Evidence that the transmembrane domain proximal region of the human T-cell leukemia virus type 1 fusion glycoprotein gp21 has distinct roles in the prefusion and fusion-activated states

To investigate the structural context of the fusion peptide region in human T-cell leukemia virus type 1 gp21, maltose-binding protein (MBP) was used as an N-terminal solubilization partner for the entire gp21 ectodomain (residues 313-445) and C-terminally truncated ectodomain fragments. The bacterial expression of the MBP/gp21 chimeras resulted in soluble trimers containing intramonomer disulfide bonds. Detergents blocked the proteolytic cleavage of fusion peptide residues in the MBP/gp21-(313-425) chimera, indicating that the fusion peptide is available for interaction with detergent despite the presence of an N-terminal MBP domain. Limited proteolysis experiments indicated that the transmembrane domain proximal sequence Thr(425)-Ala(439) protects fusion peptide residues from chymotrypsin. MBP/gp21 chimera stability therefore depends on a functional interaction between N-terminal and transmembrane domain proximal regions in a gp21 helical hairpin structure. In addition, thermal aggregation experiments indicated that the Thr(425)-Ser(436) sequence confers stability to the fusion peptide-containing MBP/gp21 chimeras. The functional role of the transmembrane domain proximal sequence was assessed by alanine-scanning mutagenesis of the full-length envelope glycoprotein, with 11 of 12 single alanine substitutions resulting in 1.5- to 4.5-fold enhancements in cell-cell fusion activity. By contrast, single alanine substitutions in MBP/gp21 did not significantly alter chimera stability, indicating that multiple residues within the transmembrane domain proximal region and the fusion peptide and adjacent glycine-rich segment contribute to stability, thereby mitigating the potential effects of the substitutions. The fusion-enhancing effects of the substitutions are therefore likely to be caused by alteration of the prefusion complex. Our observations suggest that the function of the transmembrane domain proximal sequence in the prefusion envelope glycoprotein is distinct from its role in stabilizing the fusion peptide region in the fusion-activated helical hairpin conformation of gp21.     

De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.     

Changes in the transmembrane region of the human immunodeficiency virus type 1 gp41 envelope glycoprotein affect membrane fusion.

The charged amino acids near or within the membrane-spanning region of the human immunodeficiency virus type 1 gp41 envelope glycoprotein were altered. Two mutants were defective for syncytium formation and virus replication even though levels of envelope glycoproteins on the cell or virion surface and CD4 binding were comparable to those of the wild-type proteins. Thus, in addition to anchoring the envelope glycoproteins, sequences proximal to the membrane-spanning gp41 region are important for the membrane fusion process.     

Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.     

Multimerization potential of the cytoplasmic domain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41

We previously demonstrated that an envelope mutant of human immunodeficiency virus type 1 lacking the entire cytoplasmic domain interferes in trans with the production of infectious virus by inclusion of the mutant envelope into the wild-type envelope complex. We also showed that the envelope incorporation into virions is not affected when the wild-type envelope is coexpressed with the mutant envelope. These results suggest that an oligomeric structure of the cytoplasmic domain is functionally required for viral infectivity. To understand whether the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 has the potential to self-assemble as an oligomer, in the present study we fused the coding sequence of the entire cytoplasmic domain at 3' to the Escherichia coli malE gene, which encodes a monomeric maltose-binding protein. The expressed fusion protein was examined by chemical cross-linking, sucrose gradient centrifugation, and gel filtration. The results showed that the cytoplasmic domain of gp41 assembles into a high-ordered structural complex. The intersubunit interaction of the cytoplasmic domain was also confirmed by a mammalian two-hybrid system that detects protein-protein interactions in eucaryotic cells. A cytoplasmic domain fragment expressed in eucaryotic cells was pulled down by glutathione-Sepharose 4B beads via its association with another cytoplasmic domain fragment fused to the C terminus of the glutathione S-transferase moiety. We also found that sequences encompassing the lentiviral lytic peptide-1 and lentiviral lytic peptide-2, which are located within residues 828-856 and 770-795, respectively, play a critical role in cytoplasmic domain self-assembly. Taken together, the results from the present study indicate that the cytoplasmic domain of gp41 by itself is sufficient to assemble into a multimeric structure. This finding supports the hypothesis that a multimeric form of the gp41 cytoplasmic domain plays a crucial role in virus infectivity.     

The ectodomain of the human T-cell leukemia virus type 1 TM glycoprotein is involved in postfusion events.

To examine the contribution of the transmembrane envelope glycoprotein (TM) to the infectivity of the human T-cell leukemia virus type 1 (HTLV-1), single amino acid substitutions were introduced throughout its ectodomain. The mutated envelopes were tested for intracellular maturation and for functions, including ability to elicit syncytium formation and ability to mediate cell-to-cell transmission of the virus. Three major phenotypes, defining three functionally distinct regions, were identified. (i) Mutations causing defects in intracellular maturation of the envelope precursor are mostly distributed in the central portion of the TM ectodomain, containing the immunosuppressive peptide. This region, which includes vicinal cysteines thought to form an intramolecular disulfide bridge, is probably essential for correct folding of the protein. (ii) Mutations resulting in reduced syncytium-forming ability despite correct intracellular maturation are clustered in the amino-terminal part of the TM ectodomain, within the leucine zipper-like motif. Similar motifs with a propensity to form coiled-coil structures have been implicated in the fusion process driven by other viral envelope proteins, and HTLV-1 may thus conform to this general rule for viral fusion. (iii) Mutants with increased syncytium-forming ability define a region immediately amino-terminal to the membrane-spanning domain. Surprisingly, these mutants exhibited severe defects in infectivity, despite competence for fusion. Existence of this phenotype indicates that capacity for cell-to-cell fusion is not sufficient to ensure viral entry, even in cell-to-cell transmission. The ectodomain of the TM glycoprotein thus may be involved in postfusion events required for full infectivity of HTLV-1, which perhaps represents a unique feature of this poorly infectious retrovirus.     

Membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41.

The transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1) has been implicated in the cytopathology observed during HIV infection. The first amino acids located at the amino terminus are involved in membrane fusion and syncytium formation, while sequences located at the carboxy terminus have been predicted to interact with membranes and modify membrane permeability. The HIV-1 gp41 gene has been cloned and expressed in Escherichia coli cells by using pET vectors to analyze changes in membrane permeability produced by this protein. This system is well suited for expressing toxic genes in an inducible manner and for analyzing the function of proteins that modify membrane permeability. gp41 enhances the permeability of the bacterial membrane to hygromycin B despite the low level of expression of this protein. To localize the regions of gp41 responsible for these effects, a number of fragments spanning different portions of gp41 were inducibly expressed in E. coli. Two regions of gp41 were shown to increase membrane permeability: one located at the carboxy terminus, where two highly amphipathic helices have been predicted, and another one corresponding to the membrane-spanning domain. Expression of the central region of gp41 comprising this domain was highly lytic for E. coli cells and increased membrane permeability to a number of compounds. These findings are discussed in the light of HIV-induced cytopathology and gp41 structure.     

Human T-cell leukemia virus type 1 envelope glycoprotein gp46 interacts with cell surface heparan sulfate proteoglycans

The major receptors required for attachment and entry of the human T-cell leukemia virus type 1 (HTLV-1) remain to be identified. Here we demonstrate that a functional, soluble form of the HTLV-1 surface envelope glycoprotein, gp46, fused to an immunoglobulin Fc region (gp46-Fc) binds to heparan sulfate proteoglycans (HSPGs) on mammalian cells. Substantial binding of gp46-Fc to HeLa and Chinese hamster ovary (CHO) K1 cells that express HSPGs was detected, whereas binding to the sister CHO lines 2244, which expresses no HSPGs, and 2241, which expresses no glycosaminoglycans (GAGs), was much reduced. Enzymatic removal of HSPGs from HeLa and CHO K1 cells also reduced gp46-Fc binding. Dextran sulfate inhibited gp46-Fc binding to HSPG-expressing cells in a dose-dependent manner, whereas chondroitin sulfate was less effective. By contrast, dextran sulfate inhibited gp46-Fc binding to GAG-negative cells such as CHO 2244, CHO 2241, and Jurkat T cells weakly or not at all. Dextran sulfate inhibited HTLV-1 envelope glycoprotein (Env)-pseudotyped virus infection of permissive, HSPG-expressing target cells and blocked syncytium formation between HTLV-1 Env-expressing cells and HSPG-expressing permissive target cells. Finally, HSPG-expressing cells were more permissive for HTLV-1 Env-pseudotyped virus infection than HSPG-negative cells. Thus, similar to other pathogenic viruses, HTLV-1 may have evolved to use HSPGs as cellular attachment receptors to facilitate its propagation.     

Lipid membrane fusion induced by the human immunodeficiency virus type 1 gp41 N-terminal extremity is determined by its orientation in the lipid bilayer.

The amino-terminal extremity of the human immunodeficiency virus type 1 transmembrane protein (gp41) is thought to play a pivotal role in the fusion of virus membranes with the plasma membrane of the target cell and in syncytium formation. Peptides with sequences taken from the human immunodeficiency virus type 1 gp41 fusogenic (synthetic peptides SPwt and SP-2) and nonfusogenic (SP-3 and SP-4) glycoproteins adopt mainly a beta-sheet conformation in the absence of lipid, as determined by attenuated total reflection Fourier transform infrared spectroscopy, and after interaction with large unilamellar liposomes, the beta-sheet is partly converted into an alpha-helical conformation. Peptides SPwt and SP-2 but not SP-3 or SP-4 were able to promote lipid mixing as assessed by fluorescence energy transfer assay and dye leakage in a vesicle leakage assay. By using polarized attenuated total reflection Fourier transform infrared spectroscopy, SPwt and SP-2 were found to adopt an oblique orientation in the lipid membrane whereas SP-3 and SP-4 were oriented nearly parallel to the plane of the membrane. These findings confirm the correlation between the membrane orientation of the alpha-helix and the lipid mixing ability in vitro. Interestingly, the data provide a direct correlation with the fusogenic activity of the parent glycoproteins in vivo.     

Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein.

Insertion of four amino acids into various locations within the amino-terminal halves of the human immunodeficiency virus type 1 gp120 or gp41 envelope glycoprotein disrupts the noncovalent association of these two envelope subunits (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. A. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987). To localize the determinants on the gp120 envelope glycoprotein important for subunit association, amino acids conserved among primate immunodeficiency viruses were changed. Substitution mutations affecting either of two highly conserved regions located at the amino (residues 36 to 45) and carboxyl (residues 491 to 501) ends of the mature gp120 molecule resulted in nearly complete dissociation of the envelope glycoprotein subunits. Partial dissociation phenotypes were observed for some changes affecting residues in the third and fourth conserved gp120 regions. These results suggest that hydrophobic regions at both ends of the gp120 glycoprotein contribute to noncovalent association with the gp41 transmembrane glycoprotein.     

The genetic background as a determinant of human T-cell leukemia virus type 1 proviral load

Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked with HTLV-1-associated diseases. HTLV-1 proviral load is higher in persons with adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis than in asymptomatic carriers. However there are little data available on the factors controlling HTLV-1 proviral load in carriers. To study the effect of genetic background on HTLV-1 proviral load, we employed a mouse model of HTLV-1 infection that we had established. Here we analyzed nine strains of mice and found there is a great variation of proviral load among mouse strains that is not necessarily dependent on major histocompatibility complex. The antibody response is also different among these strains. To our knowledge, this is the first demonstration of the importance of the genetic background other than major histocompatibility complex controlling the HTLV-1 proviral load.     

gp120-independent fusion mediated by the human immunodeficiency virus type 1 gp41 envelope glycoprotein: a reassessment.

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.     

Chimeric synthetic peptides from the envelope (gp46) and the transmembrane (gp21) glycoproteins for the detection of antibodies to human T-cell leukemia virus type II.

Two chimeric synthetic peptides incorporating immunodominant sequences from HTLV-II virus were synthesized. Monomeric peptides P2 and P3 represent sequences from transmembrane protein (gp21) and envelope protein (gp46) of the virus. The peptide P2 is a gp21 (370-396) sequence and the peptide P3 is a gp46 (178-205) sequence. Those peptides were arranged in a way that permits one to obtain different combinations of chimeric peptides (P2-GG-P3 and P3-GG-P2), separated by two glycine residues as spacer arms. The antigenic activity of these peptides was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels anti-HTLV-II-positive sera (n = 11), anti-HTLV-I/II-positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 2), and anti-HTLV-I-positive sera (n = 22), while specificity was evaluated with anti-HIV-positive samples (n = 19) and samples from healthy blood donors (n = 30). The efficacy of the chimeric peptides in solid-phase immunoassays was compared with the monomeric peptides and a mixture of the monomeric peptides. Higher sensitivity was observed for chimeric peptide Q5 assay. Those results may be related to a higher peptide adsorption capacity to the solid surface and for epitope accessibility to the antibodies. This chimeric peptide would be very useful for HTLV-II diagnostic.     

A chimeric human T-cell lymphotropic virus type 1 with the envelope glycoprotein of Moloney murine leukemia virus is infectious for murine cells

We constructed a chimeric human T-cell lymphotropic virus type 1 (HTLV-1) provirus in which the original envelope precursor sequence was replaced by that of ecotropic Moloney murine leukemia virus (Mo-MuLV). Chimeric particles produced by transient transfection of this chimeric provirus were infectious for murine cells, such as NIH 3T3 fibroblasts, lymphoid EL4 cells, and primary CD4(+) T lymphocytes, whereas HTLV-1 particles were not. The infectivity of chimeric particles increased 10 times when the R peptide located at the carboxy terminus of the MuLV envelope glycoprotein was deleted. Primary murine CD4(+) T lymphocytes, infected by the Delta R chimeric virus, released particles that could spread the infection to other naive murine lymphoid cells. This chimeric virus, with the Mo-MuLV envelope glycoprotein and the replication characteristics of HTLV-1, should be useful in studying the pathogenesis of HTLV-1 in a mouse model.     

Characterization of envelope glycoprotein mutants for human T-cell leukemia virus type 1 infectivity and immortalization

The human T-cell leukemia virus type 1 (HTLV-1) envelope protein is required for virus spread. This study further characterizes the role of the envelope protein in HTLV-1 immortalization. Viruses with single amino acid substitutions within the SU protein at residue 75, 81, 95, 101, 105, or 195 or with a C-terminal cytoplasmic domain truncation (CT), as well as an envelope-null (EN) virus, were generated within an infectious molecular clone, ACH. Transfection of 293T cells resulted in the release of similar amounts of virus particles from all of the mutants as determined by p19 enzyme-linked immunosorbent assay and immunoblot analysis of Gag in cell lysates and supernatants. The virus particles from all mutants except ACH-101, ACH-CT, and ACH-EN were infectious for B5 macaque cells in cell-free and cell-to-cell transmission assays and were capable of immortalizing transfected CD4(+) lymphocytes. These results indicate that HTLV-1 spread is required for immortalization.     

Role of the ectodomain of the gp41 transmembrane envelope protein of human immunodeficiency virus type 1 in late steps of the membrane fusion process

The membrane fusion process mediated by the gp41 transmembrane envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) was addressed by a flow cytometry assay detecting exchanges of fluorescent membrane probes (DiI and DiO) between cells expressing the HIV-1 envelope proteins (Env) and target cells. Double-fluorescent cells were detected when target cells expressed the type of chemokine receptor, CXCR4 or CCR5, matching the type of gp120 surface envelope protein, X4 or R5, respectively. Background levels of double-fluorescent cells were observed when the gp120-receptor interaction was blocked by AMD3100, a CXCR4 antagonist. The L568A mutation in the N-terminal heptad repeat (HR1) of gp41 resulted in parallel inhibition of the formation of syncytia and double-fluorescent cells, indicating that gp41 had a direct role in the exchange of fluorescent probes. In contrast, three mutations in the loop region of the gp41 ectodomain, located on either side of the Cys-(X)(5)-Cys motif (W596 M and W610A) or at the distal end of HR1 (D589L), had limited or no apparent effect on membrane lipid mixing between Env(+) and target cells, while they blocked formation of syncytia and markedly reduced the exchanges of cytoplasmic fluorescent probes. The loop region could therefore have a direct or indirect role in events occurring after the merging of membranes, such as the formation or dilation of fusion pores. Two types of inhibitors of HIV-1 entry, the gp41-derived peptide T20 and the betulinic acid derivative RPR103611, had limited effects on membrane exchanges at concentrations blocking or markedly reducing syncytium formation. This finding confirmed that T20 can inhibit the late steps of membrane fusion (post-lipid mixing) and brought forth an indirect argument for the role of the gp41 loop region in these steps, as mutations conferring resistance to RPR103611V were mapped in this region (I595S or L602H).     

Determination of the minimal fusion peptide of bovine leukemia virus gp30

In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide.     

DC-SIGN facilitates fusion of dendritic cells with human T-cell leukemia virus type 1-infected cells

Interactions between the oncogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) and dendritic cells (DCs) are poorly characterized. We show here that monocyte-derived DCs form syncytia and are infected upon coculture with HTLV-1-infected lymphocytes. We examined the role of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin expressed in DCs, in HTLV-1-induced syncytium formation. DC-SIGN is known to bind with high affinity to various viral envelope glycoproteins, including human immunodeficiency virus (HIV) and hepatitis C virus, as well as to the cellular receptors ICAM-2 and ICAM-3. After cocultivating DCs and HTLV-1-infected cells, we found that anti-DC-SIGN monoclonal antibodies (MAbs) were able to decrease the number and size of HTLV-1-induced syncytia. Moreover, expression of the lectin in epithelial-cell lines dramatically enhanced the ability to fuse with HTLV-1-positive cells. Interestingly, in contrast to the envelope (Env) glycoproteins of HIV and other viruses, that of HTLV-1 does not bind directly to DC-SIGN. The facilitating role of the lectin in HTLV-1 syncytium formation is mediated by its interaction with ICAM-2 and ICAM-3, as demonstrated by use of MAbs directed against these adhesion molecules. Altogether, our results indicate that DC-SIGN facilitates HTLV-1 infection and fusion of DCs through an ICAM-dependent mechanism.     

An antiviral peptide targets a coiled-coil domain of the human T-cell leukemia virus envelope glycoprotein

Retrovirus entry into cells is mediated by the viral envelope glycoproteins which, through a cascade of conformational changes, orchestrate fusion of the viral and cellular membranes. In the absence of membrane fusion, viral entry into the host cell cannot occur. For human T-cell leukemia virus type 1 (HTLV-1), synthetic peptides that mimic a carboxy-terminal region of the transmembrane glycoprotein (TM) ectodomain are potent inhibitors of membrane fusion and virus entry. Here, we demonstrate that this class of inhibitor targets a fusion-active structure of HTLV-1 envelope. In particular, the peptides bind specifically to a core coiled-coil domain of envelope, and peptide variants that fail to bind the coiled-coil lack inhibitory activity. Our data indicate that the inhibitory peptides likely function by disrupting the formation of a trimer-of-hairpins structure that is required for membrane fusion. Importantly, we also show that peptides exhibiting dramatically increased potency can be readily obtained. We suggest that peptides or peptide mimetics targeting the fusion-active structures of envelope may be of therapeutic value in the treatment of HTLV-1 infections.