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Journal of Virology, May 2008, p. 4965-4973, Vol. 82, No. 10
0022-538X/08/$08.00+0     doi:10.1128/JVI.02458-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Nonhelical Leash and {alpha}-Helical Structures Determine the Potency of a Peptide Antagonist of Human T-Cell Leukemia Virus Entry{triangledown}

Antonis Mirsaliotis, Daniel Lamb, and David W. Brighty*

Biomedical Research Centre, Ninewells Hospital and Medical School, The University, Dundee DD1 9SY, Scotland, United Kingdom

Received 15 November 2007/ Accepted 20 February 2008


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ABSTRACT
 
Viral fusion proteins mediate the entry of enveloped viral particles into cells by inducing fusion of the viral and target cell membranes. Activated fusion proteins undergo a cascade of conformational transitions and ultimately resolve into a compact trimer of hairpins or six-helix bundle structure, which pulls the interacting membranes together to promote lipid mixing. Significantly, synthetic peptides based on a C-terminal region of the trimer of hairpins are potent inhibitors of membrane fusion and viral entry, and such peptides are typically extensively {alpha}-helical. In contrast, an atypical peptide inhibitor of human T-cell leukemia virus (HTLV) includes {alpha}-helical and nonhelical leash segments. We demonstrate that both the C helix and C-terminal leash are critical to the inhibitory activities of these peptides. Amino acid side chains in the leash and C helix extend into deep hydrophobic pockets at the membrane-proximal end of the HTLV type 1 (HTLV-1) coiled coil, and these contacts are necessary for potent antagonism of membrane fusion. In addition, a single amino acid substitution within the inhibitory peptide improves peptide interaction with the core coiled coil and yields a peptide with enhanced potency. We suggest that the deep pockets on the coiled coil are ideal targets for small-molecule inhibitors of HTLV-1 entry into cells. Moreover, the extended nature of the HTLV-1-inhibitory peptide suggests that such peptides may be intrinsically amenable to modifications designed to improve inhibitory activity. Finally, we propose that leash-like mimetic peptides may be of value as entry inhibitors for other clinically important viral infections.


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INTRODUCTION
 
The entry of enveloped viruses into cells requires fusion of the viral and cellular membranes. For many viruses, membrane fusion is catalyzed by viral class I integral membrane glycoproteins in response to an activation trigger, such as receptor engagement or low pH (9). Experimentally supported models suggest a common mechanism of action for viral fusion proteins in which membrane fusion is achieved by refolding of the homotrimeric fusion protein from a metastable prefusogenic structure to a stable six-helix bundle (9, 16, 18-20, 25, 35). Following fusion protein activation, an N-terminal hydrophobic peptide is thrust into the target cell membrane, resulting in the formation of a prehairpin intermediate in which the C terminus is anchored in the viral membrane and the N terminus is embedded in the target cell membrane. The rod-like prehairpin intermediate, which is stabilized by assembly of a trimeric coiled coil, then resolves into a six-helix bundle or trimer-of-hairpins structure that brings the viral and cellular membranes into close proximity, destabilizes the lipid bilayers, and ultimately promotes membrane fusion (9, 21, 22).

The retroviral envelope glycoprotein complex consists of a trimer of surface glycoproteins (SU) held on a trimer of a class I fusion protein referred to as the transmembrane glycoprotein (TM). The structure of the human T-cell leukemia virus type 1 (HTLV-1) TM six-helix bundle has been particularly well resolved (16). For each monomer of TM, an amino-terminal hydrophobic fusion peptide is connected via a glycine-rich linker to an {alpha}-helical motif that interacts with the equivalent helix of adjacent TM monomers to form a triple-stranded coiled coil. At the base of the coiled coil, the peptide backbone folds back in a 180° loop referred to as the chain reversal region. An extended peptide sequence that includes a short C-terminal {alpha}-helix (C helix) continues in an antiparallel manner along the grooves formed on the surface of the core coiled coil (Fig. 1). The six-helix bundle of HTLV-1 TM is remarkably reminiscent of the prototypic structures identified in the fusion proteins of human immunodeficiency virus (HIV) (4) and influenza virus (25).


Figure 1
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  		FIG. 1. HTLV-1 TM and the recombinant TM fusion protein. (A) Structure of the trimer-of-hairpins motif of HTLV-1 TM. The central triple-stranded coiled coil is shown in space-filling form with the extended antiparallel peptide and C-helical region shown in color. (B) Representation of the recombinant trimeric MBP-TM fusion protein used in this study as a model of the exposed coiled coil. The structure is displayed with each monomer of TM in ribbon format, with the three MBP monomers shown as a white space-filling model. (C) Detail of the HTLV-1 TM leash and {alpha}-helical region (shown in ribbon form with side-chains represented as sticks). The structure shows the residues that are included in the N terminus of the peptide inhibitor (Pcr-400) of HTLV envelope-catalyzed membrane fusion. The amino acid coordinates denote residue positions in the envelope protein precursor; this numbering system is retained in describing the mimetic peptides for ease of comparison. Residues replaced in this study are shown in red. All structures were modeled from Protein Data Bank ID 1MG1 using MacPymol software (6a; http://www.pymol.org).

While the global architecture is conserved, there is nevertheless significant variation among the six-helix bundles of divergent virus groups. The C helix of HIV TM is extensive and runs along the length of the core coiled coil (4). By contrast, in influenza virus, the C helix is short, is situated at the membrane-distal end of the coiled coil, and is followed by an extended nonhelical peptide chain that packs into the grooves of the core coiled coil (25). An elegant study has led to the proposal that, for viruses such as influenza virus, membrane fusion is achieved by a leash-in-a-groove mechanism, whereby the extended nonhelical peptide chain acts as a leash with the amino acid side chains securing the C-terminal peptide sequences to the core coiled coil, thereby drawing the target membranes together (25).

Like the fusion protein of influenza virus, the C-terminal domain of the HTLV-1 trimer of hairpins displays leash-like properties, with an extended hydrophobic peptide chain on either side of a short {alpha}-helix (16). However, unlike influenza virus but in keeping with the TM of HIV, the C helix of HTLV-1 TM is located toward the membrane-proximal end of the core coiled coil. Thus, the HTLV-1 fusion protein exhibits features that are reminiscent of both HIV and influenza virus.

Importantly, for HIV (8, 14, 29, 36) and for HTLV-1 (26, 28), synthetic peptides that mimic the C-terminal {alpha}-helical domain are potent inhibitors of virus entry into cells. These peptides target the coiled coil of the prehairpin intermediate and likely act in a trans-dominant-negative manner to block six-helix bundle formation and therefore prevent envelope-mediated membrane fusion and virus entry (9, 23, 26, 30). Consequently, C helix-based fusion inhibitors (10, 14, 26, 36) are attractive and, in some cases, validated candidates for antiviral therapy (15).

Given the leash-like properties of the C-terminal segment of the HTLV-1 six-helix bundle and the lack of effective antiviral agents targeted to HTLV-1, we sought to explore the contribution of the leash and {alpha}-helical regions to the inhibitory properties of the HTLV-1 C helix mimetics. We demonstrate that amino acids in both the nonhelical leash and the {alpha}-helical region directly contribute to the potency of the inhibitory peptides and stabilize the interaction of the peptide with the coiled coil. Moreover, substitution of a single amino acid within the {alpha}-helical region of the mimetic peptide dramatically improves the potency of the inhibitory peptide. Our data are consistent with the view that specific amino acid side chains of the inhibitory peptide make contact with deep hydrophobic pockets in the core coiled coil and that these contacts are necessary but not sufficient for optimal inhibition of envelope-mediated membrane fusion. Finally, we suggest that specific sites of deep contact on the coiled coil define attractive targets for small-molecule antiviral drugs to combat HTLV-1 infections.


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MATERIALS AND METHODS
 
Cells and plasmids. HeLa, 293T, and HOS cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum. The plasmids pHTE-1 (7), pMAL-gp21hairpin (maltose-binding protein [MBP]-Hairpin), pMAL-gp21fishhook (MBP-Fishhook), pMAL-STOP (MBP) (26), and pNL4-3.Luc.R–.E– (5, 12) have been described previously.

Peptide synthesis. Peptides (Table 1) were synthesized using standard solid-phase 9-fluorenylmethoxy carbonyl chemistry and, unless stated otherwise, had acetylated N termini and amidated C termini. The peptides were purified by reverse-phase high-pressure liquid chromatography and verified for purity by matrix-assisted laser desorption ionization-time of flight mass spectrometry. All peptides were dissolved in dimethyl sulfoxide, the concentrations of peptide stock solutions were confirmed by absorbance at 280 nm in 6 M guanidine hydrochloride, and the peptides were used at the final concentrations indicated.


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  		TABLE 1. Peptides used in this study

Expression and purification of MBP-TM proteins. Expression and purification of the MBP-TM fusion proteins and assessment of the oligomerization status of the MBP-TM chimeras by Superdex 200 gel filtration chromatography were carried out as described previously (26). The concentrations of the fusion proteins were estimated by Bradford assay, and the recombinant proteins were stored at –80°C in phosphate-buffered saline (PBS) supplemented with 20% glycerol.

Peptide binding and competition binding assays. Peptide binding to recombinant coiled coil was examined using previously described direct or competition binding assays (23). Microtiter 96-well plates (Nunc Maxi-Sorp) were coated overnight at 4°C with MBP-Fishhook (recombinant coiled coil; 10 µg/ml) or control protein (MBP or MBP-Hairpin) in PBS, pH 7.2. The plates were washed twice with wash buffer (PBS, 0.2% Tween 20) and blocked with blocking buffer (5% nonfat powdered milk Marvel in PBS, 0.2% Tween 20) for 1 h at room temperature. The peptide Bio-Pcr-400 (19) in wash buffer was incubated with the immobilized coiled coil in the presence or absence of dithiothreitol (DTT) (5 mM), or with peptide competitors at the concentrations specified, at room temperature for 1 h. Subsequently, the plates were washed (five times) to remove unbound peptide and incubated for 1 h with 100 µl streptavidin-horseradish peroxidase (HRP) (Sigma; 1:10,000 dilution) at room temperature. The plates were washed five times to remove unbound streptavidin-HRP and two times with PBS to remove residual detergent. Finally, bound streptavidin-HRP, and therefore bound Bio-Pcr-400, was detected using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate, and the absorbance at 415 nm was read (Bio-Rad plate reader). In control assays, Bio-Pcr-400 did not bind to MBP or MBP-Hairpin (reference 23 and data not shown).

Syncytium interference assays. Syncytium interference assays were performed as described previously (13, 26). HeLa cells (3.0 x 105) transfected with the envelope expression vector pHTE-1 were added to 7.0 x 105 untransfected HeLa target cells. The effector and target cells were cocultured in the absence or presence of the P-400-related peptides at the concentrations specified. The cells were incubated for 12 to 15 h at 37°C, washed twice with PBS, and fixed in PBS plus 3% paraformaldehyde. The assays were performed in triplicate, and the number of syncytia from five low-power fields per replicate was scored by light microscopy. For some assays, syncytia were stained using Giemsa. Nonlinear regression analysis of the dose-response curves and calculation of the concentration of peptide required to give half-maximal inhibition (IC50) of syncytium formation were performed using the GraphPad Prism software package.

Viral-pseudotyping assay. 293T Cells at 70% confluence were cotransfected with pHTE and the pNL4-3.Luc.R–.E– vector (5, 12) (generously provided by Nathaniel Landau through the AIDS Research and Reference Reagent Program; 8 µg of each vector) using the FuGENE 6 transfection reagent (Roche). After 24 h, the supernatant was replaced with fresh medium containing 10 mM sodium butyrate. After a further 20 h, the supernatant was removed, and the cells were washed once with medium and incubated for 48 h in fresh medium without sodium butyrate. The viral supernatant was centrifuged at 2,500 rpm (Heraeus Instruments; Labofuge 400) for 5 min and filtered through a 0.22-µM filter. Virus-containing supernatant (400 µl) was added to 5 x 105 HOS cells in the presence or absence of peptide. After 20 h, the medium was replaced, and the HOS cells were incubated for a further 24 h to maximize retroviral-gene expression. The HOS cells were then washed once with PBS and lysed using lysis buffer (Promega). Luciferase assays were performed on the lysates using a Promega kit and a TD-20/20 luminometer (Turner Designs). Luciferase activity was normalized according to protein concentration in the lysates as determined by Bradford assay.


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RESULTS
 
N- or C-terminal deletions severely impair the activity of an inhibitory peptide. We have demonstrated that a peptide inhibitor of HTLV-1-induced membrane fusion specifically interacts with the core coiled-coil motif of HTLV-1 TM (23, 27). The synthetic peptide Pcr-400 emulates amino acid residues 400 to 429 of envelope, which includes the C-terminal leash and {alpha}-helical segments of the HTLV-1 TM ectodomain (Fig. 1). Circular-dichroism spectroscopy indicates that the peptide has the ability to form an {alpha}-helical structure in nonaqueous solution (27). The peptide binds to a trimeric recombinant coiled coil (Fig. 1B) but fails to interact with the six-helix bundle of TM (23, 27). Moreover, radical amino acid substitutions within the peptide completely abolish binding to the recombinant coiled coil and result in loss of the inhibitory properties of the peptide (23, 27). Thus, the accumulated data indicate that the inhibitory peptide faithfully mimics the extended leash and {alpha}-helical structures of the C-terminal domain of the HTLV-1 trimer of hairpins.

Since C helix mimetics are potent inhibitors of HTLV-1 entry, we wished to examine the molecular features that contribute to the specificity and inhibitory activity of the peptide. The TM crystal structure (Fig. 1) reveals that the side chains of multiple amino acids in the leash and {alpha}-helical segment contact the core coiled coil, but it was unclear which of these regions is important for the inhibitory activity of the peptide. Two derivative peptides were therefore generated that lack 10 amino acid residues at the C terminus (Pcr-{Delta}C) or 12 residues at the amino terminus (Pcr-{Delta}N); these peptides retain a central 7-amino-acid overlap. Importantly, Pcr-{Delta}C spans the N-terminal leash residues and the entire {alpha}-helical segment but lacks the C-terminal leash, whereas Pcr-{Delta}N encompasses a fragment of the {alpha}-helical region and includes the entire C-terminal leash (Table 1 and Fig. 1C). The truncated peptides were compared with the parental peptide, Pcr-400, for the ability to inhibit HTLV-1 envelope-induced syncytium formation. HeLa cells were transfected with the HTLV-1 Env expression vector pHTE-1, and the resulting transfectants were used as effector cells in cell fusion assays. Coculture of untransfected HeLa cells with Env-expressing HeLa cells resulted in extensive syncytium formation. Syncytium formation was strongly inhibited in the presence of Pcr-400, whereas control peptides failed to inhibit membrane fusion (Fig. 2). Significantly, the truncated peptides, Pcr-{Delta}C and Pcr-{Delta}N, also failed to inhibit syncytium formation at the concentrations tested (Fig. 2A and B), indicating that large N- or C-terminal deletions severely impair the inhibitory properties of the peptide. Therefore, peptides that include the entire N-terminal leash and {alpha}-helix but lack the C-terminal nonhelical peptide region are unable to inhibit Env-mediated membrane fusion. Equally, the C-terminal leash residues alone are insufficient for inhibition of fusion.


Figure 2
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  		FIG. 2. Deletions disrupt the activities of peptide inhibitors of membrane fusion. (A) HeLa target cells were cocultured with mock-transfected (top left) or envelope-expressing HeLa cells in the presence of control peptide (HTLV-derived P-80 or HIV-derived C34) or in the presence or absence of Pcr-400 or the truncated derivatives Pcr-{Delta}N and Pcr-{Delta}C. The cells were stained with Giemsa and imaged by low-power light microscopy. (B) The numbers of syncytia per low-power field were scored (means ± standard deviations of triplicate assays).

N-terminal cysteine residues are not required for inhibitory activity. The inhibitory peptide Pcr-400 includes two adjacent cysteine residues at the N terminus. The crystal structure of the trimer of hairpins reveals that the side chain of Cys401, equivalent to the second cysteine of Pcr-400, lies along a shallow groove on the surface of the coiled coil (Fig. 3A). Significantly, a prototypic peptide inhibitor based on the C-helical region of the HTLV-1 strain ATK (Patk-400, previously referred to as P-400) (23, 26) has an arginine at this position. Replacement of this arginine with cysteine was one of three substitutions that produced the more potent peptide inhibitor Pcr-400. The amino acid sequence of Pcr-400 is identical to that of HTLV-1 strain CR and closely follows the consensus sequence for all HTLV-1 strains. Given the structural information (16), the improved potency of Pcr-400 relative to Patk-400 (26), and the conservation of Cys401 among HTLV-1 isolates, it was feasible that the second cysteine residue of Pcr-400 is required for optimum binding to the coiled coil. Moreover, in TM, Cys400 and Cys401 are involved in disulfide bond formation; Cys400 forms an intrachain disulfide linkage with Cys393 of TM (16), while Cys401 is required for an intersubunit disulfide link with SU (32). Evidence indicates that disulfide isomerization is a critical event in regulating the fusogenic activity of retroviral envelope (31-33), and Cys400 and Cys401 of HTLV-1 TM play essential roles in these isomerization reactions. The presence of adjacent cysteine residues in Pcr-400 raises the possibility that the formation of disulfides between the inhibitory peptide and TM or SU contributes to the inhibition of envelope-mediated membrane fusion.


Figure 3
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  		FIG. 3. Disulfide bonding and cysteine residues are not required for the inhibitory activity of the peptide. (A) Cys401 of Env, equivalent to the second cysteine of the mimetic peptide (blue sticks), lies in a groove on the surface of the coiled coil (green; space-filling model). The arrowhead indicates the position of Leu403. (B) Comparison of the ability of an N-terminally biotinylated Pcr-400 peptide to bind to immobilized recombinant HTLV-1 coiled coil in the presence (+DTT) or absence (–DTT) of 5 mM DTT. (C) The inhibitory properties of the parental peptide (Pcr-400), the control peptide (P-80), and the alanine-substituted peptide (Pcr-CC/AA) were compared in syncytium interference assays. The error bars represent standard deviations.

However, a biologically active N-terminally biotinylated derivative of Pcr-400 bound to a recombinant trimeric coiled coil even in the presence of reducing agent (Fig. 3B), suggesting that disulfide formation between the peptide and the cysteine of recombinant TM is not required for efficient binding. Also, covalent adducts coupled directly to the peptide via the adjacent cysteine residues do not interfere with the inhibitory properties of the peptide in syncytium interference assays or with the capacity of the peptide to bind to the coiled coil in ligand-binding assays (reference 26 and our unpublished data). The importance of the adjacent cysteine residues to the activity of Pcr-400 was compared in syncytium interference assays, and in coiled-coil-binding assays, with that of a peptide (Pcr-CC/AA) in which both cysteines were replaced with alanine. Both Pcr-400 and the alanine-substituted peptide blocked envelope-mediated membrane fusion with essentially identical dose-response curves (Fig. 3C) and also bound to recombinant coiled coil in indistinguishable manners (data not shown). Taken together, the data indicate that the adjacent N-terminal cysteine side chains do not contribute in any significant way to the inhibitory activity of the peptide and that disulfide bond formation is not required for optimal inhibition of membrane fusion.

Extension of the peptide does not improve the inhibitory activity. The extended nonhelical leash and {alpha}-helical region of TM is rich in leucine and incorporates five of the eight leucine residues present in the post-chain-reversal ectodomain of gp21, but only three of these leucine residues are resolved in the crystal stucture (Fig. 1) (16). To test if extending the synthetic peptide to include the additional leucine residues would improve the binding of the peptide to the core coiled coil and thereby improve the inhibitory potency, the peptide was extended at the C-terminal end to incorporate six additional amino acids (NWDLGL), which includes two of the three remaining leucine residues of the TM ectodomain. Syncytium interference assays revealed that the extended peptide, Pcr-435, was just as active as the parental peptide, Pcr-400, and extension of the peptide did not improve either the potency or the efficacy of the inhibitor (data not shown). Therefore, all of the amino acid residues required for inhibitory activity are contained within Pcr-400, and the peptide can be extended at the C terminus without loss of function.

Potent antagonism of membrane fusion requires specific leucine residues. Our early studies (26) demonstrated that replacement of all of the leucine residues in Pcr-400 with alanine severely impaired the inhibitory activity of the peptide and disrupted peptide binding to the coiled coil (23, 27). While the leucine residues in Pcr-400 are clearly important for activity, the contribution of individual residues to peptide function has yet to be established.

Therefore, a series of derivative peptides was generated in which individual residues were replaced with alanine (Table 1). Each of the peptides was tested for the ability to inhibit membrane fusion in syncytium interference assays. Surprisingly, several of the leucine-to-alanine substitutions, specifically, Leu403Ala, Leu424Ala, and Leu429Ala, had little effect on the activities of the derived peptides, as they inhibited membrane fusion as effectively as the parental peptide (Fig. 4A). In addition, the derivative peptides Pcr-L403A, Pcr-L424A, and Pcr-L429A largely retained the ability to bind to the recombinant coiled coil in competition binding experiments (Fig. 4B). These results were particularly surprising for Pcr-L403A, as in TM, the Leu403 side chain extends down into the groove on the surface of the coiled coil and docks in a hydrophobic pocket (Fig. 4C) formed by Ala372 and Ala375 from one N helix and Ile371 and Tyr374 from the adjacent N helix, where the tyrosine and isoleucine form the C-terminal limit and base of the pocket, respectively. Due to the short side chain of the alanine substitution, the peptide derivative Pcr-L403A is most likely unable to make the relevant contact between the peptide leash region and the pocket in the core coiled coil and would be expected to leave the pocket unoccupied and more exposed to solvent. Nevertheless, in the context of the mimetic peptide, particular leucine residues in the leash region, equivalent to Leu403, Leu424, and Leu429, can be replaced, at least with alanine, without significant loss of peptide function.


Figure 4
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  		FIG. 4. Specific leucine residues in the leash and {alpha}-helical segments of the mimetic peptide are critical to inhibitory activity. (A) The parental peptide (Pcr-400) and the alanine-substituted peptides (Pcr-L403A, Pcr-L424A, and Pcr-L429A) were compared for the ability to inhibit membrane fusion in syncytium interference assays. (B) Peptide Pcr-400 and the alanine-substituted peptides were examined for the ability to bind to immobilized recombinant core coiled coil of HTLV-1 in competition with a biotinylated peptide (Bio-Pcr-400). The datum points represent the means ± standard deviations (SD) of triplicate assays. The cavities on the coiled coil of TM occupied by Leu403 of the C-terminal leash (C) and by {alpha}-helix residue Leu413 (D) and leash residue Leu419 (E) are shown (the coiled coil is represented in blue, gray, and red space-filling form with key residues labeled and shown as sticks; the TM residues mimicked by the inhibitory peptide are illustrated as green, blue, and red sticks). The arrowhead in panel E indicates V350 from the adjacent TM monomer. (F) The parental peptide (Pcr-400) and peptides carrying alanine substitutions, Pcr-L413A and Pcr-L419A, were examined for the ability to inhibit membrane fusion in syncytium interference assays. (G) Peptides Pcr-400, Pcr-L413A, and Pcr-L419A were compared for the ability to compete with Bio-Pcr-400 for core coiled-coil binding (means ± SD of triplicate assays).

Interestingly, TM residues L413 and L419 also make contact with the coiled coil (Fig. 4D and E), and in contrast to Pcr-400, the peptide derivatives with substitutions at these residues, Pcr-L413A and Pcr-L419A, were particularly poor inhibitors of membrane fusion and syncytium formation (Fig. 4F). From the dose-response curves and the calculated IC50 for each peptide (Table 2), it was estimated that there is a >20-fold loss in peptide potency for Pcr-L413A and Pcr-L419A compared to Pcr-400. Importantly, Leu413 is situated within the {alpha}-helical segment of the C helix mimetic peptide, while Leu419 is in the leash region immediately C terminal to the helix-breaking diproline motif. Thus, residues within both the leash and the {alpha}-helical segment of the peptide likely make critical contacts with the core coiled coil. In support of the data from cell-based assays, the peptides also displayed dramatically reduced abilities to bind to the recombinant coiled coil in competition binding assays (Fig. 4G). Significantly, both Leu413 and Leu419 reach down into deep hydrophobic pockets in the coiled coil (Fig. 4D and E), and these contacts appear to be required for optimum peptide binding. Although both of these leucine residues are required for maximal inhibitory activity, substitution of any individual leucine within the mimetic peptide did not completely abrogate the inhibitory activity of the peptide.


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  		TABLE 2. Calculated amounts of peptides required to give 50% inhibition of syncytium formation

Analysis of isoleucine residues in the leash and {alpha}-helical regions of Pcr-400. The contributions of two isoleucine residues to the inhibitory activity of the peptide and the ability of the peptide to bind the coiled coil were also examined. In TM, these isoleucine residues are situated at positions 405 and 412 and are therefore in the N-terminal leash and short {alpha}-helix, respectively (Fig. 1A and C). We reasoned that Ile405 would be an important contact of the leash with the core coiled coil, as the side chain of this residue reaches down into a deep pocket at the membrane-distal end of the coiled coil (Fig. 5A). The pocket is lined on one side by Asn367 and Ile371 of one helix and on the other side by Leu368 and Leu369 of the adjacent helix.


Figure 5
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  		FIG. 5. Two isoleucine residues modulate inhibitory peptide activity. (A) Ile405 of the peptide (yellow, red, and blue sticks) makes contact with a deep hydrophobic pocket on the coiled coil (blue and red space-filling model; key residues are labeled and shown as sticks; the arrowhead indicates the position of N367 of the coiled coil). (B to D) Pcr-400 and the peptides Pcr-I405A (B) and Pcr-I412A (C) were examined for the ability to inhibit membrane fusion in syncytium assays and for the ability to compete with Bio-Pcr-400 (D) for binding to immobilized core coiled coil (means ± standard deviations of triplicate assays).

A peptide was synthesized replacing the isoleucine 405 residue with alanine (Pcr-I405A). The peptide was then examined for the ability to inhibit envelope-catalyzed membrane fusion. As anticipated, the peptide carrying the Ile405Ala substitution had considerably reduced inhibitory activity in syncytium interference assays (Fig. 5B). However, compared to the 20-fold loss in activity for L413A and L419A, the loss of inhibitory activity for Pcr-I405A was more modest, representing a 10-fold reduction in activity.

Most interestingly, compared to the partially defective mimetic Pcr-I405A, the alanine-substituted peptide Pcr-I412A provided a dramatically different result. In syncytium interference assays, Pcr-I412A displayed a significantly enhanced ability to inhibit envelope-mediated membrane fusion (Fig. 5C). The calculated IC50s indicated that Pcr-I412A was 4-fold more active than the parental peptide Pcr-400, which was modeled on the HTLV-1 strain CR, and almost 50-fold more active than the original inhibitory peptide, Patk-400 (Table 2), which is based on the envelope sequence of HTLV-1 strain ATK (Table 1). Moreover, Pcr-I412A demonstrated significantly enhanced ability to compete for coiled-coil binding compared to either the parental peptide, Pcr-400, or the defective peptide, Pcr-I405A (Fig. 5D). Thus, substitution of a single amino acid within the {alpha}-helical region of the mimetic peptide results in a pronounced enhancement of inhibitory activity in membrane fusion assays and improved coiled-coil-binding properties.

Inhibition of viral entry. Syncytium interference assays provide useful information on some aspects of membrane fusion but do not reflect all of the events associated with envelope function. For example, syncytium formation does not necessarily equate to retroviral entry (2, 6, 34). Therefore, the inhibitory properties of the peptides were examined using a robust and widely used viral-infectivity assay that is based on luciferase-transducing HIV-1 particles pseudotyped with HTLV-1 envelope. HOS cells were incubated with HTLV-1 Env-pseudotyped viral particles in the presence of inactive control peptides or with the Pcr-400-derived inhibitory peptides. As anticipated, the control peptides failed to block viral entry (Fig. 6) whereas Pcr-400 potently blocked viral entry in a dose-dependent manner, as shown by the dramatic decrease in transduced luciferase activity. In agreement with the syncytium interference assays, the peptides Pcr-L413A and Pcr-L419A demonstrated significantly reduced inhibitory properties compared to the parental peptide, Pcr-400 (Fig. 6), indicating that the contacts made by these peptide residues with the coiled coil are required for maximal inhibition of viral entry. Importantly, peptide Pcr-I412A, which produced the most robust inhibition in the syncytium assays, was also the most potent inhibitor of viral entry (Fig. 6). It is apparent that, for these particular peptide inhibitors, data derived from syncytium interference assays are highly predictive of the properties of the inhibitors in blocking viral entry, so that substitutions that abolish or, alternatively, improve the capacity of the peptide to block membrane fusion have equivalent effects on viral entry. Our data indicate that the C helix-based peptides robustly block both envelope-mediated membrane fusion and infection of cells by viral particles.


Figure 6
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  		FIG. 6. Replacement of specific amino acids in C helix-based peptide inhibitors has profound effects on the inhibition of viral entry. HIV particles pseudotyped with envelope of HTLV-1 were incubated with target cells in the presence or absence of the peptide inhibitors Pcr-400, Pcr-L413A, Pcr-L419A, and Pcr-I412A at the concentrations indicated. After infection (40 to 48 h), the cells were examined for transduced luciferase activity. The datum points represent the means (± standard deviations) of triplicate assays from three independent cultures. AU, arbitrary units.


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DISCUSSION
 
Here, we demonstrated that both the C helix and the extended nonhelical leash regions contribute substantially to the potency of a peptide inhibitor of HTLV-1 entry. Our data indicate that all of the structural motifs required for optimal binding to the coiled coil are contained within the peptide inhibitor. However, large deletions of the leash and/or helical motifs severely impair peptide function and suggest that multiple contacts along the length of the peptide are required for optimal interaction with the coiled coil of fusion-active envelope. In the native trimer of hairpins, nonpolar amino acid residues of the leash and C helix reach into deep pockets or cavities in the surface of the core coiled coil (16) and are likely required for stable assembly of the trimer of hairpins. Our data indicate that equivalent contacts are made between the mimetic peptide and the coiled coil of fusion-active envelope and that such interactions are critical to the inhibitory activity of the peptide.

In particular, the leash residue, equivalent to Ile405, packs into a pocket at the C-terminal end of the coiled coil and appears to contribute substantially to the properties of the mimetic peptide, as replacement of this isoleucine with alanine markedly reduced peptide potency. By contrast, several leucine residues within the leash could be replaced without significant loss of peptide function, despite the fact that at least one of these residues, equivalent to Env Leu403, is also predicted to reach into the nonpolar pocket at the C-terminal end of the coiled coil. Significantly, the contribution of Leu403 to binding in the hydrophobic pocket is relatively small compared to the extensive contacts of Ile405 in this region.

Notably, two leucine residues, one located in the short {alpha}-helical segment and the other located in the C-terminal leash, are particularly important for binding of the peptide to the coiled coil and for inhibitory activity, and these residues are positioned toward the C-terminal end of the peptide. In TM, Leu413 and Leu419 reach into a deep and extended but largely hydrophobic cavity at the membrane-proximal end (N terminus) of the coiled coil. The data are consistent with the view that, for the peptide, the side chain of the leash residue Leu419 also docks into this hydrophobic cavity. On one side, the cavity is lined by Asp353, Val350, and Leu346 of one helix of the coiled coil, while residues Ile354, Val350, and Leu347 of the neighboring helix line the other side. In a similar manner, the Leu413 residue of the C helix also projects into an extended hydrophobic groove flanked by Ala360, Gln356, and Leu357 from one of the N helices and Ile361 and Thr358 of the adjacent helix, where Leu357 and Ile361 combine to form the base of the groove and Gln356 and Thr358 form the sides. It is likely that the contacts made by these leucine residues within the membrane-proximal cavity account for the importance of these residues in peptide binding and function. Importantly, the extensive cavity at the membrane-proximal end of the coiled coil appears to be an ideal target for low-molecular-weight inhibitors of envelope-mediated membrane fusion and HTLV-1 entry into cells. Small molecules that bind within and occupy the cavity would be expected to prevent docking of the relevant leucine side chains of TM and therefore to block formation of the trimer of hairpins and subsequent membrane fusion.

Deep hydrophobic contacts between the C-terminal sequences and the core coiled coil are a feature of the trimers of hairpins of other viral fusion proteins, including the predominantly helical structures of HIV TM (3, 4, 18) and paramyxovirus F glycoprotein (17) and the extended leash of viruses such as influenza virus (25) and HTLV-1 (16). In contrast to HTLV-1, amino acid residues in the HIV C helix mimetic that are required for optimal inhibition of HIV membrane fusion make contact within a deep cavity at the membrane-distal end of the coiled coil (3), while in keeping with HTLV-1, contacts between the leash of influenza virus and the membrane-proximal end of the coiled coil are critical to membrane fusion (25). Thus, while there is conservation of the global fold of the trimer-of-hairpins motif, there are notable differences in the ways that these structures are stabilized. Identifying the key contact residues will aid the rational development of novel peptide and small-molecule inhibitors of fusion proteins from exotic or emerging viral pathogens.

Of particular interest, a single amino acid substitution within the {alpha}-helical region yielded a significant improvement in peptide potency. Examination of the TM crystal structure provides an attractive explanation for the increased activity of Pcr-I412A. In TM, the side chain of Ile412 extends at a shallow angle to the side of the {alpha}-helix region and is oriented toward the surface of the coiled coil but away from the largely hydrophobic groove (16) (Fig. 7). In this orientation, there is an unfavorable clash between the {gamma}-2- and {delta}-methyl groups of the Ile412 side chain and the side chain of Gln356 on the surface of the coiled coil. Although alternative docking solutions are possible, our data are consistent with the view that by replacing the isoleucine of the peptide inhibitor with alanine, the steric clash is removed (Fig. 7B), thereby allowing Pcr-I412A to dock more effectively with the coiled coil than the parental peptide Pcr-400. Our results reveal that improved peptide inhibitors can be obtained and that modification of residues other than those involved in direct binding with the hydrophobic groove can enhance the biological activities of the peptides. Based on these observations and on the predicted flexibility of the extended nonhelical leash, we suggest that inhibitory peptides based on "leash-like" structures may be intrinsically amenable to structure-assisted optimization to improve both the potency and efficacy of the inhibitors.


Figure 7
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  		FIG. 7. Replacement of Ile412 with alanine improves peptide binding to the coiled coil by removal of a steric clash. (A) Image of the region mimicked by the inhibitory peptide shown in green-stick format, with the {alpha}-helical region shown as a ribbon, I412 (blue stick format, with dots to show the space filled) clashes with Gln356 (red) on the sidewall of the groove on the coiled coil (gray; space-filling form). (B) This clash is removed by the alanine substitution in peptide Pcr-I412A; the model presented is consistent with the derived data, but alternative peptide-docking solutions are possible. The arrowhead indicates the position of Leu413.

We have noted that inhibitors based on the consensus sequence of HTLV-1 TM, and which are identical to the C helix region of strain CR, are more potent than those based on strain ATK. Our data presented here and elsewhere (23, 26) and our unpublished studies indicate that the improved activity of Pcr-400 (Table 2) is due to three amino acid differences between these inhibitors (Table 1). Replacement of arginine at residue 2 of Patk-400 with cysteine in Pcr-400 overcomes a charge clash between the peptide and the coiled coil (26). The replacement of proline at residue 4 with leucine provides additional hydrophobic interactions with the coiled coil, but the principal effect is to remove a proline-induced distortion of the extended leash sequences, thereby allowing Ile405 to dock appropriately with the coiled coil. Finally, the serine at residue 11 of Pcr-400 does not contribute directly to contact with the coiled coil. Nevertheless, the serine replaces a proline residue in Patk-400 that is situated in the middle of the short {alpha}-helix. Proline is not readily accommodated within {alpha}-helices, and our studies suggest that distortion of the helical region by proline places Leu413 in a suboptimal location for interaction with the coiled coil. Therefore, no single-amino-acid substitution accounts for the improved activity of Pcr-400. Instead, the improved activity is due to the combined effects of the three amino acid differences between the prototypic peptide, Patk-400, and the improved inhibitor, Pcr-400.

Mimetic peptides based on the leash and C helix of the HTLV-1 trimer of hairpins have potential as antiviral therapeutic agents. Worldwide, HTLV-1 infections have considerable clinical impact and are associated with an aggressive adult T-cell leukemia and a progressive neurodegenerative disease, HTLV-1-associated myelopathy, or tropical spastic paraparesis. Moreover, viral replication persists in HTLV-associated disease (1, 11, 24, 37), implying that novel antiretroviral compounds targeting HTLV entry may be of therapeutic value. Our study identified a small region of the HTLV-1 envelope that could be targeted by small-molecule inhibitors, namely, the deep hydrophobic pockets at the membrane-proximal end of the coiled coil that are required for stable docking of the C-terminal {alpha}-helix and C-terminal leash of the HTLV-1 trimer of hairpins. Finally, since extended peptide motifs and short helical segments are found in a number of disparate viral fusion proteins, we suggest that mimetic peptides based on leash-like regions of other class 1 viral fusion proteins may be of therapeutic utility in the treatment of infections by novel or emerging viral pathogens.


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ACKNOWLEDGMENTS
 
We thank Jenny Woof and Alexander Schüttelkopf for helpful comments on the manuscript and Steve Everett and members of the laboratory for helpful discussions.

The Leukemia Research Fund generously supported this work through a project grant (LRF-0227) to D.W.B.


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FOOTNOTES
 
* Corresponding author. Mailing address: The Biomedical Research Centre, Ninewells, Hospital and Medical School, The University, Dundee DD1 9SY, Scotland, United Kingdom. Phone: 44 1382 660111. Fax: 44 1382 669993. E-mail: d.w.brighty{at}dundee.ac.uk Back

{triangledown} Published ahead of print on 27 February 2008. Back


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Journal of Virology, May 2008, p. 4965-4973, Vol. 82, No. 10
0022-538X/08/$08.00+0     doi:10.1128/JVI.02458-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Lamb, D., Mirsaliotis, A., Kelly, S. M., Brighty, D. W. (2009). Basic Residues Are Critical to the Activity of Peptide Inhibitors of Human T Cell Leukemia Virus Type 1 Entry. J. Biol. Chem. 284: 6575-6584 [Abstract] [Full Text]  

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