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The Biomedical Research Centre, Ninewells Hospital and Medical School, The University, Dundee DD1 9SY, UK
Correspondence
David W. Brighty
brighty{at}cancer.org.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sodroski, 1999). The receptor-stimulated changes in Env conformation promote insertion of the fusion peptide of TM into the target-cell membrane and, through a cascade of molecular rearrangements, promote membrane fusion (Eckert & Kim, 2001; Sodroski, 1999). For both human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV), this model of virus entry is supported by the observations that mutations within Env (Delamarre et al., 1994, 1997, 1999; Pique et al., 1990, 1992, 1993; Poon & Chen, 1998; Rosenberg et al., 1997), anti-Env antibodies (Baba et al., 1993; Blanchard et al., 1999; Burton et al., 2004, 2005; Desgranges et al., 1994; Kuroki et al., 1992; Palker et al., 1992; Shida et al., 1987; Tanaka et al., 1991), recombinant competitive ligands (Deen et al., 1988; Fisher et al., 1988; Jassal et al., 2001), soluble polyanions (Moulard et al., 2000; Piñón et al., 2003b) and Env-derived synthetic peptides (Brighty & Jassal, 2001; Jiang et al., 1993; Jinno et al., 1999; Piñón et al., 2003a; Sagara et al., 1996; Wild et al., 1992) all interfere with envelope-mediated membrane fusion and potently block retrovirus infection of cells. The ability to interfere with envelope activity has generated considerable hope that small-molecule antagonists of envelope function or immunological agents targeting envelope can be developed as clinically relevant therapies to combat retrovirus infections.
Recently, the crystal structure for one conformation of the extracellular region of HTLV-1 TM has been solved and reveals that, in common with other viral fusion proteins (Baker et al., 1999; Bullough et al., 1994; Chan et al., 1997; Fass et al., 1996; Kobe et al., 1999; Malashkevich et al., 1998, 1999; Rosenthal et al., 1998; Weissenhorn et al., 1998; Wilson et al., 1981), HTLV-1 TM adopts a trimer-of-hairpins motif (Fig. 1). For HTLV-1 TM (gp21), the N-terminal 
-helices of three gp21 monomers assemble to form a central, triple-stranded coiled coil. At the base of the coiled coil, each monomer forms a 180° loop stabilized by formation of a disulphide bond, thereby reversing the polypeptide-chain direction; the C-terminal sequences adopt an extended structure, including a short helical region that lies in an antiparallel manner within the grooves formed by the core coiled coil to yield the characteristic six-helix-bundle or trimer-of-hairpins conformation of retroviral TM (Fig. 1) (Kobe et al., 1999).
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Previously, our group and others have characterized synthetic peptides that potently inhibit HTLV-1 Env-mediated membrane fusion and virus entry into cells (Brighty & Jassal, 2001; Jinno et al., 1999; Piñón et al., 2003a; Sagara et al., 1996). One of the inhibitory peptides, P-400, mimics the extended C-helical region (aa 400–429) of HTLV-1 TM. By using pull-down and electrophoretic mobility-shift assays, Brighty and colleagues (Piñón et al., 2003a) demonstrated that C-helix-mimetic peptides interact directly with the core coiled-coil domain of HTLV-1 TM, and that the inhibitory properties of the peptides are correlated with their ability to bind to the core coiled coil. Whilst it is clear that the inhibitory peptides interact directly with the core coiled-coil motif and that this interaction is crucial to the peptides' inhibitory properties, it is currently unclear whether this is the only site of interaction with envelope. Nevertheless, the demonstration that P-400-related peptides inhibit membrane fusion by targeting the core coiled coil of TM raises the possibility that additional antagonists of membrane fusion targeting the core coiled coil can be identified.
To date, the assays available to monitor association of C-helix-mimetic peptides or the C-helical region of TM with the core coiled coil are laborious, qualitative rather than quantitative and not suited to the high-throughput methodologies required for screening panels of reagents for inhibitory activity. To overcome these obstacles, an in vitro assay has been developed to examine in a quantitative manner the association of C-helix peptides with recombinant HTLV-1 TM. The assay has been used to examine the specificity of binding of C-helix mimetics to derivatives of HTLV-1 TM and to investigate the inhibitory activity of a variety of TM-derived synthetic peptides. Finally, we have used the assay to identify a novel monoclonal antibody (mAb) that disrupts formation of the HTLV-1 six-helix bundle in vitro. The assay developed in this study is of utility in the biochemical analysis of peptide association with the core coiled coil and will facilitate rapid screening of reagents for the ability to disrupt formation of the six-helix bundle of HTLV-1 TM and, ultimately, to identify novel inhibitors of envelope-mediated membrane fusion.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Plasmids.
The plasmids pHTE-1 (Dokhelar et al., 1989), pMAL-gp21hairpin and pMAL-gp21fishhook, and the control maltose-binding protein (MBP) vector pMAL-stop, have been described previously (Piñón et al., 2003a). The vectors pMAL-N-helix (MBP–N-helix), encoding aa M338–W387, and pMAL-C-helix, encoding aa C400–L429, of the TM region of HTLV-1 envelope were generated by PCR amplification of the respective coding regions using primers that incorporate a 5' EcoRI and a 3' HindIII site. The PCR products were digested and the fragments were cloned into the EcoRI and HindIII sites of pMalc2.
Peptide synthesis.
Peptides (Table 1) were synthesized by using standard solid-phase Fmoc chemistry and, unless stated otherwise, have acetylated N termini and amidated C termini. The peptides were purified by reverse-phase HPLC and verified for purity by MALDI-TOF (matrix-assisted laser desorption/ionization–time-of-flight) mass spectrometry. All peptides were dissolved in DMSO, the concentration of peptide stock solutions was confirmed by measurement of A280 in 6 M guanidine hydrochloride and peptides were used at the final concentrations indicated.
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), were grown at 37 °C in the presence of ampicillin (100 µg ml–1) until the OD600 reached 0.6. Cells were induced with IPTG at a final concentration of 0.5 mM for 4 h at 37 °C. Cells were harvested, resuspended in column buffer [20 mM Tris/HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA] supplemented with PMSF (1 mM) and aprotinin (1 µg ml–1) and lysed by sonication. Clarified cell lysates were diluted 1 : 5 in column buffer and loaded onto an amylose column pre-equilibrated with column buffer. The column was washed with 12 column volumes of column buffer and the MBP and MBP–TM fusion proteins were eluted with column buffer containing 10 mM maltose. The concentration of the fusion proteins was estimated by Bradford assay. The oligomerization states of our MBP–TM chimeras were assessed by Superdex 200 gel-filtration chromatography in PBS (Piñón et al., 2003a; data not shown).
ELISA.
Microtitre 96-well plates (MaxiSorp; Nunc) were coated overnight at 4 °C with the chimeric MBP–TM fusion proteins (MBP–Fishhook, MBP–Hairpin, MBP–N-helix, MBP–C-helix or control MBP; all at 10 µg ml–1) in PBS (pH 7.2). Plates were blocked (5 % Marvel/PBS/0.2 %Tween 20) for 1 h at room temperature, washed (five times) and immobilized antigen was incubated with antibodies at the concentrations indicated for 2 h at room temperature. After washing (five times), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1 : 10 000 dilution) (Sigma) was added and the bound antibody was detected by using fresh ABTS substrate at 15 mg ml–1 [in 0.1 M citric acid, 60 % H2O2 (30 % w/v)]. After 10–20 min, colour development was stopped and A415 was read.
Peptide-binding and competition-binding assays.
Microtitre 96-well plates (MaxiSorp; Nunc) were coated overnight at 4 °C with MBP–Fishhook or the test MBP-fusion protein (10 µg ml–1) in PBS (pH 7.2). Plates were washed twice with wash buffer (PBS, 0.2 % Tween 20) and blocked with blocking buffer (5 % Marvel in PBS, 0.2 % Tween 20) for 1 h at room temperature. The peptide Bio-Pcr-400 was prepared as a stock solution in wash buffer containing 5 mM DTT. Peptide Bio-Pcr-400 was incubated with target ligands, in the presence or absence of peptide competitors at the concentrations specified, at room temperature for 1 h. When examining the ability of mAbs to block binding of Bio-Pcr-400 to the coiled coil, the peptide was added at 2.5 µg ml–1 in wash buffer without DTT. Subsequently, plates were washed (five times) to remove unbound peptide and incubated for 1 h with 100 µl streptavidin–HRP (Sigma) (1 : 10 000 dilution) at room temperature. Plates were washed five times to remove unbound streptavidin–HRP and twice with PBS to remove residual detergent. Finally, bound streptavidin–HRP and therefore bound Bio-Pcr-400 was detected by using ABTS substrate and A415 was read (Bio-Rad plate reader).
Syncytium-interference assay.
Syncytium-interference assays were performed as described previously (Jassal et al., 2001). Briefly, 3x105 HeLa cells, transfected with the envelope expression vector pHTE-1, were added to 7.0x105 untransfected HeLa target cells. The effector and target cells were co-cultured in the absence or presence of the P-400-related peptides at the concentrations specified. The cells were incubated for 12–15 h at 37 °C, washed twice with PBS and fixed in PBS/3 % formaldehyde. Assays were performed in triplicate and the number of syncytia from five low-power fields per replicate was scored by light microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) fused to MBP (Piñón et al., 2003a). One of these fusion proteins, MBP–Fishhook, faithfully mimics the central core coiled coil and disulphide-bonded loop of TM (Fig. 1b, c); the fusion protein forms stable trimers, it binds to a peptide inhibitor of envelope-mediated membrane fusion in in vitro pull-down assays and, in syncytium-interference assays, it competes with viral envelope as a target for the inhibitory peptide Pcr-400 (Piñón et al., 2003a). Having demonstrated that the core coiled-coil domain interacts directly with the inhibitory C-helix mimetic Pcr-400, we wished to examine this interaction in more detail with the specific objective of developing a facile assay that can be used to examine the binding reaction between the C-helix peptides and the core coiled coil and to identify inhibitors of the interaction between the core coiled coil and the C-helical region of HTLV-1 TM.
To examine the core coiled coil-binding properties of Pcr-400, a derivative peptide was synthesized that is modified by addition of a biotin group fused to the terminal amine of the peptide by a six-carbon spacer arm. The biotin group allows rapid capture and detection of the peptide by using immobilized streptavidin or streptavidin–HRP conjugates, respectively. The spacer arm was included to avoid steric interference between the biotin group of the modified C-helix peptide and the peptide-binding site on the core coiled coil. As covalent modification of the peptide may affect its biological activity adversely, syncytium-interference assays were used to compare the inhibitory properties of the biotinylated peptide with those of the parental peptide, Pcr-400. Briefly, HeLa cells were transfected with the HTLV-1 Env expression vector pHTE-1 and the transfectants were used as effector cells in cell-fusion assays. Co-culture of untransfected HeLa cells with Env-expressing HeLa cells induced rampant syncytium formation in untreated cultures, but syncytium formation was inhibited dramatically in the presence of the peptide Pcr-400 (Fig. 2a). This inhibition of syncytium formation was specific to Pcr-400, as an irrelevant control peptide derived from the SU region of envelope had no inhibitory effect on cell fusion (Fig. 2a). Moreover, a derivative of Pcr-400 in which aa N407 and S408 were substituted with the bulky aromatic amino acids tyrosine and phenylalanine, respectively, also failed to inhibit syncytium formation. Importantly, the biotinylated peptide Bio-Pcr-400 blocked syncytium formation just as effectively as the parental peptide, demonstrating that the biotin spacer-arm modification had no adverse effect on the biological activity of the inhibitory peptide.
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), indicating that binding to the core coiled coil was a specific property of the C-helix-mimetic peptide.
To examine the target specificity of C-helix peptides and the conformational requirements of TM for peptide binding, the association of Bio-Pcr-400 to a panel of MBP–TM and Env-derived fusion proteins was explored. Published size-exclusion chromatography data and non-denaturing PAGE analysis indicate that two of these MBP-fusion proteins, MBP–Hairpin and MBP–Fishhook, are trimeric and faithfully recapitulate the trimer-of-hairpins structure and the core coiled coil, respectively, of HTLV-1 TM (Piñón et al., 2003a). In addition, two truncated TM derivatives that are incapable of forming trimers (Fig. 3a, b; our unpublished results), but represent the monomeric N-helix and the monomeric C-helix, were also tested for the ability to bind Bio-Pcr-400. In these assays, Bio-Pcr-400 bound specifically only to the trimeric recombinant core coiled coil (Fig. 3c). Bio-Pcr-400 did not bind to the control MBP-carrier protein and, most significantly, we observed little or no binding to the recombinant fusion protein that mimics the trimer-of-hairpins motif of HTLV-1 TM. In addition, Bio-Pcr-400 failed to bind to truncated TM derivatives that represent monomeric forms of the N-helical or C-helical regions of TM. Moreover, as inhibitory peptides may interact with more than one region of envelope (Liu et al., 2005), binding of the biotinylated peptide to intact SU (gp46–Fc; Jassal et al., 2001) was explored by pull-down and enzyme-linked binding assays; however, to date, no additional interactions have been detected (data not shown).
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As expected, the unmodified C-helix mimetic Pcr-400 was found to be a highly effective competitive inhibitor of Bio-Pcr-400 binding to the core coiled coil (Fig. 4a). This competitive inhibition was specific, because a control peptide, C-34, which mimics the C-helical region of HIV-1 envelope, or P-80, an irrelevant peptide derived from the SU region of HTLV-1 Env, failed to inhibit binding of Bio-Pcr-400 (Fig. 4a). In contrast, a C-helix peptide based on the variant HTLV-1 strain ATK (Patk-400), which is a relatively weak inhibitor of membrane fusion catalysed by envelope from the HTLV-1 strain CR, blocked binding of Bio-Pcr-400 to the core coil, but was not as effective as the homologous peptide inhibitor Pcr-400 (Fig. 4b). Moreover, the biologically inactive mutant peptide Pcr-NS407YF also failed to block binding of Bio-Pcr-400 to the core coiled coil (Fig. 4b). The ability of peptides derived from the N-helical regions to competitively block binding of Bio-Pcr-400 to the core coil was also examined. Synthetic peptides P-338 and P-360, which mimic overlapping elements of the N-helical region of HTLV-1 TM, also failed to inhibit binding of Bio-Pcr-400 to the core coiled coil, suggesting that the C-helical peptides have little or no affinity for truncated forms of the N-helical region of TM (Fig. 4b). These data also suggest that the N-helix-mimetic peptides are unlikely to intercalate into, or disrupt the conformation of, the preformed core coiled coil. In keeping with these data, the N-helical-mimetic peptides do not inhibit syncytium formation or membrane fusion catalysed by HTLV-1 envelope (data not shown).
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Seven mAbs reactive with HTLV-1 envelope protein and two control mAbs, one reactive with the V3 loop of HIV-1 envelope and the other reactive with the MBP-carrier protein, were examined for the ability to inhibit binding of Bio-Pcr-400 to the core coiled coil. The vast majority of these mAbs, even at high concentration (5 µg ml–1), were unable to block binding of the bioactive peptide to the recombinant core coiled-coil region of HTLV-1 TM (Fig. 5). However, mAb 18-8 antagonized binding of Bio-Pcr-400 to the core coiled coil in a concentration-dependent manner (Fig. 5). Thus, in vitro, mAb 18-8 is able to interfere with formation of the six-helix-bundle structure of HTLV-1 TM.
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Derivatives of TM fused to MBP were examined for reactivity with mAb 18-8 (Fig. 6). The mAb did not bind to the control fusion partner MBP, and little or no binding of mAb 18-8 was observed with the intact trimer-of-hairpins form of TM. Moreover, mAb 18-8 failed to bind to the N-helix derivative (MBP–N-helix) or the trimeric core coiled coil. These data indicate that mAb 18-8 does not recognize an epitope within the N-helical and disulphide-bonded loop regions of TM. Instead, strong reactivity of mAb 18-8 was observed with the MBP–C-helix fusion protein, indicating that the mAb probably recognizes an epitope contained within, or at least partially overlapping, the C-helical region of HTLV-1 TM. Moreover, recognition of this epitope appears to be conformation-dependent, as mAb 18-8 failed to bind to the trimer-of-hairpins form of recombinant TM.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Association of Pcr-400 with the core coiled coil has been demonstrated in pull-down assays and electrophoretic mobility-shift assays (Piñón et al., 2003a), but the interaction of the inhibitory peptide with other regions of envelope had not been explored. Here, by using a quantitative binding assay, it is demonstrated that binding of the inhibitory peptide to HTLV-1 TM is highly selective, and efficient binding is observed only for the trimeric coiled-coil motif of TM. No binding is observed to the trimer-of-hairpins form of TM, suggesting that, once formed, the six-helix-bundle structure is extremely stable and that synthetic peptides are unable to displace the C-helical region of TM from the grooves in the core coiled coil. Therefore, the peptide inhibitors must bind to fusion-active envelope prior to formation of the six-helix-bundle structure. Importantly, amino acid side chains of each N-helix monomer make direct contact with the C-helical region of TM and, in the trimeric state, these contacts are critical for C-helix binding (Piñón et al., 2003a; Kobe et al., 1999); despite the importance of these side-chain interactions, monomeric forms of the N-helical region are unable to bind the inhibitory peptides. Thus, trimerization is a critical structural requirement for association of the inhibitory peptides with the fusion-active intermediates of HTLV-1 TM. From the data presented here, the coiled coil appears to be the sole target for binding of the C-helix mimetic Pcr-400, as binding to other regions of HTLV TM is not observed. In contrast, for HIV, the well-characterized peptide inhibitor T20 appears to interact with multiple regions of HIV envelope, including the core coiled coil, the membrane-proximal region of gp41 and a CD4-induced co-receptor-binding region on gp120 (Liu et al., 2005). It is worth noting that, for HTLV-1 and the closely related Bovine leukemia virus, accumulating evidence indicates that an interaction occurs between the chain-reversal region of TM and SU, and that this involves formation of a disulphide bond between a CX6CC motif in TM and a reactive CXXC motif in SU (Johnston & Radke, 2000; Wallin et al., 2004). Although Pcr-400 encodes both Cys400 and Cys401, which are required for TM–TM and TM–SU disulphide interactions, we have been unable to detect any interaction of Pcr-400 with SU in our binding and pull-down assays. Thus, for the HTLV-inhibitory peptide Pcr-400, the potential spectrum of binding sites appears to be restricted to the trimeric core coiled coil of the pre-hairpin intermediate of TM.
A notable aspect of the assay described here is that association of the biotinylated peptide with the core coiled coil to yield a six-helix bundle is exquisitely sensitive to competitive inhibition. Control or mutant peptides that fail to inhibit HTLV-1 syncytium formation also fail to compete with Bio-Pcr-400 for association with the coiled coil. Conversely, and as expected, the untagged P-400 peptides based on sequences of HTLV-1 strains ATK and CR are efficient competitors of Bio-Pcr-400 binding to the core coiled coil. Moreover, the homologous CR-derived peptide is the most active inhibitor of syncytium formation and is by far the most efficient competitive inhibitor in the plate-binding assays. Thus, the assay exhibits properties that make it ideal for high-thoughput screening of reagents to identify inhibitors of the six-helix-bundle conformation of TM.
Interestingly, synthetic peptides that mimic fragments of the N-helical region of HTLV-1 TM failed to compete with the core coiled coil for binding to Bio-Pcr-400. Moreover, none of the N-helix-mimetic peptides described so far inhibits envelope-mediated membrane fusion (this study; Sagara et al., 1996). In contrast, synthetic peptides that correspond to overlapping fragments of the HIV-1 N-helix are effective inhibitors of HIV-induced membrane fusion (Eckert & Kim, 2001; Wild et al., 1992). A distinguishing feature of the HIV-derived N-helical peptides is that truncated forms of the HIV-1 N-helical region have a tendency to form trimeric structures (Louis et al., 2003; Lu & Kim, 1997; Lu et al., 1995). From the accumulating data, it is tempting to speculate that the HTLV-1 N-helical peptides are unable to inhibit membrane fusion due to an inability to form stable trimeric coiled coils. In keeping with this view, the data presented here demonstrate that monomeric forms of the HTLV-1 N-helices fail to bind C-helix peptides in direct binding assays. It is not clear why truncated forms of HTLV-1 TM that encode the entire leucine/isoleucine heptad-repeat region, or fragments thereof, lack the propensity to form stable trimers, but this aspect of TM function is currently being investigated.
The inhibition of HTLV-1 Env-induced membrane fusion by C-helix peptides suggests that strategies designed to elicit neutralizing antibodies reactive with the core coiled coil or C-helix of TM would be of value in the development of an HTLV-1 vaccine. The development of a plate-based assay for association of C-helix peptides with the core coiled coil has permitted a survey of mAbs for the ability to prevent six-helix-bundle formation. This strategy successfully identified a mAb that, in a dose-dependent manner, prevents association of the C-helix peptide Pcr-400 with the core coiled coil. Epitope mapping confirmed that the mAb recognizes a motif that is contained within, or overlaps with, the C-helical region of TM. Given the reactivity of mAb 18-8, it is likely that the antibody binds directly to the C-helical peptides and prevents their association with the coiled coil sterically. It is therefore surprising that, whilst the antibody is able to prevent six-helix-bundle formation in vitro, it is unable, even at high concentrations, to block envelope-mediated membrane fusion in syncytium-interference assays or virus entry in pseudotyping assays. Whilst several plausible explanations could account for the lack of neutralizing activity displayed by mAb 18-8, by far the simplest explanation is that the epitope recognized by this antibody is inaccessible during the fusion process.
Precedents for lack of neutralizing activity of antibodies targeting the fusion-dependent structures of retroviral TM have been documented. In the case of HIV, a mAb (NC-1) reactive with the gp41 six-helix-bundle structure failed to neutralize virus infection (de Rosny et al., 2004), whilst polyclonal antibodies directed to the trimeric N-helix peptides demonstrated inhibitory activity only under very selective conditions (Golding et al., 2002). Taken together, the published studies and the data presented here imply that it will be technically challenging to generate antibodies that possess the required target specificity and neutralizing activity against retrovirus TM proteins. Nevertheless, a notable success has been reported. By using phage-display technology, Miller et al. (2005) isolated a single-chain antibody, denoted D5, that recognizes trimeric N-helical peptides that mimic the HIV core coiled coil. When converted to a full IgG molecule, N-helix-binding activity was retained, D5 IgG blocked HIV envelope-mediated membrane fusion and the antibody neutralized a range of HIV-1 clinical isolates (Miller et al., 2005), confirming elegantly that neutralization of virus infection by antibodies targeted to the pre-hairpin intermediate of TM is achievable. It remains to be determined whether neutralizing antibodies reactive with the pre-hairpin intermediate of HTLV-1 TM can be identified, but the identification of an antibody that recognizes the C-helical region of TM and antagonizes six-helix-bundle formation in vitro indicates that an extensive search for such antibodies is warranted.
Retrovirus entry into cells is emerging as a validated target for antiretroviral therapy (Kilby & Eron, 2003; Kilby et al., 1998; Lalezari et al., 2003) and inhibition of HTLV-1 entry by neutralizing antibodies or small-molecule antagonists of envelope function appears to be a viable objective for therapeutic intervention in HTLV-1-associated disease. From this study, it is clear that antibodies targeting the relevant pre-hairpin structures of HTLV-1 TM can be identified, but it remains to be determined whether antibodies with the appropriate neutralizing activity can be generated. Nevertheless, the development of a quantitative assay for binding of C-helix peptides to the core coiled coil will facilitate the characterization of pre-hairpin reactive antibodies, permit further biochemical analysis of inhibitory peptides and, ultimately, be of utility in the identification of small-molecule antagonists of envelope function.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Baker, K. A., Dutch, R. E., Lamb, R. A. & Jardetzky, T. S. (1999). Structural basis for paramyxovirus-mediated membrane fusion. Mol Cell 3, 309–319.[CrossRef][Medline]
Blanchard, S., Astier-Gin, T., Tallet, B., Moynet, D., Londos-Gagliardi, D. & Guillemain, B. (1999). Amino acid changes at positions 173 and 187 in the human T-cell leukemia virus type 1 surface glycoprotein induce specific neutralizing antibodies. J Virol 73, 9369–9376.
Brighty, D. W. & Jassal, S. R. (2001). The synthetic peptide P-197 inhibits human T-cell leukemia virus type 1 envelope-mediated syncytium formation by a mechanism that is independent of Hsc70. J Virol 75, 10472–10478.
Bullough, P. A., Hughson, F. M., Skehel, J. J. & Wiley, D. C. (1994). Structure of influenza haemagglutinin at the pH of membrane fusion. Nature 371, 37–43.[CrossRef][Medline]
Burton, D. R., Desrosiers, R. C., Doms, R. W., Koff, W. C., Kwong, P. D., Moore, J. P., Nabel, G. J., Sodroski, J., Wilson, I. A. & Wyatt, R. T. (2004). HIV vaccine design and the neutralizing antibody problem. Nat Immunol 5, 233–236.[CrossRef][Medline]
Burton, D. R., Stanfield, R. L. & Wilson, I. A. (2005). Antibody vs. HIV in a clash of evolutionary titans. Proc Natl Acad Sci U S A 102, 14943–14948.
Chan, D. C., Fass, D., Berger, J. M. & Kim, P. S. (1997). Core structure of gp41 from the HIV envelope glycoprotein. Cell 89, 263–273.[CrossRef][Medline]
Deen, K. C., McDougal, J. S., Inacker, R., Folena-Wasserman, G., Arthos, J., Rosenberg, J., Maddon, P. J., Axel, R. & Sweet, R. W. (1988). A soluble form of CD4 (T4) protein inhibits AIDS virus infection. Nature 331, 82–84.[CrossRef][Medline]
Delamarre, L., Pique, C., Pham, D., Tursz, T. & Dokhelar, M. C. (1994). Identification of functional regions in the human T-cell leukemia virus type I SU glycoprotein. J Virol 68, 3544–3549.
Delamarre, L., Rosenberg, A. R., Pique, C., Pham, D. & Dokhelar, M. C. (1997). A novel human T-leukemia virus type 1 cell-to-cell transmission assay permits definition of SU glycoprotein amino acids important for infectivity. J Virol 71, 259–266.[Abstract]
Delamarre, L., Pique, C., Rosenberg, A. R., Blot, V., Grange, M. P., Le Blanc, I. & Dokhelar, M. C. (1999). The Y-S-L-I tyrosine-based motif in the cytoplasmic domain of the human T-cell leukemia virus type 1 envelope is essential for cell-to-cell transmission. J Virol 73, 9659–9663.
de Rosny, E., Vassell, R., Jiang, S., Kunert, K. & Weiss, C. D. (2004). Binding of the 2F5 monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type 1 gp41: implications for fusion-inducing conformational changes. J Virol 78, 2627–2631.
Desgranges, C., Souche, S., Vernant, J. C., Smadja, D., Vahlne, A. & Horal, P. (1994). Identification of novel neutralization-inducing regions of the human T cell lymphotropic virus type I envelope glycoproteins with human HTLV-I-seropositive sera. AIDS Res Hum Retroviruses 10, 163–173.[Medline]
Dokhelar, M. C., Pickford, H., Sodroski, J. & Haseltine, W. A. (1989). HTLV-1 p27rex regulates gag and env protein expression. J Acquir Immune Defic Syndr 2, 431–440.
Eckert, D. M. & Kim, P. S. (2001). Mechanisms of viral membrane fusion and its inhibition. Annu Rev Biochem 70, 777–810.[CrossRef][Medline]
Fass, D., Harrison, S. C. & Kim, P. S. (1996). Retrovirus envelope domain at 1.7 Å resolution. Nat Struct Biol 3, 465–469.[CrossRef][Medline]
Fisher, R. A., Bertonis, J. M., Meier, W., Johnson, V. A., Costopoulos, D. S., Liu, T., Tizard, R., Walker, B. D., Hirsch, M. S. & other authors (1988). HIV infection is blocked in vitro by recombinant soluble CD4. Nature 331, 76–78.[CrossRef][Medline]
Golding, H., Zaitseva, M., de Rosny, E., King, L. R., Manischewitz, J., Sidorov, I., Gorny, M. K., Zolla-Pazner, S., Dimitrov, D. S. & Weiss, C. D. (2002). Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates. J Virol 76, 6780–6790.
Jassal, S. R., Lairmore, M. D., Leigh-Brown, A. J. & Brighty, D. W. (2001). Soluble recombinant HTLV-1 surface glycoprotein competitively inhibits syncytia formation and viral infection of cells. Virus Res 78, 17–34.[CrossRef][Medline]
Jiang, S., Lin, K., Strick, N. & Neurath, A. R. (1993). HIV-1 inhibition by a peptide. Nature 365, 113.[Medline]
Jinno, A., Haraguchi, Y., Shiraki, H. & Hoshino, H. (1999). Inhibition of cell-free human T-cell leukaemia virus type 1 infection at a postbinding step by the synthetic peptide derived from an ectodomain of the gp21 transmembrane glycoprotein. J Virol 73, 9683–9689.
Johnston, E. R. & Radke, K. (2000). The SU and TM envelope protein subunits of bovine leukemia virus are linked by disulfide bonds, both in cells and in virions. J Virol 74, 2930–2935.
Kilby, J. M. & Eron, J. J. (2003). Novel therapies based on mechanisms of HIV-1 cell entry. N Engl J Med 348, 2228–2238.
Kilby, J. M., Hopkins, S., Venetta, T. M., DiMassimo, B., Cloud, G. A., Lee, J. Y., Alldredge, L., Hunter, E., Lambert, D. & other authors (1998). Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nat Med 4, 1302–1307.[CrossRef][Medline]
Kobe, B., Center, R. J., Kemp, B. E. & Poumbourios, P. (1999). Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins. Proc Natl Acad Sci U S A 96, 4319–4324.
Kuroki, M., Nakamura, M., Itoyama, Y., Tanaka, Y., Shiraki, H., Baba, E., Esaki, T., Tatsumoto, T., Nagafuchi, S. & Nakano, S. (1992). Identification of new epitopes recognized by human monoclonal antibodies with neutralizing and antibody-dependent cellular cytotoxicity activities specific for human T cell leukemia virus type 1. J Immunol 149, 940–948.[Abstract]
Lalezari, J. P., Henry, K., O'Hearn, M., Montaner, J. S., Piliero, P. J., Trottier, B., Walmsley, S., Cohen, C., Kuritzkes, D. R. & other authors (2003). Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in North and South America. N Engl J Med 348, 2175–2185.
Liu, S., Lu, H., Niu, J., Xu, Y., Wu, S. & Jiang, S. (2005). Different from the HIV fusion inhibitor C34, the anti-HIV drug Fuzeon (T-20) inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120. J Biol Chem 280, 11259–11273.
Louis, J. M., Nesheiwat, I., Chang, L., Clore, G. M. & Bewley, C. A. (2003). Covalent trimers of the internal N-terminal trimeric coiled-coil of gp41 and antibodies directed against them are potent inhibitors of HIV envelope-mediated cell fusion. J Biol Chem 278, 20278–20285.
Lu, M. & Kim, P. S. (1997). A trimeric structural subdomain of the HIV-1 transmembrane glycoprotein. J Biomol Struct Dyn 15, 465–471.[Medline]
Lu, M., Blacklow, S. C. & Kim, P. S. (1995). A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat Struct Biol 2, 1075–1082.[CrossRef][Medline]
Malashkevich, V. N., Chan, D. C., Chutkowski, C. T. & Kim, P. S. (1998). Crystal structure of the simian immunodeficiency virus (SIV) gp41 core: conserved helical interactions underlie the broad activity of gp41 peptides. Proc Natl Acad Sci U S A 95, 9134–9139.
Malashkevich, V. N., Scheider, B. J., McNally, M. L., Milhollen, M. A., Pang, J. X. & Kim, P. S. (1999). Core structure of the envelope glycoprotein GP2 from Ebola virus at 1.9-Å resolution. Proc Natl Acad Sci U S A 96, 2662–2667.
Miller, M. D., Geleziunas, R., Bianchi, E., Lennard, S., Hrin, R., Zhang, H., Lu, M., An, Z., Ingallinella, P. & other authors (2005). A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope. Proc Natl Acad Sci U S A 102, 14759–14764.
Moulard, M., Lortat-Jacob, H., Mondor, I., Roca, G., Wyatt, R., Sodroski, J., Zhao, L., Olson, W., Kwong, P. D. & Sattentau, Q. J. (2000). Selective interactions of polyanions with basic surfaces on human immunodeficiency virus type 1 gp120. J Virol 74, 1948–1960.
Palker, T. J., Riggs, E. R., Spragion, D. E., Muir, A. J., Scearce, R. M., Randall, R. R., McAdams, M. W., McKnight, A., Clapham, P. R. & other authors (1992). Mapping of homologous, amino-terminal neutralizing regions of human T-cell lymphotropic virus type I and II gp46 envelope glycoproteins. J Virol 66, 5879–5889.
Piñón, J. D., Klasse, P. J., Jassal, S. R., Welson, S., Weber, J., Brighty, D. W. & Sattentau, Q. J. (2003a). Human T-cell leukemia virus type 1 envelope glycoprotein gp46 interacts with cell surface heparan sulfate proteoglycans. J Virol 77, 9922–9930.
Piñón, J. D., Kelly, S. M., Price, N. C., Flanagan, J. U. & Brighty, D. W. (2003b). An antiviral peptide targets a coiled-coil domain of the human T-cell leukemia virus envelope glycoprotein. J Virol 77, 3281–3290.
Pique, C., Tursz, T. & Dokhelar, M. C. (1990). Mutations introduced along the HTLV-I envelope gene result in a non-functional protein: a basis for envelope conservation? EMBO J 9, 4243–4248.[Medline]
Pique, C., Pham, D., Tursz, T. & Dokhelar, M. C. (1992). Human T-cell leukemia virus type I envelope protein maturation process: requirements for syncytium formation. J Virol 66, 906–913.
Pique, C., Pham, D., Tursz, T. & Dokhelar, M. C. (1993). The cytoplasmic domain of the human T-cell leukemia virus type I envelope can modulate envelope functions in a cell type-dependent manner. J Virol 67, 557–561.
Poon, B. & Chen, I. S. (1998). Identification of a domain within the human T-cell leukemia virus type 2 envelope required for syncytium induction and replication. J Virol 72, 1959–1966.
Rosenberg, A. R., Delamarre, L., Pique, C., Pham, D. & Dokhelar, M.-C. (1997). The ectodomain of the human T-cell leukemia virus type 1 TM glycoprotein is involved in postfusion events. J Virol 71, 7180–7186.[Abstract]
Rosenthal, P. B., Zhang, X., Formanowski, F., Fitz, W., Wong, C. H., Meier-Ewert, H., Skehel, J. J. & Wiley, D. C. (1998). Structure of the haemagglutinin-esterase-fusion glycoprotein of influenza C virus. Nature 396, 92–96.[CrossRef][Medline]
Sagara, Y., Inoue, Y., Shiraki, H., Jinno, A., Hoshino, H. & Maeda, Y. (1996). Identification and mapping of functional domains on human T-cell lymphotropic virus type 1 envelope proteins by using synthetic peptides. J Virol 70, 1564–1569.[Abstract]
Shida, H., Tochikura, T., Sato, T., Konno, T., Hirayoshi, K., Seki, M., Ito, Y., Hatanaka, M., Hinuma, Y. & other authors (1987). Effect of the recombinant vaccinia viruses that express HTLV-I envelope gene on HTLV-I infection. EMBO J 6, 3379–3384.[Medline]
Sodroski, J. G. (1999). HIV-1 entry inhibitors in the side pocket. Cell 99, 243–246.[CrossRef][Medline]
Tanaka, Y., Zeng, L., Shiraki, H., Shida, H. & Tozawa, H. (1991). Identification of a neutralization epitope on the envelope gp46 antigen of human T cell leukemia virus type I and induction of neutralizing antibody by peptide immunization. J Immunol 147, 354–360.[Abstract]
Wallin, M., Ekstrom, M. & Garoff, H. (2004). Isomerization of the intersubunit disulphide-bond in Env controls retrovirus fusion. EMBO J 23, 54–65.[CrossRef][Medline]
Weissenhorn, W., Carfi, A., Lee, K. H., Skehel, J. J. & Wiley, D. C. (1998). Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain. Mol Cell 2, 605–616.[CrossRef][Medline]
Wild, C., Oas, T., McDanal, C., Bolognesi, D. & Matthews, T. (1992). A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition. Proc Natl Acad Sci U S A 89, 10537–10541.
Wilson, I. A., Skehel, J. J. & Wiley, D. C. (1981). Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 Å resolution. Nature 289, 366–373.[CrossRef][Medline]
Received 17 July 2006;
accepted 30 September 2006.
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