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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: HIV-1 inhibition • lipopeptides • coiled coil • T-20 • virus entry • envelope protein • heptad repeat • peptide-membrane interaction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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).
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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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
).
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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
).
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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.
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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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), 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 
-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.
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ACKNOWLEDGMENTS |
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Received for publication March 20, 2007. Accepted for publication May 17, 2007.
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