptide, also known as T20 or Enfuvirtide, which inhibits fusion at nanomolar concentrations. The N- and C-terminal regions of the peptide have been shown to be crucial for its activity, since mutations and truncations to either of them, abolished inhibition (16 , 17) . The common model for its inhibitory function assumes blocking core formation by binding the endogenous NHR region in the prehairpin conformation. Nevertheless, accumulating evidence suggests that the C-terminal sequence is probably involved in an additional, distinct mode of action (18 19 20 21 22) . Substitution for D-amino acids in this region did not interfere with the ability of the peptide to inhibit fusion, indicating involvement of this region in membrane binding (23 24 25) . Regardless of the mode of action of DP178, applying variants of DP178 as fusion inhibitors can be utilized to monitor their effect on endogenous structures during a dynamic ongoing fusion process.

In this study, we sought to establish the C-terminal boundary of the endogenous core structure, and for this purpose, we determined the minimal DP178 inhibitory sequence that enables the creation of a stable, functional core in situ. Additionally, to ascertain the contribution of the C-terminal to the fusion process, we designed DP178 variants in which the truncated C-terminus was replaced by fatty acids. Such substitution can enable us to characterize the contribution of the actual sequence vs. its hydrophobicity and to obtain shorter lipopeptides as potential fusion inhibitors with reduced manufacturing costs. In this study, we utilized C- and N-terminally truncated DP178 peptides, as well as their C- and N-terminally conjugated fatty acid variants. Two different cell-cell fusion systems were employed and one of them was further developed into a new triple staining assay to enable detection of the concentration of the peptides on the cells. The results are discussed in light of the HIV-1 fusion mechanism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
F-Moc amino acids, including lysine with an MTT side chain-protecting group and F-Moc Rink Amide MBHA resin, were purchased from Novabiochem AG (Laufelfinger, Switzerland). Other peptide synthesis reagents and fatty acids, namely, octanoic acid (C8), dodecanoic acid (C12), and hexadecanoic acid (C16), were purchased from Sigma Chemical Co. (Kibutz Beit Haemek, Israel). DiD (DiIC18 (5) or 1,1'-dioctadecyl-3,3,3',3',-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt), DiI (1,1'-dioctadecyl-3,3,3',3',0 tetramethylinocarbocyanine perchlorate) lypophilic fluorescent probes and NBD-F, fluoride (4-fluoro-7-nitrobenzofurazan) were obtained from Biotium (Hayward, CA, USA). The XTT Proliferation Assay kit, and the Steady-Glow Luciferase detection kit were purchased from Biological Industries Israel (Beit Haemek).

Cell lines and reagents
Cell culture reagents and media were purchased from Biological Industries Israel (Beit Haemek LTD). All cell lines were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH HL2/3 cells expressing cleaved HIV-1 molecular clone HXB2/3gpt were from Dr. Barbara Felber and Dr. George Pavlakis. TZM-bl indicator cells, which express high levels of CD4 and CCR5 along with endogenously expressed CXCR4, were from Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme Inc. (Durham, NC, USA). Jurkat E6–1 cells were from Dr. Arthur Weiss (26) , and Jurkat HXBc2 (4) cells expressing HIV-1 HXBc2 Rev and ENV proteins were from Dr. Joseph Sodroski. Cells were cultured every 3 to 4 days and maintained in the appropriate medium at 37°C with 5% CO2 in a humidified incubator. For ENV expression, Jurkat HXBc2 (4) cells were transferred to medium without tetracycline 3 days prior to experiments.

Peptide synthesis, fatty acid conjugation, and fluorescent labeling
DP178wt, DP, DP20, DP15, 22DP, and 20DP were synthesized on Rink Amide MBHA resin by using the F-moc strategy, as described previously (27) . All peptides contain a lysine residue at their C-terminus with an MTT side chain protecting group, enabling the conjugation of a fatty acid that required a special deprotection step under mild acidic conditions (2x1 min of 5% TFA in DCM and 30 min of 1% TFA in DCM). Conjugation of a fatty acid to the N terminus was performed using standard F-moc chemistry. The addition of the NBD (emission-530 excitation-467) fluorescent probe to the N terminus of selected peptides was performed in DMF + 2% DIEA solution for 1 h. All peptides were cleaved from the resin by a TFA:DDW:TES (93.1:4.9:2 (v/v)) mixture and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) to >95% homogeneity. The molecular weight of the peptides was confirmed by platform LCA electrospray mass spectrometry.

Determining the relative hydrophobicity of the peptides
Each peptide was loaded under the same conditions onto a RP-HPLC C4 column, and their retention times were recorded.

Reporter gene fusion assay
Fusion was monitored based on activation of HIV LTR-driven luciferase cassette in TZM-bl (Target) cells by HIV-1 Tat from the HL2/3 (Effector) cells. The fusion assay was performed in a serum-free medium to minimize nonspecific association of peptides with serum proteins. The peptides were solubilized in DMSO at various dilutions. The TZM-bl cells, plated in 96-well clusters (2.5x104 per well) overnight at 37°C, were placed in 50 µl of serum-free medium per well prior to fusion assays. The target cells were cocultured with 50 µl HL2/3 cells (2.5x104 per well in serum-free medium) for 6 h at 37°C in the presence or absence of various peptide concentrations (1 µl peptide per well). Efficiency of fusion was determined by measuring luciferase activity in the wells following the instructions of the manufacturer using the steady-glow luciferase determination kit (Promega, Madison, WI, USA). The light intensity was measured using a VICTOR2 1420 multilabel counter (Wallac, PerkinElmer Life Sciences, Wellesley, MA, USA). Background luminescence in TZM-bl cells was determined without the addition of HL2/3 cells. The assay was also performed in medium containing serum for the three most active peptides: DP178, DP-C12, and DP-C16.

Triple staining flow cytometry fusion assay
The protocol utilizing Jurkat E6–1 and Jurkat HXBc2 cells for a cell-cell fusion assay was previously described (28) . In short, Jurkat E6–1 and Jurkat HXBc2 cells were labeled with DiI and DiD lypophilic fluorescent probes, respectively. The two cell populations were coincubated for 6 h in a ratio of 1:1 in the presence of different concentrations of the inhibitory peptides. Cells coincubated without the presence of peptides served as optimal fusion reference. Unlabeled cells that were handled similarly served as an intrinsic fluorescence control. Cells labeled separately with DiI or DiD were used to compensate for the optimal separation of fluorescent signals. Jurkat HXBc2 cells labeled with DiI were coincubated with Jurkat HXBc2 cells labeled with DiD for a fusion background that was subtracted from the measurements of the experiment. The following alterations were applied to the original protocol: 1) 5 µl of a 1 mg/ml DiI or DiD solution in dimethyl sulfoxide (DMSO) was added to 1 ml of 4 x 106 cells/ml Jurkat E6–1 or Jurkat HXBc2 cells, respectively. 2) Data from at least 600,000 events for each well were collected on FACSort upgraded to a FACSCalibur cell analyzer (Becton Dickinson, Franklin Lakes, NJ, USA) and further analyzed.

For triple staining, the same experiment was performed in the presence of NBD-labeled peptides. Cells labeled separately with DiI or DiD and unlabeled cells in the presence of an NBD labeled peptide were used to compensate for the optimal separation of the three fluorescent signals. Analysis of the data enabled us to assign a labeled peptide to different cell populations.

Cytotoxicity assays (XTT proliferation assay)
Aliquots of 2.5 x 104 Jurkat E6–1 cells were distributed onto a 96-well plate (Falcon) in the presence of various peptide concentrations. Wells in the last two columns served as blank (medium only), and 100% survival controls (cells and medium only), respectively. The plate was then incubated for 24 h before adding to each well the XTT reaction solution (sodium 3'-[1-(phenyl-aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate and N-methyl dibenzopyrazine methyl sulfate, mixed in a proportion of 50:1). Following an incubation of 4–5 h, the optical density was read at a 450-nm wavelength in an enzyme-linked immunoabsorbent assay plate reader. LC50 (concentration at which 50% of the cells die) was determined relative to the control, 2.5 x 104 cells in 100 µl medium without serum. All assays were performed in duplicate.

Hemolysis of human red blood cells
Fresh hRBCs were rinsed two times with PBS (35 mM phosphate buffer, 0.15 M NaCl, pH 7.3), followed by centrifugation for 7 min at 2000 rpm and resuspension in PBS. Peptides, at various concentrations, were dissolved in PBS and added to 50 µl of the stock hemolysis of human red blood cell (hRBC) solution in PBS for a final volume of 100 µl (final erythrocyte concentration, 4% (v/v)). The resulting suspension was incubated with agitation for 60 min at 37°C. The samples were then centrifuged at 2000 rpm for 7 min. The release of hemoglobin was monitored by measuring the absorbance of the supernatant at 540 nm. Controls for zero hemolysis (blank) and 100% hemolysis consisted of hRBCs suspended in PBS and PBS with 1% Triton, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Determining the minimal functional inhibitory core structure
First, to determine the minimal DP178 sequence that enables the creation of a stable, functional core in situ, we synthesized C-terminally truncated DP178 peptides. Additionally, to examine the crucial component-sequence vs. hydrophobicity, we conjugated a fatty acid to their nicked C-terminus (see Fig. 2 A). All peptides were examined in a cell-cell fusion inhibition assay. DP and DP-C12 exhibited very low and strong inhibition activity; IC50 of 2864 nM and 9.9 nM, respectively. In contrast, DP20 and DP15, as well as their fatty acid conjugates, did not exhibit any fusion inhibition ability (see Fig. 2B ). Furthermore, truncating the DP peptide further at its N terminus (see Fig. 2A ) also abolished activity. To determine whether the five C-terminal amino acids of the DP peptide are crucial for the creation of the core structure or whether DP20 is too short and cannot reach its target site, we replaced four C-terminal amino acids of DP with glycine residues, as well as conjugated a longer fatty acid to DP20 (see Fig. 2C ). DP20G4, DP20G4-C12, and DP20-C16 did not exhibit significant inhibitory activity (Fig. 2C ). We concluded that the sequence of the DP peptide is the minimal sequence that enables the creation of a functional core; the C-terminal amino acids of DP are crucial for its inhibitory activity, and the role of the hydrophobic C-terminus can be compensated by another, unrelated hydrophobic moiety.


Figure 2
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  		Figure 2. Determination of the minimal functional inhibitory core structure. A) Sequences of the peptides synthesized are marked. Each peptide was also conjugated to a fatty acid with 12 carbon atoms in its chain (C12). Each conjugate is designated by the name of the peptide followed by C12, for example, DP15-C12. The C-terminal amino acid in all peptides is lysine, which enables the conjugation of a fatty acid to its side chain. B) Fusion inhibition curves are presented for two representative peptides, as well as their fatty acid conjugates. Top: active peptides DP and DP-C12 appear. Bottom: inactive peptides DP20 and DP20-C12. C) Peptides (DP20G4, DP20G4-C12, and DP20-C16) designed to determine whether the C-terminal amino acids of DP are crucial for creation of a functional core, are illustrated in the top panel, whereas their fusion inhibition curves are presented in the bottom panel. C16 is the abbreviation used for a fatty acid with 16 carbon atoms in its chain.

Importance of the fatty acid carbon chain length
To analyze the significance of the fatty acid carbon chain length, we conjugated fatty acids with increasing lengths to the DP peptide (see Fig. 3 A). Their inhibitory capability correlated to the length of the fatty acid chain, namely, DP was the least active, DP-C8 had medium activity, DP-C12 had strong activity, and DP-C16 was the most active, with almost identical inhibitory capability as the wt DP178 peptide (see Fig. 3B and Table 1 ).


Figure 3
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  		Figure 3. Importance of fatty acid chain length. A) Illustration of the conjugated peptides prepared. C8, C12, and C16, fatty acids with 8, 12, and 16 carbon atoms in their chains, respectively. B) Fusion inhibition curves for the different peptides. DP, DP-C8, DP-C12, DP-C16, and DP178 are represented by closed squares, diamonds, circles, triangles, and open squares, respectively. Symbols represent the raw data, while the dotted lines represent the fitted curves.


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  		Table 1. Inhibitory activity and hydrophobicity values for the DP peptides

Concentration of the peptides on the membrane of the cells
We speculated that the fatty acid allows anchoring of the peptides to the membrane surface and that the extent of anchoring correlates to the length of the fatty acid carbon chain. To test this possibility, we used a previously described quantitative fluorescence cytometric assay (28) . It enables the quantitative analysis of fusion and its inhibition by coincubating target and effector cells fluorescently labeled with two distinct lypophilic probes in the presence or absence of inhibitory peptides. Fusion is followed by monitoring doubly labeled cells, while inhibition is followed by monitoring the decrease in the doubly labeled cell population. We further developed the assay into a new triple-staining flow cytometry assay. This was achieved by monitoring fusion and its inhibition in the presence of inhibitory peptides labeled with a third fluorescent probe. Results were monitored by flow cytometry and further analyzed. The assay allows us to determine the IC50 of the peptide, as well as the assignment of labeled peptides to cells. The results indicate that the concentration of the peptides on the cells increases in relation to the length of the conjugated fatty acid (see Fig. 4 ).


Figure 4
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  		Figure 4. Concentration of the peptides on cells. Assigning NBD-labeled peptides to cells by employing a new triple staining flow cytometry assay. DP, DP-C8, DP-C12, DP-C16, and DP178 are represented by closed squares, diamonds, circles, triangles, and open squares, respectively. DP and DP-C8 peptides overlay each other.

Importance of the orientation of the peptide toward the endogenous NHR region
Another set of DP peptides with fatty acid conjugated to their N terminus was prepared to examine the importance of the orientation of the peptide toward the endogenous NHR region (see Fig. 5 A). The different activities of the peptides are presented in Fig. 5B-D . The fusion inhibition capability of the N-terminally conjugated DP peptides correlated to the fatty acids carbon chain length, namely, C8-DP was the least active, C12-DP had intermediate activity, and C16-DP was the most active (see Table 1 ). When comparing them to the C-terminally conjugated DP peptides, we observed a reduction of activity; C8-DP has approximately the same activity as DP-C8, C12-DP lost ~10-fold of DP-C12 activity, and C16-DP lost ~30-fold of DP-C16 activity (see Table 1 ). Furthermore, we prepared an additional peptide with an eight-carbon chain fatty acid conjugated both to the C and N terminus to further examine the importance of hydrophobicity vs. orientation by comparing its activity to DP peptides with a 16-carbon chain fatty acid conjugated to the N/C terminal. DP-C16, C16-DP, and C8-DP-C8 exhibited different inhibition activities (see Fig. 5 and Table 1 ). To conclude, the length of the fatty acid carbon chain is critical, and it is correlated to the inhibitory activity; furthermore, the orientation of the peptides is important for their activity pattern.


Figure 5
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  		Figure 5. Importance of the orientation of the peptide. A) Illustration of the DP peptide, as well as its N-terminal fatty acid conjugates. B) Fusion inhibition curves for DP peptides conjugated to a fatty acid with 8 carbon atoms in its chain. DP-C8, C8-DP, and C8-DP-C8 are presented by closed diamonds, open diamonds, and crosses respectively. C) Fusion inhibition curves for DP peptides conjugated to a fatty acid with 12 carbon atoms in its chain. DP-C12 and C12-DP are represented by closed and open circles, respectively. D) Fusion inhibition curves for DP peptides conjugated to a fatty acid with 16 carbon atoms in its chain. DP-C16 and C16-DP are represented by closed and open triangles, respectively. Symbols represent the raw data, while the dotted lines represent the fitted curves for all of the panels in this figure.

Comparison between the hydrophobicities of the peptides
RP-HPLC was employed in order to examine whether the different fatty acids confer different hydrophobicities according to the fatty acid chain length of the conjugated peptides. The retention times of the peptides are presented in Table 1 . When the fatty acid is conjugated to the C terminal, there is a correlation between the fatty acid carbon chain length and the conjugated hydrophobicity of the peptides; the retention times for DP, DP-C8, DP-C12, and DP-C16 are 19.2, 22.3, 25.7, and 29.9 min. A similar correlation was observed for the N-terminal conjugates (see Table 1 ). Note that the N-terminal conjugated peptides are more hydrophobic than the C-terminal conjugated peptides and that DP178 wt is as hydrophobic as C8-DP-C8. In summary, the hydrophobicity correlates to the fatty acid length in either the C or N terminally conjugated peptides.

Drug potential characteristics of the lipopeptides
A series of experiments were performed to test the potential of DP-C12 and DP-C16 as anti-HIV-1 drugs in comparison to DP178. In a cytotoxicity assay DP-C16 exhibited the same cytotoxicity for Jurkat (human) T-cells as DP178, whereas DP-C12 had reduced cytotoxicity (LC50 of DP178, DP-C12, and DP-C16 was ~12.5, >50, and ~12.5 µM, respectively). None of them were hemolytic to red blood cells up to 50 µM concentration. The fusion inhibition assay was also performed with medium containing serum, and the inhibitory capability was not affected. These experiments revealed that DP-C12 and DP-C16 seem to have the same anti-HIV-1 fusion potential as DP178.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Since the sequence of DP178 originates from the gp41 sequence (see Fig. 1 ), understanding its inhibitory mechanism will enable us to deduce the function of the parallel endogenous region. In this study, we exploited variants of DP178 that underwent truncations and conjugations to fatty acids with varying lengths, in order to establish the C-terminal boundary of the endogenous core structure, and to better understand the contribution of the C-terminal of DP178 to its inhibitory activity. Previously, it was demonstrated that the hydrophobic C-terminal of DP178 is crucial for its activity; the peptide lost inhibitory activity following truncation or mutation of only four C-terminally hydrophobic amino acids (16) . Strikingly, we demonstrated here that the whole C-terminal region can be replaced by an unrelated hydrophobic moiety. We identified a truncated variant, DP, which following its conjugation to a fatty acid, regained the wt inhibitory activity. Since further attempts to truncate the C or N-terminal regions resulted in abolishing activity, the DP sequence seems to be the minimal sequence able to create a functional core with the endogenous gp41 protein in situ. This is the first indication of the exact C-terminal boundary of the core structure in an ongoing fusion process.

The variants exhibited a direct correlation between the length of the conjugated fatty acid and their inhibitory activity. Additionally, the length of their conjugated fatty acid correlated to their concentration on the cells, as revealed by a newly developed triple fluorescent assay. Taking these results into account, apparently the fatty acid enables the conjugated peptide to bind the membrane surface of the cells, which increases the concentration of the conjugated peptide near the fusion sites. N-terminal conjugates have a reversed orientation in regard to the endogenous gp41 and therefore were not expected to inhibit fusion. However, N-terminal conjugates exhibited inhibitory activity, although, to a significantly lesser degree than the C-terminal conjugates. The extent of activity loss correlated to the fatty acid length. A possible explanation for the results with the N-terminally conjugated peptides is that there is equilibrium between soluble and membrane-anchored conjugated peptides, which depends on the length of the conjugated fatty acid. This equilibrium increases the concentration of the peptides on the cells whether the fatty acid is conjugated to the C- or N terminus of the peptide. As the length of the fatty acid increases, the equilibrium shifts toward fatty acid anchored to the membrane of the cell. However, it seems that permanent anchoring was not reached since even C16-DP was still active. Apparently, the variants can inhibit either in their soluble form (demonstrated by the N-terminal conjugates) or by a combination of their soluble and anchored forms (demonstrated by the enhanced activity of the C-terminally conjugated variants in comparison to the N-terminally conjugated variants). Interestingly, in a recently published paper, it was demonstrated that the inhibitory potency of the 5-helix inhibitor depends on its association rate rather than its binding affinity, and it was suggested that antiviral potency might be enhanced by increasing the local inhibitor concentration near the viral membrane (29) .

Previously, a fatty acid (C8) was conjugated to an inactive SIV-DP178 variant mutated in its four C-terminal amino acids (30) . After C-terminal conjugation, the activity of the mutant recovered, whereas after N-terminal conjugation, it did not. In contrast, when the C-terminal of DP178 (results of the present study) is completely omitted, the N-terminal conjugation (C8) regains partial activity, since the activity depends solely on the concentration of the peptide on the cells. Taking both studies into account, it appears that the C-terminal of DP178, specifically the three aromatic residues region, is involved in binding either the membrane surface or the endogenous gp41. Recently, two studies using cryoelectron microscopy tomography revealed the structures of ENV spikes on the membranes of SIV and HIV-1 virions; the first (31) supports binding of the PTM region to the membrane of the virus, whereas the second (32) supports intersubunit interactions of this region. In either case, we propose that the C-terminal part of DP178 provides an anchor that enables the N-terminal of the peptide to create a stable core with the NHR region. When mutated, C-terminal fatty acid conjugation can compensate for the C-terminal contribution by anchoring the mutated peptide to the membrane. N-terminal conjugation (in the context of the full DP178) cannot compensate since even if the peptide can flip to the right orientation, the mutated amino acids probably interfere with the tight connection needed to establish the interaction with the NHR region. A remote yet an interesting approach to increase the affinity of the N-terminal region of DP178 to the endogenous NHR uses mutations of the endogenous sequence to include unnatural amino acids. Thus, a rigid {alpha}-helical secondary structure of this region was obtained (33) .

The cost of manufacturing peptides rises exponentially with their increasing length. Thus, for therapeutic applications, the aim is to attain the shortest peptide possible. We tested the potential of DP-C12 and DP-C16 as anti-HIV-1 fusion drugs in comparison to DP178 by using several assays: their cytotoxicity to human T cells, their hemolytic effect on red blood cells, and their ability to inhibit fusion in the presence of serum or while dissolved in PBS. The results were similar to those of DP178, suggesting that both peptides are potential HIV-1 fusion inhibitors.

In summary, we established that the C-terminal of DP178 functions mainly as a hydrophobic anchor enabling core prevention by the N-terminal of DP178. A better understanding of the mechanism will enable the design of potentially improved drugs. Because the HIV-1 fusion mechanism is shared by many other enveloped viruses, as well as by intracellular vesicle fusion, these results might contribute to their research and therapeutic efforts. Importantly, we demonstrated for the first time the C-terminal boundary of the core structure in an ongoing dynamic fusion process.


   ACKNOWLEDGMENTS
 
We thank Shai Kaplan for his excellent counsel, and Batya Zarmi for her valuable help with peptide purification. This study was supported by the Israel Science Foundation. Y.S. has the Harold S. and Harriet B. Brady Professorial Chair in Cancer Research.

Received for publication March 20, 2007. Accepted for publication May 17, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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